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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03941</article-id><article-id pub-id-type="doi">10.7554/eLife.03941</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biochemistry</subject></subj-group><subj-group subj-group-type="heading"><subject>Human biology and medicine</subject></subj-group></article-categories><title-group><article-title>The glucuronyltransferase B4GAT1 is required for initiation of LARGE-mediated α-dystroglycan functional glycosylation</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-16383"><name><surname>Willer</surname><given-names>Tobias</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16384"><name><surname>Inamori</surname><given-names>Kei-ichiro</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="aff" rid="aff5"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16385"><name><surname>Venzke</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16386"><name><surname>Harvey</surname><given-names>Corinne</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16387"><name><surname>Morgensen</surname><given-names>Greg</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16388"><name><surname>Hara</surname><given-names>Yuji</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="aff" rid="aff6"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-19649"><name><surname>Beltrán Valero de Bernabé</surname><given-names>Daniel</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16448"><name><surname>Yu</surname><given-names>Liping</given-names></name><xref ref-type="aff" rid="aff7"/><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16392"><name><surname>Wright</surname><given-names>Kevin M</given-names></name><xref ref-type="aff" rid="aff8"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-15214"><name><surname>Campbell</surname><given-names>Kevin P</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff2"/><xref ref-type="aff" rid="aff3"/><xref ref-type="aff" rid="aff4"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Molecular Physiology and Biophysics</institution>, <institution>University of Iowa, Carver College of Medicine</institution>, <addr-line><named-content content-type="city">Iowa City</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Neurology</institution>, <institution>University of Iowa, Carver College of Medicine</institution>, <addr-line><named-content content-type="city">Iowa City</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Department of Internal Medicine</institution>, <institution>University of Iowa, Carver College of Medicine</institution>, <addr-line><named-content content-type="city">Iowa City</named-content></addr-line>, <country>United States</country></aff><aff id="aff4"><institution>Howard Hughes Medical Institute, University of Iowa, Carver College of Medicine</institution>, <addr-line><named-content content-type="city">Iowa City</named-content></addr-line>, <country>United States</country></aff><aff id="aff5"><institution content-type="dept">Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology</institution>, <institution>Tohoku Pharmaceutical University</institution>, <addr-line><named-content content-type="city">Komatsushima</named-content></addr-line>, <country>Japan</country></aff><aff id="aff6"><institution content-type="dept">Department of Synthetic Chemistry and Biological Chemistry</institution>, <institution>Graduate School of Engineering, Kyoto University</institution>, <addr-line><named-content content-type="city">Kyoto</named-content></addr-line>, <country>Japan</country></aff><aff id="aff7"><institution content-type="dept">Medical Nuclear Magnetic Resonance Facility</institution>, <institution>University of Iowa, Carver College of Medicine</institution>, <addr-line><named-content content-type="city">Iowa City</named-content></addr-line>, <country>United States</country></aff><aff id="aff8"><institution content-type="dept">Vollum Institute</institution>, <institution>Oregon Health and Science University</institution>, <addr-line><named-content content-type="city">Portland</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Pfeffer</surname><given-names>Suzanne R</given-names></name><role>Reviewing editor</role><aff><institution>Stanford University</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>kevin-campbell@uiowa.edu</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>03</day><month>10</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03941</elocation-id><history><date date-type="received"><day>09</day><month>07</month><year>2014</year></date><date date-type="accepted"><day>01</day><month>10</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Willer et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Willer et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03941.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="article-reference" xlink:href="10.7554/eLife.03943"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03941.001</object-id><p>Dystroglycan is a cell membrane receptor that organizes the basement membrane by binding ligands in the extracellular matrix. Proper glycosylation of the α-dystroglycan (α-DG) subunit is essential for these activities, and lack thereof results in neuromuscular disease. Currently, neither the glycan synthesis pathway nor the roles of many known or putative glycosyltransferases that are essential for this process are well understood. Here we show that FKRP, FKTN, TMEM5 and B4GAT1 (formerly known as B3GNT1) localize to the Golgi and contribute to the O-mannosyl post-phosphorylation modification of α-DG. Moreover, we assigned B4GAT1 a function as a xylose β1,4-glucuronyltransferase. Nuclear magnetic resonance studies confirmed that a glucuronic acid β1,4-xylose disaccharide synthesized by B4GAT1 acts as an acceptor primer that can be elongated by LARGE with the ligand-binding heteropolysaccharide. Our findings greatly broaden the understanding of α-DG glycosylation and provide mechanistic insight into why mutations in B4GAT1 disrupt dystroglycan function and cause disease.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.001">http://dx.doi.org/10.7554/eLife.03941.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03941.002</object-id><title>eLife digest</title><p>Dystroglycan is a protein that is critical for the proper function of many tissues, especially muscles and brain. Dystroglycan helps to connect the structural network inside the cell with the matrix outside of the cell. The extracellular matrix fills the space between the cells to serve as a scaffold and hold cells together within a tissue. It is well established that the interaction of cells with their extracellular environments is important for structuring tissues, as well as for helping cells to specialize and migrate. These interactions also play a role in the progression of cancer.</p><p>As is the case for many proteins, dystroglycan must be modified with particular sugar molecules in order to work correctly. Enzymes called glycosyltransferases are responsible for sequentially assembling a complex array of sugar molecules on dystroglycan. This modification is essential for making dystroglycan ‘sticky’, so it can bind to the components of the extracellular matrix. If sugar molecules are added incorrectly, dystroglycan loses its ability to bind to these components. This causes congenital muscular dystrophies, a group of diseases that are characterized by a progressive loss of muscle function.</p><p>Willer et al. use a wide range of experimental techniques to investigate the types of sugar molecules added to dystroglycan, the overall structure of the resulting ‘sticky’ complex and the mechanism whereby it is built. This reveals that a glycosyltransferase known as B3GNT1 is one of the enzymes responsible for adding a sugar molecule to the complex. This enzyme was first described in the literature over a decade ago, and the name B3GNT1 was assigned, according to a code, to reflect the sugar molecule it was thought to transfer to proteins. However, Willer et al. (and independently, Praissman et al.) find that this enzyme actually attaches a different sugar modification to dystroglycan, and so should therefore be called B4GAT1 instead.</p><p>Willer et al. find that the sugar molecule added by the B4GAT1 enzyme acts as a platform for the assembly of a much larger sugar polymer that cells use to anchor themselves within a tissue. Some viruses–including Lassa virus, which causes severe fever and bleeding–also use the ‘sticky’ sugar modification of dystroglycan to bind to and invade cells, causing disease in humans. Understanding the structure of this complex, and how these sugar modifications are added to dystroglycan, could therefore help to develop treatments for a wide range of diseases like progressive muscle weakening and viral infections.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.002">http://dx.doi.org/10.7554/eLife.03941.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>glycosylation</kwd><kwd>B4GAT1</kwd><kwd>B3GNT1</kwd><kwd>LARGE</kwd><kwd>alpha-dystroglycan</kwd><kwd>basement membrane</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>Paul D. Wellstone Muscular Dystrophy Cooperative Research Center - 1U54NS053672</award-id><principal-award-recipient><name><surname>Campbell</surname><given-names>Kevin P</given-names></name><name><surname>Willer</surname><given-names>Tobias</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>American Recovery and Reinvestment Act (ARRA) - 1 RC2 NS069521-01</award-id><principal-award-recipient><name><surname>Campbell</surname><given-names>Kevin P</given-names></name><name><surname>Willer</surname><given-names>Tobias</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100005202</institution-id><institution>Muscular Dystrophy Association</institution></institution-wrap></funding-source><award-id>238219</award-id><principal-award-recipient><name><surname>Campbell</surname><given-names>Kevin P</given-names></name><name><surname>Willer</surname><given-names>Tobias</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000011</institution-id><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Campbell</surname><given-names>Kevin P</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Post-phosphoryl modification of α-dystroglycan requires the glucuronyltransferase B4GAT1; this enzyme synthesizes the acceptor glycan that serves as a primer for the glycosyltransferase LARGE to synthesize the laminin-binding glycan.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Dystroglycan (DG) is a highly glycosylated basement membrane receptor involved in a variety of physiological processes including maintenance of the skeletal muscle-cell membrane integrity and establishment of the structure and function of the central nervous system (<xref ref-type="bibr" rid="bib4">Barresi and Campbell, 2006</xref>). DG is composed of a cell-surface α-subunit and a transmembrane β-subunit. α-DG acts as a receptor for laminin-G domain-containing extracellular matrix (ECM) proteins such as laminin, agrin, perlecan and neurexin (<xref ref-type="bibr" rid="bib4">Barresi and Campbell, 2006</xref>). In addition, it serves as a cellular receptor and entry site for most Old World arenaviruses, including the highly pathogenic Lassa virus (LASV) and Clade C New Word arenaviruses (<xref ref-type="bibr" rid="bib12">Cao et al., 1998</xref>). LASV is the causative agent of severe hemorrhagic fever in humans, a disease that has a mortality rate of ∼15% resulting in several thousand deaths each year.</p><p>α-DG effectiveness as a receptor is dependent on complex post-translational modifications. Besides numerous modifications with <italic>N</italic>-glycans and mucin-type <italic>O</italic>-glycans, a highly complicated series of additions to a phosphorylated <italic>O</italic>-mannosyl glycan moiety in the N-terminal region of the mucin domain are essential for ligand binding (<xref ref-type="bibr" rid="bib30">Kanagawa et al., 2004</xref>; <xref ref-type="bibr" rid="bib24">Hara et al., 2011</xref>). Defects in the proper post-translational processing of α-DG result in loss of receptor function, and in a broad spectrum of congenital muscular dystrophies (CMDs) that are accompanied by a variety of brain and eye malformations. Collectively, these dystrophies are classified as dystroglycanopathies (<xref ref-type="bibr" rid="bib4">Barresi and Campbell, 2006</xref>). To date, over 17 genes have been reported to be directly or indirectly involved in this ‘functional glycosylation’ of α-DG, and have been linked to human disease when mutated (<xref ref-type="bibr" rid="bib42">Mercuri and Muntoni, 2012</xref>; <xref ref-type="bibr" rid="bib7">Bonnemann et al., 2014</xref>).</p><p>Recent gene discovery efforts revealed several novel dystroglycanopathy genes with unknown function (<xref ref-type="bibr" rid="bib53">Vuillaumier-Barrot et al., 2012</xref>; <xref ref-type="bibr" rid="bib11">Buysse et al., 2013</xref>; <xref ref-type="bibr" rid="bib28">Jae et al., 2013</xref>). In our previous work we were able to assign functions to the POMGNT2 (Protein O-linked mannose N-acetylglucosaminyltransferase 2) (GTDC2), B3GALNT2 and POMK (Protein O-mannose kinase) (SGK196) gene products, which contribute to the synthesis of a phosphorylated, O-mannosyl-linked trisaccharide on α-DG (<xref ref-type="bibr" rid="bib62">Yoshida-Moriguchi et al., 2013</xref>) (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). This so-called Core M3 structure (GalNAc-β3-GlcNAc-β4-Man-α Ser/Thr) is synthesized in the endoplasmic reticulum (ER), and it is thought to be a platform for further functional modification of α-DG as it passes through the secretory pathway. The glycosyltransferase LARGE was shown to synthesize and transfer repeating units of [–3-xylose–α1,3-glucuronic acid-β1–] to α-DG (<xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>). This heteropolymer is postulated to be the terminal glycan moiety anchored by the Core M3 structure. It resembles the ligand-binding glycan and its length correlates with the affinity of α-DG for its ligands (<xref ref-type="bibr" rid="bib22">Goddeeris et al., 2013</xref>). However, how the laminin-binding glycan synthesized by LARGE is attached to the Core M3 structure, and which glycans or other molecules contribute to and form part of this linker structure, remains unknown.</p><p>We set out to elucidate the structure and monosaccharide composition of the α-DG post-phosphoryl modification, applying a strategic and multifaceted experimental approach starting from the terminal end synthesized by LARGE. We used glycosylation-deficient cells, in vitro enzyme assays, deglycosylation strategies, and NMR (Nuclear Magnetic Resonance)-based structure analysis as experimental tools.</p><p>This strategy revealed a β1,4 glucuronyltransferase activity for B4GAT1. We present experimental evidence that this enzyme B4GAT1, which was previously described in the literature as B3GNT1 (<xref ref-type="bibr" rid="bib48">Sasaki et al., 1997</xref>) in fact encodes for a β1,4 glucuronyltransferase and not a β1,3 N-acetylglucosaminyltransferase as previously thought. This activity contributes to production of the post-phosphoryl glycan linker by transferring a glucuronic acid (GlcA) residue onto a xylose (Xyl) acceptor. It thereby forms a glucuronyl-β1,4-xylosyl disaccharide, the direct acceptor required by the glycosyltransferase LARGE to initiate formation of the terminal heteropolysaccharide that is involved in ligand binding. B4GAT1 enzymatic activity, of both a recombinant form and the endogenous protein in mouse embryonic fibroblasts (MEFs), was further characterized using a newly developed HPLC (High-Performance Liquid Chromatography)-based assay for B4GAT1 activity.</p><p>Our findings contribute to the current understanding of α-DG posttranslational processing, providing mechanistic insights regarding the pathomechanism underlying α-DG glycosylation-deficient CMDs and revealing new therapeutic avenues for blocking entry of pathogenic LASV viruses.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>α-DG O-mannosyl post-phosphoryl modification occurs in the Golgi</title><p>Our previous work showed that the ER-resident enzymes POMGNT2 (GTDC2), B3GALNT2 and POMK (SGK196) contribute to synthesis of the phosphorylated Core M3 trisaccharide on α-DG, a moiety that is required as platform for further modification with the LARGE mediated laminin-binding glycan (<xref ref-type="bibr" rid="bib62">Yoshida-Moriguchi et al., 2013</xref>). However, a number of additional genes, namely <italic>FKTN</italic> (Fukutin) (<xref ref-type="bibr" rid="bib32">Kobayashi et al., 1998</xref>; <xref ref-type="bibr" rid="bib15">de Bernabe et al., 2003</xref>), <italic>FKRP</italic> (Fukutin-related protein) (<xref ref-type="bibr" rid="bib9">Brockington et al., 2001</xref>; <xref ref-type="bibr" rid="bib6">Beltran-Valero de Bernabe et al., 2004</xref>) <italic>TMEM5</italic> (<xref ref-type="bibr" rid="bib53">Vuillaumier-Barrot et al., 2012</xref>) and <italic>B4GAT1</italic> <italic>(</italic><italic>B3GNT1</italic><italic>)</italic> (<xref ref-type="bibr" rid="bib58">Wright et al., 2012</xref>; <xref ref-type="bibr" rid="bib11">Buysse et al., 2013</xref>; <xref ref-type="bibr" rid="bib49">Shaheen et al., 2013</xref>) are known to be crucial for proper α-DG glycosylation, yet how they contribute has not yet been determined (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). To investigate if these unassigned genes are involved in the pre- or post-phosphorylation process of Core M3, we expressed Fc-tagged recombinant α-DG (DGFc340) in [<sup>32</sup>P] orthophosphate-labeled control and glycosylation-deficient cells. DGFc340 is a secreted α-DG deletion construct that contains only the minimal region of the α-DG mucin-like domain (aa 316–340), that is required for its functional glycosylation followed by a C-terminal fusion tag encoding the heavy-chain constant (Fc) moiety of human IgG1 (to enable purification of the secreted recombinant protein) (<xref ref-type="bibr" rid="bib24">Hara et al., 2011</xref>). Although only a small subpopulation of the expressed DGFc protein enters the pathway for functional maturation it was demonstrated that this truncated α-DG fusion protein is a valuable tool to study α-DG functional glycosylation (<xref ref-type="bibr" rid="bib24">Hara et al., 2011</xref>).</p><p>The goal was to test if DGFc340 can be [<sup>32</sup>P] phosphorylated in fibroblasts with defects in various dystroglycanopathy genes (<xref ref-type="table" rid="tbl1">Table 1</xref>). In our experiment, fibroblasts with defects in <italic>FKTN</italic>, <italic>FKRP</italic>, <italic>TMEM5</italic>, <italic>B4GAT1</italic> <italic>(</italic><italic>B3GNT1</italic><italic>)</italic> and <italic>LARGE</italic>, but not in the phosphate kinase <italic>POMK,</italic> were able to produce radioactively labeled DGFc340 (<xref ref-type="fig" rid="fig1">Figure 1A</xref>), indicating that FKTN, FKRP, TMEM5, B4GAT1 and LARGE are involved downstream of POMK in the Core M3 post-phosphorylation process.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.003</object-id><label>Table 1.</label><caption><p>Summary of features of control and glycosylation-deficient cell lines</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.003">http://dx.doi.org/10.7554/eLife.03941.003</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Mutant gene</th><th>Clinical phenotype</th><th>Cell type</th><th>Nucleotide variant</th><th>Amino acid</th><th>reference</th></tr></thead><tbody><tr><td><bold>Control (human)</bold></td><td>none</td><td>Human skin fibroblast</td><td/><td/><td>CRL-2127 (ATCC)</td></tr><tr><td><bold><italic>POMK</italic></bold></td><td>WWS/MEB</td><td>Human skin fibroblast</td><td>14bp homozygous deletion (c.720_733delGCTGGTG<break/>AGTGCG)], homozygous</td><td>p.Leu241Profs*26</td><td>(<xref ref-type="bibr" rid="bib62">Yoshida-Moriguchi et al., 2013</xref>)</td></tr><tr><td rowspan="2"><bold><italic>FKTN</italic></bold></td><td rowspan="2">WWS</td><td rowspan="2">Human skin fibroblast</td><td>c.385delA</td><td>p.I129fsX1</td><td>GM16192</td></tr><tr><td>c.1176C > A, heterozygous</td><td>p.Y392X</td><td>(Coriell Cell Repository)</td></tr><tr><td><bold><italic>FKRP</italic></bold></td><td>WWS</td><td>Human skin fibroblast</td><td>c.1A > G, homozygous</td><td>p.M1V</td><td>(<xref ref-type="bibr" rid="bib52">Van Reeuwijk et al., 2010</xref>)</td></tr><tr><td><bold><italic>TMEM5</italic></bold></td><td>WWS</td><td>Human skin fibroblast</td><td>c.1101 G > A, homozygous</td><td>p.G333R</td><td>unpublished</td></tr><tr><td><bold><italic>B4gat1 (</italic></bold><italic>B3gnt1</italic><bold><italic>)</italic></bold></td><td>CMD</td><td>MEF</td><td>c.464T > C, compound het with LacZ null allele, <italic>B4gat1</italic><sup><italic>LacZ/M155T</italic></sup></td><td>p.M155T</td><td>(<xref ref-type="bibr" rid="bib58">Wright et al., 2012</xref>)</td></tr><tr><td><bold><italic>Large</italic></bold><sup><bold><italic>myd</italic></bold></sup></td><td>CMD</td><td>MEF</td><td>deletion of exons 5−7, homozygous</td><td/><td>(<xref ref-type="bibr" rid="bib23">Grewal et al., 2001</xref>)</td></tr><tr><td><bold>Control (mouse)</bold></td><td>none</td><td>MEF</td><td/><td/><td/></tr></tbody></table></table-wrap><fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.004</object-id><label>Figure 1.</label><caption><title>Postulated α-DG modifying enzymes are involved in post-phosphorylation processes in the Golgi prior to LARGE.</title><p>(<bold>A</bold>) Phosphorylation of Fc-tagged DGFc340 in the context of α-DG glycosylation defects. Fc-tagged DGFc340 was produced in [<sup>32</sup>P] orthophosphate-labeled fibroblasts from control and glycosylation-deficient patients and mice (<xref ref-type="table" rid="tbl1">Table 1</xref>). The DGFc340 was isolated from the culture medium by using protein A-agarose and the samples were separated by SDS PAGE. Gels were stained with Coomassie brilliant blue (CBB) and analyzed by phosphorimaging (<sup>32</sup>P). (<bold>B</bold>) Subcellular localization of α-DG modifying putative glycosyltransferases, as assessed by immunofluorescence. HEK293T cells stably expressing c-Myc-tagged proteins were stained with anti-Myc (red), anti-Giantin (Golgi marker, green) and 4 ́,6-diamidino-2-phenylindole (DAPI, nuclei, blue). Individual stainings for c-Myc and Giantin are shown in greyscale and a merged image is shown in color. Scale bars indicate 10 µm. (<bold>C</bold>) Quantitative On-Cell protein blot analysis of LARGE-induced α-DG glycosylation hyperglycosylation in glycosylation-deficient cells. α-DG glycosylation status was tested with and without forced LARGE overexpression by adenovirus mediated gene transfer. The On-Cell Western blots were probed with an antibody against the glycosylated form of α-DG (IIH6). IIH6 On-Cell quantitative data were normalized with DRAQ5 cell DNA dye (n = 3). Error bars, SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.004">http://dx.doi.org/10.7554/eLife.03941.004</ext-link></p></caption><graphic xlink:href="elife03941f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03941.005</object-id><label>Figure 1—figure supplement 1.</label><caption><title>α-DG functional glycosylation and known proteins contributing to its synthesis.</title><p>α-DG core M3 functional glycosylation can be divided in 2 major processing steps. O-mannosyl pre-phosphoryl modification which is carried out by enzymes in the endoplasmic reticulum (ER) (highlighted in the blue box) and O-mannosyl post-phosphoryl modification by known or putative glycosyltransferases in the Golgi (highlighted in the red box). Both gene products with known function (black) and gene products with currently unidentified function (red) are indicated. The putative glycosyltransferases B4GAT1 (B3GNT1), FKTN, FKRP and TMEM5 are proposed to act prior to LARGE which adds a GlcA-Xyl heteropolymer that is responsible for ligand binding. However based on current knowledge it cannot be completely ruled out that they are involved in the modification of the LARGE glycan repeat itself to modulate ligand binding.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.005">http://dx.doi.org/10.7554/eLife.03941.005</ext-link></p></caption><graphic xlink:href="elife03941fs001"/></fig></fig-group></p><p>Immunofluorescence examination of HEK293T (Human Embryonic Kidney) cells stably transfected with Myc-tagged constructs of this set of proteins, revealed that they co-localize with the Golgi-resident marker protein Giantin (<xref ref-type="bibr" rid="bib37">Linstedt and Hauri, 1993</xref>) (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). Previously, Golgi localization was also demonstrated for FKRP (<xref ref-type="bibr" rid="bib20">Esapa et al., 2002</xref>), FKTN (<xref ref-type="bibr" rid="bib20">Esapa et al., 2002</xref>; <xref ref-type="bibr" rid="bib59">Xiong et al., 2006</xref>), B4GAT1 (B3GNT1) (<xref ref-type="bibr" rid="bib11">Buysse et al., 2013</xref>) and LARGE (<xref ref-type="bibr" rid="bib10">Brockington et al., 2005</xref>). These results indicate that most if not all of the α-DG O-mannosyl post-phosphoryl processing is carried out by Golgi-resident enzymes.</p><p>The laminin-binding glycan repeat generated by LARGE is hypothesized to be the terminal glycan structure of the α-DG O-mannosyl post-phosphoryl modification (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). This would suggest that FKTN, FKRP, TMEM5 and B4GAT1 contribute to a post-phosphoryl linker structure, that can serve as an acceptor for the modification with LARGE. Previous work by Kuga et al., (<xref ref-type="bibr" rid="bib33">Kuga et al., 2012</xref>) also had indicated that FKTN and FKRP are part of the α-DG O-mannosyl post-phosphoryl modification pathway. To test our hypothesis, we infected a panel of glycosylation-deficient cells with a LARGE expressing adenovirus construct and analyzed the glycosylation status of α-DG and the degree of hyperglycosylation by On-Cell immunoblotting with monoclonal antibody IIH6, which recognizes the α-DG laminin-binding glycan transferred by LARGE (<xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>; <xref ref-type="bibr" rid="bib22">Goddeeris et al., 2013</xref>). As expected, overexpression of LARGE did not produce the IIH6-positive glycan or significantly bypass the glycosylation defect in either <italic>FKTN-</italic>, <italic>FKRP-</italic>, <italic>TMEM5-</italic> or <italic>B4GAT1</italic><italic>-</italic>deficient cells (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), supporting the notion that these encoded proteins work prior to LARGE in the O-mannosyl post-phosphoryl modification process.</p><p>In summary, our data suggest that Golgi-localized putative glycosyltransferases FKTN, FKRP, TMEM5 and B4GAT1 are essential for the synthesis of a linker structure that is connecting the α-DG O-mannosyl phosphate platform with the terminal laminin binding glycan added by LARGE.</p></sec><sec id="s2-2"><title>Glucuronic acid serves as an acceptor sugar for LARGE polymer initiation with Xylose</title><p>To further elucidate the structure of the α-DG post-phosphoryl modification, we examined how synthesis of the terminal LARGE glycan was initiated. Although LARGE is known to be a dual glycosyltransferase that synthesizes repeating units of [–3-xylose–α1,3-glucuronic acid-β1–] on α-DG, the identities of both the initiating sugar and the acceptor sugar for this laminin-binding polymer remained unknown. To determine which sugar is initially transferred by LARGE, we developed an in vitro glycosylation assay using a recombinant soluble (transmembrane domain deleted) form of LARGE (LARGEdTM) and the acceptor protein DGFc340. DGFc340 isolated from the culture medium of <italic>Large</italic><sup><italic>myd</italic></sup> (<italic>Large</italic>-deficient) MEFs lacks the LARGE modification on phosphorylated O-mannosyl glycans, and is hypothesized to terminate in a glycan acceptor structure that can be recognized by LARGE (<xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>) (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). To determine which of the sugars in the polymer is initially transferred by LARGE we incubated DGFc340 isolated from the <italic>Large</italic><sup><italic>myd</italic></sup> MEF culture medium with LARGEdTM and [<sup>14</sup>C]-labeled UDP-xylose (Xyl) and/or UDP-glucuronic acid (GlcA) radionucleotide sugar donors. The glycosyl-transfer reaction was measured as the transfer of radioactivity onto the DGFc340 acceptor glycoprotein. As negative control we used a DGFc340 mutant construct (T317A/T319A), which lacks the O-mannosylation sites that are the crucial acceptor platform for subsequent synthesis of the laminin-binding glycan (<xref ref-type="bibr" rid="bib24">Hara et al., 2011</xref>). When the radionucleotide sugars were tested individually in the LARGEdTM in vitro assay, the addition of [<sup>14</sup>C] UDP-Xyl, but not that of [<sup>14</sup>C] UDP-GlcA radionucleotides, resulted in radioactive labeling of the DGFc340 acceptor (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). However, in the presence of both UDP-Xyl and UDP-GlcA, the transfer of radioactivity was significantly increased consistent with the fact that the LARGE glycan is a heteropolysaccharide (<xref ref-type="fig" rid="fig2">Figure 2A/B</xref>). These results indicate that xylose is the initial sugar transferred by LARGE, and that this is followed by the transfer of GlcA to form the repeating [–3-xylose–α1,3-glucuronic acid-β1–] heteropolymer.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.006</object-id><label>Figure 2.</label><caption><title>β-GlcA serves as an acceptor sugar for LARGE modification starting with xylose.</title><p>(<bold>A</bold>) Schematic diagram showing the α-DG post-phosphoryl modification in the context of control and glycosylation defects. LARGE adds the ligand-binding glycan to α-DG via a proposed glucuronic acid (GlcA) acceptor. LARGEdTM catalytic domains Xyl-T (orange) and GlcA-T (blue) are highlighted in color. Depicted are also the hypothesized terminal sugar structures of glycosylation-deficient cell lines <italic>Large</italic><sup><italic>myd</italic></sup> (<italic>Large</italic>-deficient) and pgsI-208 (UDP-xylose deficient). Cleavage of terminal β-GlcA by exoglycosidase β-glucuronidase (β-GUS) in <italic>Large</italic><sup><italic>myd</italic></sup> is indicated (scissor symbol). (<bold>B</bold>) Transfer of [<sup>14</sup>C] radiolabeled Xyl and GlcA to DGFc340 by LARGEdTM. Fc-tagged DGFc340 was produced in <italic>Large</italic><sup><italic>myd</italic></sup> (<italic>Large</italic>-deficient) MEF cells and isolated from the culture medium using protein A-agarose. The protein A-bound DGFc340 was used as acceptor in a LARGEdTM reaction with radiolabeled [<sup>14</sup>C] UDP-Xyl and/or [<sup>14</sup>C] UDP-GlcA sugar donors. The figure represents the transfer of radiolabeled saccharides onto the donor DGFc340 (n = 3). Error bars represent SD (<bold>C</bold>) β-Glucuronidase pre-treatment of DGFc340 from <italic>Large</italic><sup><italic>myd</italic></sup> deficient cells impairs LARGEdTM modification. Protein A-bound DGFc340 (acceptor) isolated from transfected <italic>Large</italic><sup><italic>myd</italic></sup> MEFs was digested with β-glucuronidase (β-GUS) prior to the LARGEdTM (enzyme) reaction, which included UDP-Xyl and UDP-GlcA as sugar (donors). After incubation with LARGEdTM DGFc340 (acceptor protein) was subjected to protein blotting with antibodies against the glycosylated form of α-DG (IIH6), against Fc and against LARGE (Rb331). (<bold>D</bold>) The ability of LARGEdTM to modify DGFc340 is impaired in the context of sugar donor-deficient CHO mutant cell lines. Fc-tagged DGFc340 was produced in various glycosylation-deficient Lec CHO cells and isolated from the culture medium using protein A-agarose. As in (<bold>C</bold>) protein A-bound DGFc340 acceptor was used in a LARGEdTM reaction and analyzed by protein blotting.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.006">http://dx.doi.org/10.7554/eLife.03941.006</ext-link></p></caption><graphic xlink:href="elife03941f002"/></fig></p><p>Next we wanted to identify the acceptor glycan used by LARGE to initiate formation of the laminin-binding glycan. Since the Xyl-T (xylosyltransferase) activity of LARGE has acceptor specificity for β-linked GlcA during heteropolymer formation, we hypothesized that β-linked GlcA might be the initial acceptor for the glycan added by LARGE. To test this, we pre-treated DGFc340 from <italic>Large</italic><sup><italic>myd</italic></sup> MEF cells with β-glucuronidase (β-GUS) (<xref ref-type="fig" rid="fig2">Figure 2A/C</xref>), and assessed its modification by LARGEdTM in an in vitro assay. Subsequent immunoblotting with the LARGE glycan-specific antibody (IIH6) revealed that the pretreatment of <italic>Large</italic><sup><italic>myd</italic></sup> DGFc340 with β-glucuronidase resulted in a strong reduction of the IIH6 signal (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). These data indicate that LARGE uses a β-linked GlcA residue as an acceptor sugar to initiate synthesis of the polymeric glycan.</p></sec><sec id="s2-3"><title>Xylose is part of the α-DG O-mannosyl post-phosphoryl modification</title><p>To determine which monosacharides contribute to synthesis of the O-mannosyl post-phosphoryl acceptor for the LARGE glycan, we performed a LARGEdTM assay with DGFc340 acceptor isolated from a panel of sugar nucleotide-deficient CHO (Chinese hamster ovary) cells (<xref ref-type="bibr" rid="bib51">Stanley, 1985</xref>; <xref ref-type="bibr" rid="bib31">Kingsley et al., 1986</xref>). LARGEdTM was able to efficiently modify DGFc340 from Pro5 (wild-type), Lec2 (CMP-sialic acid-deficient), Lec8 (UDP-galactose-deficient) and Lec13 (GDP-fucose-deficient) cells, suggesting that sialic acid, galactose and fucose do not contribute to functional glycosylation of α-DG (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). ldlD cells deficient for UDP-galactose (UDP-Gal) and UDP-N-acetylgalactosamine (UDP-GalNAc) demonstrated reduced acceptor activity for LARGEdTM, which can be explained by the fact that B3GALNT2 requires UDP-GalNAc for synthesis of the initial Core M3 structure. Similarly, DGFc340 from Lec15 cells, which are deficient for Dol-P-Man synthesis, did not serve as LARGEdTM acceptor because POMT1 (Protein <italic>O</italic>-mannosyltransferase) and POMT2 require the Dol-P-Man sugar donor to initiate the O-mannosyl Core M3 structure. Most interestingly, in the LARGEdTM in vitro assay DGFc340 from UDP-Xyl-deficient pgsI-208 CHO cells was not modified (<xref ref-type="fig" rid="fig2">Figure 2D</xref>). It had also been shown that ectopic LARGE expression in pgsI-208 CHO cells did not induce α-DG hyperglycosylation (<xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>; <xref ref-type="bibr" rid="bib1">Ashikov et al., 2013</xref>), consistent with the fact that UDP-Xyl is essential for synthesis of the LARGE glycan in vivo. However, in our LARGEdTM in vitro assay, pgsI-208 DGFc340 was also not modified by LARGEdTM, despite the presence of both of the required sugar nucleotides, UDP-GlcA and UDP-Xyl. This clearly demonstrated that one or more xylose residues are required on α-DG before it can be functionally glycosylated by LARGE (<xref ref-type="fig" rid="fig2">Figure 2A</xref>).</p></sec><sec id="s2-4"><title>B4GAT1 is a glucuronyltransferase with specificity for the β-xylose acceptor</title><p>Having identified a β-linked glucuronic acid as the terminal acceptor saccharide for LARGE and xylose as component of the α-DG post-phosphoryl glycan modification, we next sought to determine which enzyme is responsible for the hypothesized glucuronyltransferase activity. Among the group of unassigned genes (<italic>FKTN, FKRP, TMEM5, B4GAT1</italic>) only the <italic>B4GAT1</italic> gene product showed homology to glucuronyl-transferases. In particular it shares 44% similarity with the LARGE GlcA-T (Glucuronyltransferase) domain (CAZy: GT49; <xref ref-type="fig" rid="fig3">Figure 3A</xref>). This designated B4GAT1 as a promising candidate for the GlcA-T transferase upstream of LARGE. To test this hypothesis, we generated a 6xHis-tagged soluble construct of B4GAT1 (transmembrane domain deleted, B4GAT1dTM), expressed it in HEK293T cells and purified the recombinant enzyme from the culture medium (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). We then conducted a transfer assay with B4GAT1dTM as the enzyme source, UDP-GlcA as the sugar donor and fluorescently labeled β-xyloside (4-methylumbelliferyl-β-D-xyloside, Xyl-β-MU) as the acceptor. The reaction products were separated by high-performance liquid chromatography (HPLC). A unique product peak was detected only when UDP-GlcA was used as donor (<xref ref-type="fig" rid="fig3">Figure 3B</xref>, <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2A</xref>). We also tested the acceptor specificity, which revealed that B4GAT1dTM GlcA-T activity has low preference for α-linked Xyl, but showed >10- fold higher preference and specificity towards β-linked Xyl acceptors (<xref ref-type="fig" rid="fig3">Figure 3C</xref>, <xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2A</xref>). The fact that the LARGE glycan disaccharide Xyl-α1,3-GlcA-MU was a very weak acceptor for B4GAT1dTM GlcA-transfer suggests that B4GAT1 overexpression does not interfere with LARGE mediated synthesis of the laminin-binding glycan. A characterization of the B4GAT1 GlcA-T activity revealed a metal dependence for manganese (Mn<sup>2+</sup>) divalent cations (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2B</xref>) and a pH-optimum near pH 7.0 (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2C</xref>). The product peak obtained from the enzymatic reaction of B4GAT1dTM with β-Xyl-MU acceptor was isolated, and its analysis by NMR revealed that the GlcA residue was β-linked to the four position of the xylose β-MU (<xref ref-type="fig" rid="fig3s3">Figure 3—figure supplement 3</xref>, <xref ref-type="supplementary-material" rid="SD1-data">Figure 3—source data 1</xref>). Thus, B4GAT1 possesses xylose β1,4-glucuronyltransferase (GlcA-T) activity and it is specific for the substrate β-linked Xyl.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.007</object-id><label>Figure 3.</label><caption><title>B4GAT1 has xylose β1,4 glucuronyltransferase activity.</title><p>(<bold>A</bold>) Schematic representation of LARGE and B4GAT1 functional domains. GlcA-T (blue), Xyl-T (orange) and transmembrane domain (black) are indicated. (<bold>B</bold>) Representative HPLC profiles of the reaction product generated in the absence (top) and presence (bottom) of a UDP-GlcA sugar (donor) in a reaction mix containing Xyl-β-MU (acceptor) and B4GAT1dTM (enzyme). Samples were separated on an LC-18 column. P, product. S, unreacted substrate. Dotted line, %B buffer. (<bold>C</bold>) Comparison of B4GAT1dTM GlcA-T activity with respect to various xylose-MU acceptor sugars. Relative activity (%) with respect to Xyl-β-MU acceptor (specific activity: 0.2 µmol/h/mg) is shown (n = 3). Error bars represent SD. (<bold>D</bold>) Comparison of LARGEdTM Xyl-T activity with respect to various monosaccharide and disaccharide GlcA-MU acceptor sugars. Relative activity (%) with respect to intrinsic LARGE polymer specific activity with GlcA-β1,3-Xyl-α-MU disaccharide acceptor (0.08 µmol/h/mg) (n = 3). Error bars represent SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.007">http://dx.doi.org/10.7554/eLife.03941.007</ext-link></p><p><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03941.008</object-id><label>Figure 3—source data 1.</label><caption><p>Chemical shifts (ppm) of the signals in the <sup>1</sup>H and <sup>13</sup>C NMR spectra of the enzymatic reaction product of GlcA-β1,4-Xyl-β-MU of the glycosyltransferase B4GAT1.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.008">http://dx.doi.org/10.7554/eLife.03941.008</ext-link></p></caption><media mime-subtype="docx" mimetype="application" xlink:href="elife03941s001.docx"/></supplementary-material></p></caption><graphic xlink:href="elife03941f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03941.009</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Purification of B4GAT1dTM.</title><p>(<bold>A</bold>) Schematic representation of B4GAT1 and the B4GAT1dTM construct used in the enzymatic activity assay. The transmembrane (TM) sequence was replaced with a 3xFLAG-TEV tag sequence and the C-terminus was modified with a Myc-6xHis-tag. (<bold>B</bold>) Purification of recombinant B4GAT1dTM from bioreactor culture medium. The recombinant protein was expressed in HEK293T cells and purified from the culture medium using Talon metal-affinity resin. The bioreactor medium samples before (start) and after the purification (void) as well as the eluted purified protein (elution) were analyzed by immunoblotting with anti-Myc (4A6) antibody. CBB, stained with Coomassie Brilliant blue.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.009">http://dx.doi.org/10.7554/eLife.03941.009</ext-link></p></caption><graphic xlink:href="elife03941fs002"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03941.010</object-id><label>Figure 3—figure supplement 2.</label><caption><title>Basic characterization of the xylose β1,4-glucuronyltransferase activity of B4GAT1.</title><p>(<bold>A</bold>) Donor sugar specificity of B4GAT1dTM. Representative data from two independent assays, demonstrating relative activity (%) of B4GAT1dTM (enzyme) GlcA-T toward Xyl-α-MU and Xyl-β-MU (acceptor) when tested with various sugar nucleotides (donor). The specific activity set as 100% for acceptor Xyl-β-MU was 0.2 µmol/hr/mg. No sugar other than GlcA was transferred to the acceptors to a significant extent. (<bold>B</bold>) Metal dependence of the B4GAT1dTM GlcA-T activity. Activity assay was carried out in the presence or absence of each metal ion or EDTA (10 mM), and results are shown as relative activity (%). The GlcA-T activity in the presence of Mn<sup>2+</sup> (specific activity: 0.26 µmol/h/mg) was arbitrarily set at 100% (n = 3). n.d., not detected. Error bars represent SD (<bold>C</bold>) pH optimum of B4GAT1dTM GlcA-T activity. Data from three independent experiments are shown as relative activity (%). The highest activity (specific activity: 0.22 µmol/h/mg) in the dataset was arbitrarily set at 100%. The GlcA-T assays were carried out using Xyl-β-MU as acceptor. The buffers used were: acetate for pH 4.5––5.5 (open circle), MES for pH 5.5––6.5 (closed circle), MOPS for pH 6.5––7.5 (open square) and Tris–HCl for pH 7.5––8.5 (closed square). The details of the conditions are presented in the Materials and methods section. Error bars represent SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.010">http://dx.doi.org/10.7554/eLife.03941.010</ext-link></p></caption><graphic xlink:href="elife03941fs003"/></fig><fig id="fig3s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03941.011</object-id><label>Figure 3—figure supplement 3.</label><caption><title>NMR analysis reveals that B4GAT1 is a β1,4 glucuronyltransferase.</title><p>(<bold>A</bold>) HMQC spectrum (top) and overlay of HMQC (black) and HMBC (green) spectra (bottom) for the B4GAT1 enzymatic reaction product. The cross-peaks are labeled with a first letter representing the subunit designated in <bold><italic>C</italic></bold> and the rest of the label representing the position on that subunit. The observed interglycosidic cross-peak BH1/AC4 in the HMBC spectrum clearly demonstrates the presence of a 1→4 interglycosidic linkage between the residues B and A. The cross-peak marked with a star represents an impurity. (<bold>B</bold>) TOCSY (top) and ROESY (bottom) spectra of the B4GAT1 enzymatic reaction product collected with a mixing time of 77 and 300 ms, respectively. The cross-peaks are labeled as in (<bold>A</bold>). The observed interglycosidic ROEs are indicated in green circles. The ROE data indicate that both residues exist in β-configurations. (<bold>C</bold>) Schematic depiction of the disaccharide structure produced by B4GAT1, with the sugar units labeled A and B.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.011">http://dx.doi.org/10.7554/eLife.03941.011</ext-link></p></caption><graphic xlink:href="elife03941fs004"/></fig><fig id="fig3s4" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03941.012</object-id><label>Figure 3—figure supplement 4.</label><caption><title>Test B4GAT1 for GlcNAc transferase activity with iGnT substrate Gal-β1,4-GlcNAc-β-MU.</title><p>(<bold>A</bold>) Using B4GALT1 we synthesized the hypothesized iGnT substrate Gal-β1,4-GlcNAc-β-MU by transferring a β1,4 Galactose to the acceptor GlcNAc-β-MU. The purified Gal-β1,4-GlcNAc-β-MU disaccharide was further analyzed by NMR. (<bold>B</bold>) HMQC spectrum. The folded peak is shown in blue. The cross peaks are labeled with a first letter representing the subunit as designated in the structure shown above the spectra and the rest of the label representing the position on that subunit. Overlay of HMQC (black and blue) and HMBC (green) spectra. The strong cross peak labeled as BH1/AC4 was detected in the HMBC spectrum, demonstrating the presence of a 1→4 interglycosidic linkage between Gal and GlcNAc. (<bold>C</bold>) TOCSY spectrum collected with a mixing time of 77 ms. ROESY spectrum collected with a mixing time of 300 ms. The observed strong NOEs from BH1 to BH3 and BH5 (cross peaks labeled as BH1/BH3 and BH1/BH5) demonstrate that the Gal has a β-configuration. Similarly, the observed strong NOEs from AH1 to AH3 and AH5 (cross peaks labeled as AH1/AH3 and AH1/AH5) demonstrate that the GlcNAc has a β-configuration. A strong interglycosidic NOE was observed between BH1 and AH4 (green circle), which is consistent with the 1 to 4 linkage as determined from the HMBC spectrum. (<bold>D</bold>) Representative HPLC profiles of the reaction product generated in the absence (blue) and presence (red) of a UDP-GlcNAc sugar (donor) in a reaction mix containing Gal-β1,4-GlcNAc-β-MU (acceptor) and B4GAT1dTM (enzyme). Samples were separated on an LC-18 column. S, unreacted substrate. Dotted line, %B buffer.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.012">http://dx.doi.org/10.7554/eLife.03941.012</ext-link></p></caption><graphic xlink:href="elife03941fs005"/></fig></fig-group></p></sec><sec id="s2-5"><title>The substrate specificity of LARGE Xyl-T is not dependent on the glycosidic bond of the β-GlcA acceptor</title><p>It had previously been shown that, during synthesis of the LARGE heteropolysaccharide, β1,3-linked GlcA serves as the acceptor for LARGE Xyl-T (<xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>). In the current study we found that a β1,4-linked GlcA transferred by B4GAT1 serves as the acceptor glycan for initiation of synthesis of the LARGE glycan, via the addition of a xylose. To assess if LARGE can use one or the other glycosidic linkage β-linked GlcA acceptor with higher efficiency, we tested LARGEdTM Xyl-T activity on two disaccharides GlcA-β1,4-Xyl-β-MU and GlcA-β1,3-Xyl-α-MU along with the monosaccharide GlcA-β-MU (4-methylumbelliferyl-β-D-glucuronide). As shown in <xref ref-type="fig" rid="fig3">Figure 3D</xref>, LARGE did not distinguish between GlcA-β1,3-Xyl and GlcA-β1,4-Xyl, as similar activities were measured in the presence of both disaccharide acceptors. However, the length of the acceptor appears to be important, since the disaccharide acceptor showed 25-fold higher activity than the monosaccharide acceptor (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). In summary, it is currently unknown why the β1,4-linked GlcA acceptor in the initial LARGE acceptor primer and the β1,3-linked GlcA in the terminal LARGE glycan have different linkages, and how each linkage contributes spatially to the overall structure, while LARGE shows similar activity towards both acceptors.</p></sec><sec id="s2-6"><title>MEFs from <italic>B4gat1</italic>-deficient mice lack endogenous B4GAT1 activity</title><p>To elucidate further the role of B4GAT1 in vivo, we isolated MEFs from control, <italic>Large</italic><sup><italic>myd</italic></sup> (<italic>Large</italic>-deficient) and <italic>B4gat1</italic>-deficient mice (<xref ref-type="bibr" rid="bib58">Wright et al., 2012</xref>) (<xref ref-type="table" rid="tbl1">Table 1</xref>) and analyzed the glycosylation status of α-DG. Immunoblotting revealed that whereas control MEFs were positive for functional glycosylation of, and laminin-binding by, α-DG from <italic>Large</italic><sup><italic>myd</italic></sup> MEFs completely lacked both features (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Also, <italic>B4gat1</italic>-deficient MEFs demonstrated strongly reduced but detectable residual functional glycosylation and laminin binding, and normal levels of hypoglycosylated α-DG core protein (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Adenovirus-mediated ectopic expression of B4GAT1 did not affect the glycosylation status of α-DG in control and <italic>Large</italic><sup><italic>myd</italic></sup> MEFs but, as expected, was able to rescue the α-DG glycosylation defect in <italic>B4gat1</italic>-deficient MEFs (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). As demonstrated previously (<xref ref-type="bibr" rid="bib5">Barresi et al., 2004</xref>; <xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>; <xref ref-type="bibr" rid="bib56">Willer et al., 2012</xref>), forced adenovirus-mediated ectopic expression of LARGE in control and <italic>Large</italic><sup><italic>myd</italic></sup> MEFs induces α-DG hyperglycosylation. In contrast, <italic>B4gat1</italic>-deficient cells showed only a low level of α-DG hyperglycosylation after LARGE overexpression (<xref ref-type="fig" rid="fig4">Figure 4A</xref>), suggesting that in the context of mutant B4GAT1, only few acceptor sites for LARGE modification are available. Finally, ectopic co-expression of B4GAT1 and LARGE resulted in α-DG hyperglycosylation in all three tested MEF lines (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). This result is consistent with the hypothesis that B4GAT1 acts prior to the glycosyltransferase LARGE.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.013</object-id><label>Figure 4.</label><caption><title><italic>B4gat1</italic>-deficient MEFs have impaired α-DG functional glycosylation and endogenous B4GAT1 activity.</title><p>(<bold>A</bold>) Functional glycosylation and complementation analysis of α-DG in wild-type, <italic>Large-</italic> and <italic>B4gat1</italic>-deficient MEFs. Immunoblots and laminin overlay assay of WGA-enriched cell lysates extracted from WT, <italic>Large</italic><sup><italic>myd</italic></sup> (<italic>Large</italic>-deficient) and <italic>B4gat1</italic><italic>-</italic>deficient MEFs . As indicated MEFs were uninfected (mock) or infected with adenovirus constructs expressing B4GAT1, LARGE or both (B + L). Antibodies used were: glyco α-DG (IIH6), core α-DG, core β-DG (AP83), anti-V5 and anti-LARGE (Rb331). (<bold>B</bold>) Comparison of endogenous B4GAT1 GlcA-T activity in control, <italic>Large-</italic> and <italic>B4gat1</italic>-deficient MEFs. Additionally, <italic>B4gat1</italic>-deficient MEFs (<italic>B4gat1</italic><sup><italic>LacZ/M155T</italic></sup>) complemented with control B4GAT1 expressing adenovirus (Ad5) were tested. Cell lysates were used as enzyme source to measure endogenous B4GAT1 activity. Relative activity (%) with respect to control MEFs specific activity (91.6 pmol/h/mg) is shown (n = 3). Error bars represent SD. (<bold>C</bold>) Comparison of endogenous LARGE GlcA-T activity in control, <italic>Large-</italic> and <italic>B4gat1</italic>-deficient MEFs. Additionally, <italic>Large</italic>-deficient MEFs complemented with control <italic>LARGE</italic> expressing adenovirus (Ad5) were tested. WGA enriched glycoprotein samples were used as enzyme source to measure endogenous LARGE activity. Relative activity (%) with respect to control MEFs specific activity (0.52 pmol/h/mg) is shown (n = 3). Error bars represent SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.013">http://dx.doi.org/10.7554/eLife.03941.013</ext-link></p></caption><graphic xlink:href="elife03941f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03941.014</object-id><label>Figure 4—figure supplement 1.</label><caption><title>B4gat1-deficient MEFs have impaired endogenous B4GAT1 activity.</title><p>Representative HPLC profiles of the reaction product are shown. (<bold>A</bold>/<bold>B</bold>) Endogenous B4GAT1 enzyme activity of cell lysates from wild-type (<bold>A</bold>) and B4gat1-deficient (<bold>B</bold>) MEFs . B4gat1-deficient MEFs (B4gat1LacZ<sup><italic>/M155T</italic></sup>) show some residual activity <3% (asterisk). (<bold>C/D</bold>) Endogenous LARGE enzyme activity of WGA-enriched cell lysates from control (<bold>C</bold>) and <italic>Large</italic><sup><italic>myd</italic></sup> (<bold>D</bold>) (<italic>Large</italic>-deficient) MEFs. <italic>Large</italic><sup><italic>myd</italic></sup> MEFs lack LARGE activity and do not show any residual activity (asterisk). The details of the conditions are provided in the Materials and methods section.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.014">http://dx.doi.org/10.7554/eLife.03941.014</ext-link></p></caption><graphic xlink:href="elife03941fs006"/></fig></fig-group></p><p>Next, to determine if endogenous B4GAT1 activity was detectable in MEF cells, we subjected samples from control, <italic>B4gat1</italic> and <italic>Large</italic><sup><italic>myd</italic></sup> MEFs to the B4GAT1 enzyme activity assay, using Xyl-β-MU as the acceptor. Whereas control and <italic>Large</italic><sup><italic>myd</italic></sup> samples showed comparable B4GAT1 transferase activity, only low residual activity (<3%) was detectable in <italic>B4gat1</italic>-deficient cells, and this loss could be restored by ectopic expression of B4GAT1 (<xref ref-type="fig" rid="fig4">Figure 4B</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A/B</xref>). Similarly, when we tested LARGE GlcA-T activity in control and glycosylation-deficient MEFs, only <italic>Large</italic><sup><italic>myd</italic></sup> MEFs lacked LARGE GlcA-T activity; the control and <italic>B4gat1</italic><italic>-</italic>deficient cells were normal (<xref ref-type="fig" rid="fig4">Figure 4C</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1C/D</xref>). These results suggest that the enzymatic activities of B4GAT1 and LARGE are independent and that each is unaffected by mutations in the gene product of the other.</p></sec><sec id="s2-7"><title>B4GAT1 mutations affect the subcellular localization and activity of B4GAT1</title><p>To date, several disease-causing <italic>B4GAT1</italic> (formerly termed <italic>B3GNT1</italic>) mutations have been reported in human patients (<xref ref-type="bibr" rid="bib11">Buysse et al., 2013</xref>; <xref ref-type="bibr" rid="bib49">Shaheen et al., 2013</xref>), in an N-ethyl-N-nitrosourea (ENU)-induced mutant mouse model (<xref ref-type="bibr" rid="bib58">Wright et al., 2012</xref>) and in a genetic screen for modifiers of LASV entry (<xref ref-type="bibr" rid="bib28">Jae et al., 2013</xref>). To test how reported <italic>B4GAT1</italic> missense mutations affect the intracellular localization of B4GAT1 and its enzymatic activity, we cloned three mutant B4GAT1-Myc expression constructs (<xref ref-type="fig" rid="fig5">Figure 5A</xref>): Mut1 (N390D) represents a mutation identified in a patient with Walker-Warburg syndrome (<xref ref-type="bibr" rid="bib11">Buysse et al., 2013</xref>); Mut2 (D227N/D229N) is a mutation in the glycosyltransferase signature DXD motif (<xref ref-type="bibr" rid="bib54">Wiggins and Munro, 1998</xref>; <xref ref-type="bibr" rid="bib3">Bao et al., 2009</xref>); and Mut3 (M155T) mimics a mutant allele identified in a <italic>B4gat1</italic>-deficient mouse model with axon guidance defects (<xref ref-type="bibr" rid="bib58">Wright et al., 2012</xref>). Immunoblot analysis confirmed that expression levels were similar for the Myc-tagged B4GAT1 control and all three mutant constructs Mut1-Mut3 in stably expressing HEK293T cells (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Previously, it had been shown that B4GAT1 localizes to the <italic>trans</italic>-Golgi near the TGN (<italic>trans</italic>-Golgi network) (<xref ref-type="bibr" rid="bib3">Bao et al., 2009</xref>; <xref ref-type="bibr" rid="bib35">Lee et al., 2009</xref>; <xref ref-type="bibr" rid="bib11">Buysse et al., 2013</xref>). In our immunofluorescence analysis, we found both the control and mutant construct Mut1 to exhibit normal Golgi localization and to co-localize with the Golgi marker Giantin (<xref ref-type="fig" rid="fig5">Figure 5C</xref>). B4GAT1 mutations in constructs Mut2 and Mut3, however, resulted in a high degree of mislocalization to the ER, as judged by overlap of the signal with the ER marker ERp72 (<xref ref-type="fig" rid="fig5">Figure 5D</xref>), indicating that the B4GAT1 mutant proteins are misfolded and retained in the ER. Analysis of B4GAT1 enzyme activity in lysates from cells stably overexpressing these constructs revealed strongly reduced activity in the cases of all three mutants, of less than 5% compared to activity levels in wild-type control (<xref ref-type="fig" rid="fig5">Figure 5E</xref>). Similarly, none of the B4GAT1 mutant constructs was able to complement and rescue the α-DG glycosylation defect in <italic>B4gat1</italic>-deficient MEFs (<xref ref-type="fig" rid="fig5">Figure 5F</xref>). These findings confirm that the identified B4GAT1 mutations are pathological and have a direct negative impact on B4GAT1 activity regardless of their subcellular localization. Additionally, the finding that the B4GAT1 DXD motif is essential further supports a role for B4GAT1 as a glycosyltransferase, since the DXD motif is thought to be involved in binding carbohydrate sugar-nucleoside diphosphates in manganese-dependent glycosyltransferases (<xref ref-type="bibr" rid="bib54">Wiggins and Munro, 1998</xref>).<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.015</object-id><label>Figure 5.</label><caption><title>Expression analysis and GlcA-T enzyme activity of B4GAT1 mutant constructs.</title><p>(<bold>A</bold>) Schematic presentation shows B4GAT1 enzyme product with functional domains and B4GAT1 mutations Mut1-Mut3 are indicated. (<bold>B</bold>) Expression analysis of B4GAT1-Myc control and mutant constructs in stable HEK293T cells. Immunoblotting of cell lysates from HEK293T cells stably overexpressing wild-type B4GAT1-Myc and mutant constructs (Mut1, Mut2 and Mut3) with anti-Myc antibody and β-Actin (loading control). (<bold>C/D</bold>) Subcellular localization of B4GAT1-Myc control and mutant constructs in stable HEK293T cells (see <bold>B</bold>). B4GAT1<italic>-</italic>Myc constructs were stained with anti-Myc (red), (<bold>C</bold>) anti-Giantin (Golgi marker, green), (<bold>D</bold>) anti-ERp72 (ER marker, green) and 4 ́,6-diamidino-2-phenylindole (DAPI, nuclei, blue). Individual stainings for c-Myc Giantin and ERp72 are shown in greyscale, and merged images are shown in color. Scale bars indicate 10 µm. (<bold>E</bold>) B4GAT1 enzyme activity in cell lysates from stable HEK293T cells overexpressing B4GAT1-Myc wild-type and B4GAT1<italic>-</italic>Myc mutant constructs (Mut1-Mut3). Relative activity (%) with respect to B4GAT1wild-type specific activity (19.8 nmol/hr/mg) is shown (n = 3). Error bars represent SD, Statistical analyses were performed by two-tail Student's <italic>t</italic> test. **p < 0.001. (<bold>F</bold>) Complementation of <italic>B4gat1</italic>-deficient (<italic>B4gat1</italic><sup><italic>LacZ/M155T</italic></sup>) MEF cells with B4GAT1-Myc control and mutant constructs. <italic>B4gat1</italic>-deficient MEFs were nucleofected with a wild-type or mutant B4GAT1 expression construct. α-DG functional glycosylation was analyzed by On-Cell-Western analysis. α-DG functional glycosylation was detected with α-DG glyco (IIH6) antibody.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.015">http://dx.doi.org/10.7554/eLife.03941.015</ext-link></p></caption><graphic xlink:href="elife03941f005"/></fig></p></sec><sec id="s2-8"><title>β-Xylose serves as an endogenous acceptor for B4GAT1</title><p>To further characterize the endogenous acceptor for B4GAT1, we first tested if B4GAT1dTM was able to use DGFc340 from control, <italic>Large</italic><sup><italic>myd</italic></sup> and <italic>B4gat1</italic>-deficient MEFs as an acceptor. Similar to the LARGE acceptor experiment in <xref ref-type="fig" rid="fig2">Figure 2B</xref>, we used radiolabeled [<sup>14</sup>C] UDP-GlcA sugar donor and measured transfer of [<sup>14</sup>C] to the protein A-bound DGFc340 acceptor. As expected, DGFc340 isolated from <italic>B4gat1</italic>-deficient cells was the only acceptor that incorporated substantial levels of the radioactive label (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). This confirmed that only the DGFc340 acceptor from <italic>B4gat1</italic>-deficient cells resembled the terminal acceptor glycan suitable for B4GAT1dTM to add GlcA. Our B4GAT1dTM in vitro enzyme assay demonstrated acceptor specificity for β-linked xylose (<xref ref-type="fig" rid="fig3">Figure 3C</xref>). To corroborate the hypothesis that β-linked xylose also serves as the endogenous B4GAT1 α-DG acceptor we pre-treated DGFc340 from <italic>B4gat1</italic>-deficient cells with β-xylosidase and measured the transfer of [<sup>14</sup>C] GlcA by B4GAT1dTM. After β-xylosidase treatment, the ability of <italic>B4gat1</italic>-deficient DGFc340 to act as an acceptor was strongly reduced; this constitutes indirect evidence that a β-linked xylose is indeed the postulated endogenous acceptor for B4GAT1 (<xref ref-type="fig" rid="fig6">Figure 6B</xref>), and that a yet unidentified xylosyltransferase acts upstream of B4GAT1.<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.016</object-id><label>Figure 6.</label><caption><title>β-xylose is the endogenous acceptor for B4GAT1.</title><p>(<bold>A</bold>) B4GAT1dTM enzymatic transfer of [<sup>14</sup>C] radiolabeled GlcA to DGFc340. Fc-tagged DGFc340 (acceptor) was produced in control, <italic>Large</italic><sup><italic>myd</italic></sup> (<italic>Large</italic>-deficient) and <italic>B4gat1</italic>-deficient MEFs and isolated from the culture medium using protein A-agarose. The protein A-bound Fc340 was used as acceptor in a B4GAT1dTM (enzyme) reaction with radiolabeled [<sup>14</sup>C] UDP-GlcA sugar (donor). The figure represents the transfer of radiolabeled GlcA onto the donor DGFc340 (n = 3). Error bars represent SD. Statistical analyses were performed by two-tail Student's <italic>t</italic> test. **p < 0.001. (<bold>B</bold>) β-Xylosidase pre-treatment impairs B4GAT1dTM transfer of [<sup>14</sup>C] radiolabeled GlcA. DGFc340 (acceptor) from <italic>B4gat1</italic>-deficient MEFs was digested with β-xylosidase prior to the B4GAT1dTM (enzyme) transfer reaction with [<sup>14</sup>C] UDP-GlcA sugar (donor). The figure represents the transfer of radiolabeled GlcA onto the donor DGFc340 (n = 3). Error bars represent SD Statistical analyses were performed by two-tail Student's <italic>t</italic> test. **p < 0.001.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.016">http://dx.doi.org/10.7554/eLife.03941.016</ext-link></p></caption><graphic xlink:href="elife03941f006"/></fig></p></sec><sec id="s2-9"><title>NMR-studies confirm that B4GAT1 synthesizes the acceptor glycan for LARGE</title><p>To further corroborate our finding that the glycosyltransferase LARGE utilizes a glucuronic acid-β1,4-xylose-β disaccharide acceptor as a primer to elongate it with its dual glycosyltransferase and polymerizing activity, we performed NMR structural studies. In our approach towards confirming each individual glycosidic linkage, we first synthesized the tetrasacharide GlcA-Xyl-GlcA-Xyl-MU, starting with the monosaccharide acceptor Xyl-β-MU and extending it in a stepwise manner using recombinant B4GAT1dTM and LARGEdTM as enzymes sources (<xref ref-type="fig" rid="fig7">Figure 7A</xref>).<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.017</object-id><label>Figure 7.</label><caption><title>NMR analyses of the tetrasacharide generated from B4GAT1dTM and LARGEdTM enzymatic reactions.</title><p>(<bold>A</bold>) Schematic depiction of the tetrasaccharide structure produced by the sequential reactions of B4GAT1dTM followed by LARGEdTM with the sugar units labeled A-D to indicate the order of their addition. (<bold>B</bold>) Overlay of the HMQC (black) and HMBC (green) spectra of the tetrasaccharide. All cross-peaks in the HMQC spectrum are labeled. Three interglycosidic cross-peaks detected in the HMBC spectrum are also labeled and indicated with red circles. The peaks are labeled with a first letter representing the subunit designated in <bold><italic>A,</italic></bold> and the rest of the label representing the position on that subunit. (<bold>C</bold>) TOCSY spectrum (top) and ROESY spectrum (bottom) of the tetrasaccharide. The TOCSY and ROESY spectra were collected with mixing time of 77 and 300 ms, respectively. The cross-peaks are labeled as in <bold>B</bold>. The observed interglycosidic ROEs are indicated with green circles.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.017">http://dx.doi.org/10.7554/eLife.03941.017</ext-link></p><p><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.03941.018</object-id><label>Figure 7—source data 1.</label><caption><p>Chemical shifts (ppm) of the signals in the <sup>1</sup>H and <sup>13</sup>C NMR spectra of the tetrasaccharide of GlcA-β1,3-Xyl-α1,3-GlcA-β1,4-Xyl-β-MU produced by B4GAT1 and LARGE.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.018">http://dx.doi.org/10.7554/eLife.03941.018</ext-link></p></caption><media mime-subtype="docx" mimetype="application" xlink:href="elife03941s002.docx"/></supplementary-material></p></caption><graphic xlink:href="elife03941f007"/></fig></p><p>The <sup>1</sup>H and <sup>13</sup>C resonances of the isolated tetrasaccharide product were assigned by using heteronuclear multiple quantum coherence (HMQC), heteronuclear 2-bond correlation (H2BC), and heteronuclear multiple bond coherence (HMBC) spectra (<xref ref-type="fig" rid="fig7">Figure 7B</xref>, <xref ref-type="supplementary-material" rid="SD2-data">Figure 7—source data 1</xref>). The detection of the interglycosidic cross-peaks of BH1/AC4, CC1/BH3, and DH1/CC3 in the HMBC spectrum (<xref ref-type="fig" rid="fig7">Figure 7B</xref>) clearly indicates the presence of a 1→4 interglycosidic linkage between sugar residues B and A, a 1→3 interglycosidic linkage between residues C and B, and a 1→3 interglycosidic linkage between residues D and C, respectively. A strong rotating-frame Overhauser effect (ROE) was observed from the H1 to H3 and H5 protons of residues A, B, and D in the ROE spectroscopy (ROESY) spectrum (<xref ref-type="fig" rid="fig7">Figure 7C</xref>), demonstrating that they have a β-configuration. The observed strong ROE from the residue C H1 proton to its own H2, but not to H3 and/or H5 demonstrates that the residue C has an α-configuration. The inter-residue ROEs observed in the ROESY spectrum are also consistent with the interglycosidic linkage assignments determined from the HMBC spectrum. Therefore, the tetrasaccharide has the glycosidic linkage structure GlcA-β1,3-Xyl-α1,3-GlcA-β1,4-Xyl-β-MU (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). These studies show that B4GAT1 possesses β1,4 glucuronyltransferase activity, and that LARGE can elongate this primer structure by adding repeating units [-3-Xyl-α1,3-GlcA-β1-] to produce a heteropolysaccharide. To further illustrate the complexity of assembling the functional glycan of α-DG, we summarize the current knowledge about the α-DG sugar structures and the contributing genes/enzymes in <xref ref-type="fig" rid="fig8">Figure 8</xref>.<fig-group><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.03941.019</object-id><label>Figure 8.</label><caption><title>Model of proposed α-DG O-mannosyl laminin-binding glycan structure and the enzymes that contribute to its synthesis.</title><p>Post-phosphoryl modification of α-DG requires B4GAT1 (β1,4 glucuronyltransferase); this enzyme generates the acceptor glycan, which serves as a primer for the glycosyltransferase LARGE to initiate synthesis of the laminin-binding glycan. Both gene products with known function (black) and gene products with currently unidentified function (red) are indicated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.019">http://dx.doi.org/10.7554/eLife.03941.019</ext-link></p></caption><graphic xlink:href="elife03941f008"/></fig><fig id="fig8s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03941.020</object-id><label>Figure 8—figure supplement 1.</label><caption><title>B4GAT1 <italic>and LARGE</italic> expression in human tissues.</title><p>qPCR revealing ubiquitous B4GAT1 and <italic>LARGE</italic> expression in all tissues analyzed, with highest expression of <italic>LARGE</italic> in brain and heart. cDNA was synthesized using random primers and oligo(dT) on commercially available human tissues RNAs. For each tissue, B4GAT1 and <italic>LARGE</italic> were specifically amplified, in triplicate, in the presence of SYBRgreen, and their expressions was normalized to that of the 28S RNA (normalization control). The expression in each tissue is referenced with respect to that in brain. Analyzed tissues: Br (brain), Ey (eye), He (heart), Ki (kidney), Li (liver), Lu (lung), Pa (pancreas), MG (mammary gland), Ov (ovary), Pl (placenta), Pr (prostate), SM (skeletal muscle), Sp (spleen), Te (testis), Th (thymus). Error bars represent.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03941.020">http://dx.doi.org/10.7554/eLife.03941.020</ext-link></p></caption><graphic xlink:href="elife03941fs007"/></fig></fig-group></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>In this study we used a multidisciplinary approach to investigate how the assembly of the α-DG LARGE glycan is initiated, and found that it requires B4GAT1-dependent synthesis of a novel glucuronyl-xylosyl acceptor primer. We show that B4GAT1 is a xylose β1,4-glucuronyltransferase, and that it is involved in synthesizing the glycan primer that subsequently can be elongated by LARGE with the ligand-binding glycan. B4GAT1 was initially cloned and described by Sasaki et al., (<xref ref-type="bibr" rid="bib48">Sasaki et al., 1997</xref>) as β1,3-N-acetylglucosaminyltransferase (iGnT, β3GNT1 or B3GNT1), which is essential for the synthesis of poly-N-acetyllactosamine. Furthermore, the B3GNT1 enzyme was proposed to contribute to the i antigen synthesis pathway by transferring N-acetylglucosamine onto a β-galactose acceptor with β1,3 linkage (<xref ref-type="bibr" rid="bib48">Sasaki et al., 1997</xref>). In contrast our data reveal a β1,4-glucuronyltransferase activity, which we have designated B4GAT1. We tested B4GAT1dTM with UDP-GlcNAc and the proposed Gal-β1,4-GlcNAc-β-MU acceptor, but we were not able to validate any N-acetylglucosaminyltransferase activity (<xref ref-type="fig" rid="fig3s4">Figure 3—figure supplement 4</xref>). Therefore, we propose to rename the enzyme B4GAT1, as a new member of the glucuronyltransferase family of proteins. To date, only 2 other enzymes are known to have β1,4-glucuronyltransferase activity. These are EXT1 and EXT2, and both are involved in the synthesis of heparan sulphate proteoglycans (<xref ref-type="bibr" rid="bib36">Lidholt and Lindahl, 1992</xref>).</p><p>Our findings regarding assembly of the LARGE glycan reveal striking similarities to the unique mechanism underlying the synthesis of proteoglycans. Both glycan polymers consist of repeating disaccharides that are synthesized by glycosyltransferases with dual glycosyltransferase activities (<xref ref-type="bibr" rid="bib21">Esko et al., 2009</xref>; <xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>). Furthermore, in both cases assembly of the terminal heteropolymer glycan is initiated by a disaccharide primer, which is part of a larger glycan linker that anchors the polysaccharide to a protein backbone. Future studies are needed to elucidate the full α-DG glycan structure and determine the roles of the putative glycosyltransferases FKTN, FKRP and TMEM5 in anchoring the α-DG ligand-binding glycan moiety to the phosphorylated Core M3 structure. At this point it cannot be ruled out that other, currently unidentified, genes also contribute to synthesis of the functional α-DG glycan.</p><p>Similar to their counterparts in other dystroglycanopathy genes, <italic>B4GAT1</italic> <italic>(</italic><italic>B3GNT1</italic><italic>)</italic> loss-of-function mutations in human patients result in Walker-Warburg Syndrome (WWS) (<xref ref-type="bibr" rid="bib11">Buysse et al., 2013</xref>; <xref ref-type="bibr" rid="bib49">Shaheen et al., 2013</xref>), the most severe condition in a range of clinically defined CMDs that are accompanied by brain and eye malformations. Milder <italic>B4GAT1</italic> mutations with residual enzyme activity are expected to cause a milder Limb Girdle Muscular Dystrophy (LGMD) phenotype, but patients with such mutations have not yet been described.</p><p><italic>B4gat1 (</italic><italic>B3gnt1</italic><italic>)</italic>-null mutations in mice result in early embryonic lethality, at ∼ E9.5 (<xref ref-type="bibr" rid="bib58">Wright et al., 2012</xref>), as is the case for reported null mutations in <italic>Dag1</italic> (<xref ref-type="bibr" rid="bib57">Williamson et al., 1997</xref>), <italic>Pomt1</italic> (<xref ref-type="bibr" rid="bib55">Willer et al., 2004</xref>), and <italic>Fukutin</italic> (<xref ref-type="bibr" rid="bib34">Kurahashi et al., 2005</xref>). Proper α-DG glycosylation is essential for early embryonic development in the mouse, including formation of the basement membrane, as defects in the Reichert's membrane are the suspected cause of death in α-DG glycosylation-deficient mice (<xref ref-type="bibr" rid="bib57">Williamson et al., 1997</xref>; <xref ref-type="bibr" rid="bib55">Willer et al., 2004</xref>; <xref ref-type="bibr" rid="bib34">Kurahashi et al., 2005</xref>). However, an ENU-based genetic screen for abnormal CNS axonal tracks identified a viable <italic>B4gat1</italic> <italic>(</italic><italic>B3gnt1</italic><italic>)</italic> dystroglycanopathy mouse model carrying a p.M155T <italic>B4gat1</italic> mutation (<xref ref-type="bibr" rid="bib58">Wright et al., 2012</xref>). The majority of compound heterozygous mice with both a <italic>LacZ</italic> (<italic>B4gat1</italic><sup><italic>LacZ</italic></sup>) null allele and a hypomorphic p.M155T (<italic>B4gat1</italic><sup><italic>M155T</italic></sup>) allele die perinatally, but a few survive and develop a characteristic CMD phenotype. In this study we used MEFs isolated from the <italic>B4gat1</italic><sup><italic>LacZ/M155T</italic></sup> mice and measured endogenous B4GAT1 activity. As expected the <italic>B4gat1</italic>-deficient MEFs were hypomorphic, producing low-level residual B4GAT1 activity (<3% relative to levels in wild-type control) (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). This corroborates that our B4GAT1 assay can be a valuable diagnostic tool for measuring endogenous activity in patient cells and tissues. The residual B4GAT1 enzyme activity in the <italic>B4gat1</italic>-deficient MEFs was also reflected when the α-DG glycosylation status was analyzed biochemically, by immunoblotting. Although B4GAT1 endogenous activity was very low, it was sufficient to synthesize low amounts of functionally active α-DG that was capable of binding the ligand laminin (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). This finding accounts for the difference between the early embryonic lethal phenotype in <italic>B4gat1</italic> null (<italic>B4gat1</italic><sup><italic>LacZ/LacZ</italic></sup>) mice and the slightly milder phenotype in <italic>B4gat1</italic> hypomorphic (<italic>B4gat1</italic><sup><italic>LacZ/M155T</italic></sup>) mice (<xref ref-type="bibr" rid="bib58">Wright et al., 2012</xref>).</p><p>It is worth noting that α-DG glycosylation is highly tissue specific as well as highly dependent on the developmental stage of the cells/tissue (<xref ref-type="bibr" rid="bib4">Barresi and Campbell, 2006</xref>). To date, it is not fully understood what causes the tissue-specific differences in α-DG processing, which are reflected as differences in its molecular weight and its ability to bind laminin (<xref ref-type="bibr" rid="bib22">Goddeeris et al., 2013</xref>). LARGE, the key contributor to assembly of the terminal laminin-binding glycan, and B4GAT1 as the upstream priming enzyme are broadly expressed at the RNA level (<xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1</xref>). Although both genes are similarly expressed in most tissues they are strikingly different in heart with <italic>LARGE</italic> expression being high and <italic>B4GAT1</italic> being low. Based on published case reports it does not appear that <italic>LARGE</italic> patients (<xref ref-type="bibr" rid="bib39">Longman et al., 2003</xref>; <xref ref-type="bibr" rid="bib13">Clarke et al., 2011</xref>; <xref ref-type="bibr" rid="bib41">Meilleur et al., 2014</xref>) are more prone to cardiac defects than other dystroglycanopathy patients. Also lower <italic>B4GAT1</italic> expression in the heart does not present a significant bottleneck for α-DG functional glycosylation as heart α-DG has full ligand binding ability (<xref ref-type="bibr" rid="bib22">Goddeeris et al., 2013</xref>). Therefore, the functional consequences of such uncoordinated expression of <italic>B4GAT1</italic> and <italic>LARGE</italic> are currently unknown. It is more likely that other gene products involved in α-DG functional glycosylation can become limiting factors and that the integration of all involved players account for the tissue-specific differences of this complex and highly controlled synthesis pathway. Furthermore, whether α-DG that is not properly glycosylated possesses an as yet unidentified ligand binding activity remains unclear. LARGE has been shown to be highly tunable in the context of cancer, T-cell development and muscle regeneration (<xref ref-type="bibr" rid="bib16">de Bernabe et al., 2009</xref>; <xref ref-type="bibr" rid="bib38">Liou et al., 2010</xref>; <xref ref-type="bibr" rid="bib22">Goddeeris et al., 2013</xref>). Repression of <italic>LARGE</italic> expression is responsible for the defects in DG-mediated cell adhesion that are observed in epithelium-derived cancer cells, and point to a defect of its glycosylation as a factor in cancer progression (<xref ref-type="bibr" rid="bib16">de Bernabe et al., 2009</xref>). Similarly, it was demonstrated that expression of <italic>B4GAT1</italic> (<italic>B3GNT1</italic>) is absent in a IIH6-negative subpopulation (PC3-L) of an otherwise IIH6-positive human prostate cancer cell line (PC3). The loss of <italic>B4GAT1</italic> expression and laminin-binding by α-DG in these cells was inversely correlated with the observed malignancy and tumor progression of the prostate cancer when these cells were transplanted into SCID mice (<xref ref-type="bibr" rid="bib3">Bao et al., 2009</xref>). In general, these results emphasize that proper α-DG glycosylation plays a critical role in tumor suppression.</p><p>Previous data suggested that B4GAT1 (B3GNT1) may be an integral component of various enzyme complexes, working with various glycosyltransferases that are functionally associated and involved in the same biosynthetic pathway. For example, it might work with B4GALT1 (<xref ref-type="bibr" rid="bib35">Lee et al., 2009</xref>) in the synthesis of poly-N-acetyllactosamine and with LARGE (<xref ref-type="bibr" rid="bib3">Bao et al., 2009</xref>) in synthesis of the α-DG laminin-binding glycan. It was also hypothesized that B4GAT1 may regulate LARGE, as B4GAT1 overexpression promoted formation of the LARGE-generated laminin-binding glycan (<xref ref-type="bibr" rid="bib3">Bao et al., 2009</xref>). However, in light of data presented in this study, in particular the finding that endogenous LARGE activity is not affected in <italic>B4gat1</italic>-deficient cells and vice versa, it seems more likely that B4GAT1 and LARGE have independent enzyme activities (<xref ref-type="fig" rid="fig4">Figure 4B/C</xref>).</p><p>In an effort to provide additional direct evidence and further corroboration of our conclusion that a xylose present in the α-DG O-mannosyl post-phosphoryl glycan linker serves as endogenous acceptor for B4GAT1, we performed radioactive metabolic cell labeling with [3H]-xylose. The goal was to show radioactive labeling of DGFc340 expressed in <italic>B4gat1</italic>-deficient MEFs with [3H]-xylose, which could subsequently be released by β-xylosidase treatment. However, this type of metabolic cell labeling proved to be technically challenging since only an insignificant amount (∼0.01%) of the total [3H]-xylose radioactivity was incorporated into the secreted DGFc340 fusion protein even after 4 day long-term labeling (data not shown). It is known that xylose uptake from the media into cells is poor (<xref ref-type="bibr" rid="bib50">Snider et al., 2002</xref>), which in our case becomes the limiting factor and made this experimental approach not feasible. Nevertheless, we feel confident that the sum of our indirect data including B4GAT1 Xyl-β-MU acceptor specificity (<xref ref-type="fig" rid="fig3">Figure 3C</xref>), pgsI-208 DGFc340 LARGE acceptor test (<xref ref-type="fig" rid="fig2">Figure 2D</xref>), β-xylosidase B4GAT1 acceptor treatment (<xref ref-type="fig" rid="fig6">Figure 6B</xref>) and finally the in vitro synthesis of a GlcA-β1,3-Xyl-α1,3-GlcA-β1,4-Xyl-β-MU tetrasaccharide by the sequential action of B4GAT1 and LARGE (<xref ref-type="fig" rid="fig7">Figure 7</xref>) provide strong and convincing evidence that β-xylose is indeed the endogenous acceptor for B4GAT1.</p><p>In conclusion, our study has identified B4GAT1 as a xylose β1,4-glucuronyltransferase, and revealed that it contributes to the O-mannosyl post-phosphoryl glycan linker of α-DG by synthesizing a glucuronyl-xylosyl disaccharide. This is the crucial acceptor primer that is targeted by the glycosyltransferase LARGE to initiate formation of a heteropolysaccharide on α-DG that is involved in its binding to ligands. As <italic>B4GAT1</italic>-deficiency was linked to laminin-binding defects of α-DG in a variety of contexts, our new findings will shed light on the mechanism underlying α-DG glycosylation-deficient CMDs (<xref ref-type="bibr" rid="bib11">Buysse et al., 2013</xref>; <xref ref-type="bibr" rid="bib49">Shaheen et al., 2013</xref>) and tumors (<xref ref-type="bibr" rid="bib3">Bao et al., 2009</xref>), and is expected to also open new therapeutic avenues for blocking the entry of pathogenic arenaviruses, including the hemorrhagic LASV into human cells (<xref ref-type="bibr" rid="bib28">Jae et al., 2013</xref>).</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Subjects and samples</title><p>All tissues and patient cells were obtained and tested according to the guidelines set out by the Human Subjects Institutional Review Board of the University of Iowa; informed consent was obtained from all subjects or their legal guardians (See <xref ref-type="table" rid="tbl1">Table 1</xref>).</p></sec><sec id="s4-2"><title>Cell cultures</title><p>Cells were maintained at 37°C and 5% CO<sub>2</sub> in Dulbecco's modified Eagle's medium (DMEM) plus fetal bovine serum (FBS: 10% in the case of HEK293T cells, 20% in the case of fibroblasts from patient skin) and 2 mM glutamine, 0.5% penicillin-streptomycin (Invitrogen, Carlsbad, CA). Pro5 (wild-type) and the glycosylation-deficient CHO (Lec cells) mutant cell lines termed Lec2 and Lec8 were purchased from ATCC (<xref ref-type="bibr" rid="bib46">Patnaik and Stanley, 2006</xref>). The Lec15.2 (<xref ref-type="bibr" rid="bib40">Maeda et al., 1998</xref>) and ldlD (<xref ref-type="bibr" rid="bib31">Kingsley et al., 1986</xref>) cell lines were kindly provided by Monty Krieger, the Lec13 (<xref ref-type="bibr" rid="bib45">Ohyama et al., 1998</xref>) cells by Pamela Stanley and the pgsI-208 (<xref ref-type="bibr" rid="bib2">Bakker et al., 2009</xref>) cells by Jeff Esko. These CHO cells were grown and maintained in F12 nutrition mix medium with 10% fetal bovine serum (Invitrogen) at 37°C and 5% CO<sub>2</sub>. MEFs were generated from E13.5 embryos (<xref ref-type="table" rid="tbl1">Table 1</xref>) as previously described (<xref ref-type="bibr" rid="bib60">Xu et al., 2005</xref>) and were maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, and 0.5% penicillin-streptomycin at 37°C in 5% CO<sub>2</sub>.</p></sec><sec id="s4-3"><title>[<sup>32</sup>P] orthophosphate labeling of cells</title><p>Phosphorylation of α-DG in glycosylation-deficient fibroblasts was determined based on the incorporation of [<sup>32</sup>P] into a secreted Fc-tagged α-DG recombinant protein, as described elsewhere (<xref ref-type="bibr" rid="bib61">Yoshida-Moriguchi et al., 2010</xref>).</p></sec><sec id="s4-4"><title>Adenovirus generation and gene transfer</title><p>E1-deficient recombinant adenoviruses (Ad5CMV-DGFc340, Ad5CMV-DGFc340mut (T317A/T319A) and Ad5CMV-<italic>LARGE</italic>/RSVeGFP) were generated by the University of Iowa Gene Transfer Vector Core and have been described previously (<xref ref-type="bibr" rid="bib5">Barresi et al., 2004</xref>). The constructs used to generate the E1-deficient recombinant adenoviruses Ad5CMV-DGFc340 and Ad5CMV-DGFc340mut (T317A/T319A) were made from pcDNA3-DGFc340 and DGFc340mut (T317A/T319A) (<xref ref-type="bibr" rid="bib24">Hara et al., 2011</xref>). pcDNA3-DGFc340 vectors were digested with KpnI/XbaI, and the resulting fragments were ligated into a KpnI/XbaI-digested pacAd5-CMV-KNpA vector. Similarly, Ad5CMV-B4GAT1-V5/RSVeGFP was generated by PCR amplifying a 1.3 kb C-terminal V5-tagged open reading frame fragment corresponding to mouse <italic>B4gat1</italic> (<italic>B3gnt1</italic><italic>,</italic> NM_175383) and cloning it into the XhoI/NotI polylinker region of pAd5CMVK-NpA. The following primers were used to amplify, by PCR, <italic>B4gat1</italic>-V5: 6823 forward (5′-aga<bold>ctcgag</bold>accATGcaaatgtcctacgccat-3′, XhoI adapter is bolded, start ATG is shown in capital letters) and 6822 reverse (5′-tat<bold>gcggccgc</bold>CTACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCgcatcggtggggagagttgg-3′; the NotI adapter is bolded and the V5-tag is shown in capital letters). Cultured cells were infected with viral vector for 12 hr, at an MOI of 400. We examined cultures 3–5 days after treatment. We used nucleofection as nonviral method for transferring genes into MEF cells. The Human Dermal Fibroblast Nucleofector kit was used according to an optimized protocol provided by the manufacturer (Amaxa Biosystems, Germany).</p></sec><sec id="s4-5"><title>Glycoprotein enrichment and biochemical analysis</title><p>WGA-enriched glycoproteins from frozen samples and cultured cells were processed as previously described (<xref ref-type="bibr" rid="bib43">Michele et al., 2002</xref>). Immunoblotting was carried out on polyvinylidene difluoride (PVDF) membranes as previously described (<xref ref-type="bibr" rid="bib43">Michele et al., 2002</xref>). Blots were developed with IR-conjugated secondary antibodies (Pierce Biotechnology, Rockford, IL) and scanned with an Odyssey infrared imaging system (LI-COR Bioscience, Lincoln, NE). Laminin overlay assays were performed as previously described (<xref ref-type="bibr" rid="bib43">Michele et al., 2002</xref>).</p><p>The monoclonal antibodies to the fully glycosylated form of α-DG (IIH6) (<xref ref-type="bibr" rid="bib19">Ervasti and Campbell, 1991</xref>), and also the polyclonal antibodies rabbit β-dystroglycan (AP83) (<xref ref-type="bibr" rid="bib18">Duclos et al., 1998</xref>) and anti-LARGE (Rb331) (<xref ref-type="bibr" rid="bib30">Kanagawa et al., 2004</xref>) were characterized previously. G6317 (core-αDG) from rabbit antiserum was raised against a keyhole limpet hemocyanin (KLH)-conjugated synthetic peptide of human dystroglycan (<xref ref-type="bibr" rid="bib56">Willer et al., 2012</xref>). Mouse monoclonal anti-Myc (clone 4A6) antibodies were purchased from Millipore (Billerica, MA), mouse monoclonal anti-β-Actin (Clone AC-74) antibodies were purchased from Sigma (St. Louis, MO) and mouse monoclonal anti-V5 antibodies were purchased from Invitrogen.</p></sec><sec id="s4-6"><title>Immunofluorescence microscopy</title><p>HEK293T cells expressing Myc-tagged glycosyltranferases were fixed with 4% paraformaldehyde in PBS, and then permeabilized with 0.2% Triton X-100 in PBS for 10 min on ice. After blocking with 3% BSA in PBS, the slides were incubated with anti-c-Myc antibody (4A6, Millipore) anti-Giantin (abcam, United Kingdom) or anti-ERp72 antibody (Calbiochem, San Diego, CA) for 18 hr at 4°C. The cells were incubated with an appropriate secondary antibody conjugated to Alexa488 or Alexa555 fluorophore after washing with PBS. 4′,6′-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) was used for nuclear staining. Images were observed using a Zeiss Axioimager M1 fluorescence microscope (Carl Zeiss, Thornwood, NY).</p></sec><sec id="s4-7"><title>On-Cell complementation and Western Blot assay</title><p>The On-Cell complementation assay was performed as described previously (<xref ref-type="bibr" rid="bib56">Willer et al., 2012</xref>). In brief, 2 × 10<sup>5</sup> cells were seeded into a 48-well dish. The next day the cells were infected with 200 MOI of Ad5CMV-<italic>LARGE1/eGFP</italic> in growth medium. Three days later, the cells were washed in TBS and fixed with 4% paraformaldehyde in TBS for 10 min. After blocking with 3% dry milk in TBS +0.1% Tween (TBS-T), the cells were incubated with primary antibody (glyco α-DG, IIH6) in blocking buffer overnight. To develop the On-Cell Western blots we conjugated goat anti-mouse IgM (Millipore) with IR800CW dye (LI-COR), subjected the sample to gel filtration, and isolated the labeled antibody fraction. After staining with IR800CW secondary antibody in blocking buffer, we washed the cells in TBS and scanned the 48-well plate using an Odyssey infrared imaging system (LI-COR). For cell normalization, DRAQ5 cell DNA dye (Biostatus Limited, United Kingdom) was added to the secondary antibody solution.</p></sec><sec id="s4-8"><title>Cloning of C-terminal Myc-tagged B4GAT1, TMEM5, FKTN, FKRP and LARGE</title><p>Open reading frames (ORF) were PCR amplified using the following primer sequences:</p><p>m<italic>B4gat1</italic> (1.3 kb), pTW324: forward 5′-aagGGATCCacc<bold>atg</bold>caaatgtcctacgccatccg-3′ (BamHI restriction site is shown in capital letters and start ATG is bolded) and reverse 5′-aga<bold>gcggccgc</bold><underline>CTACAAGTCTTCTTCAGAAATAAGTTTTTGTTC</underline><bold>GCTAGC</bold>cccgcatcggtggggagagttgggg-3′(NotI restriction site is bolded, Myc-tag sequence is underlined and NheI restriction site is shown in capital bold letters).</p><p>h<italic>FKTN</italic> (1.4 kb), pTW322: forward 5′-taaAGATCTacc<bold>atg</bold>agtagaatcaataagaacgtggttttg-3′ (BglII restriction site is shown in capital letters and start ATG is bolded) and reverse 5′-ttcGCTAGCcccatataactggataacctcatcccactc-3′ (NheI restriction site is shown in capital letters).</p><p>m<italic>Fkrp</italic> (1.5 kb), pTW323: forward 5′-taaGGATCCacc<bold>atg</bold>cggctcacccgctgctg-3′ (BamHI restriction site is shown in capital letters and start ATG is bolded) and reverse 5′-ttcGCTAGCcccaccgcctgtcaagcttaagagtgc-3′ (NheI restriction site is shown in capital letters).</p><p>m<italic>Tmem5</italic> (1.3 kb) pTW330: forward 5′-taaGGATCCacc<bold>atg</bold>cggctgacgcggacacg-3′ (BamHI restriction site is shown in capital letters and start ATG is bolded) and reverse 5′-ttcGCTAGCcccaactttattattaataaaaaatgaactttc -3′(NheI restriction site is shown in capital letters).</p><p>m<italic>Large</italic> (2.3 kb), pTW355: forward 5′-taaAGATCTacc<bold>atg</bold>ctgggaatctgcagagggag-3′ (BglII restriction site is shown in capital letters and start ATG is bolded) and reverse 5′-ttcGCTAGCcccgctgttgttctcagctgtgagatatttc-3′ (NheI restriction site is shown in capital letters).</p><p>First a BamHI/NotI digested PCR fragment from m<italic>B4gat1</italic> was cloned into the BamHI/NotI multiple cloning site (MCS) of a pIRES-puro3-derived vector, in which the NheI site in the MCS was deleted. Subsequently all other genes were digested with either BamHI and NheI or BglII and NheI, and subcloned into a BamHI and NheI-digested m<italic>B4gat1</italic>-myc pIRES-puro (pTW324) construct.</p></sec><sec id="s4-9"><title>Cloning of B4GAT1<italic>-</italic>Myc Mut 1-3 mutant constructs</title><p>To generate the mouse B4GAT1-Myc Mut1-Mut3 mutant expression constructs, we used the same forward primer A (5′-aagGGATCCaccatgcaaatgtcctacgccatccg-3′) and a reverse primer D (5′- agagcggccgcCTACAAGTCTTCTTCAGAAATAAGTTTTTGTTCGCTAGCcccgcatcggtggggagagttgggg-3′) that were used to clone m<italic>B4GAT1</italic>-Myc (see m<italic>B4gat1</italic>-Myc pTW324 cloning). Primers A and D bind at the 5′-end and 3′-end of the m<italic>B4gat1</italic> coding region. For each mutation we designed overlapping forward (B1-3) and reverse (C1-3) primers that included the respective mutation (shown in bold capital letters):</p><p><bold>mB4GAT1-Mut1</bold> (c.1168A > G, p.N390D): B1 5′-ccaaaaggaggctgaa<bold>G</bold>accagcgcaataagatc-3′ and C1: 5′-gatcttattgcgctggt<bold>C</bold>ttcagcctccttttgg-3′.</p><p><bold>mB4GAT1-Mut2</bold> (c.679/685 G > A, p.D227N/D229N): B2 5′- ggccaactacgccctggtgatt<bold>A</bold>atgtg<bold>A</bold>acatggtgcccagcgaagggc-3′ and C2 5′- gcccttcgctgggcaccatgt<bold>T</bold>cacat<bold>T</bold>aatcaccagggcgtagttggcc-3′.</p><p><bold>mB4GAT1-Mut3</bold> (c.464T > C, M155T): B3 5′- gcgctagggtcgcca<bold>C</bold>gcacctcgtgtgcccctc-3′ and C3 5′- gaggggcacacgaggtgc<bold>G</bold>tggcgaccctagcgc.</p><p>Using the m<italic>B4gat1</italic>-Myc (pTW324) expression construct as template, we PCR amplified 5′-fragments, using primer pairs A/B1-3 and 3′-fragments using C1-3/D, respectively. The PCR products were isolated and used as the template DNAs in the second round of amplification with primer pair A-D. The 1.3 kb final PCR product was purified and digested with BamHI/NheI and then ligated into pTW324 digested with the same enzymes. The sequence of the insert DNA was confirmed by Sanger sequencing.</p></sec><sec id="s4-10"><title>Cloning of B4GAT1dTM</title><p>The construct expressing B4GAT1 without its transmembrane region was generated by amplifying a 1.1 kb cDNA fragment of mouse <italic>B4gat1</italic> (<italic>B3gnt1</italic>, acc.# NM_175383) from mB4GAT1-Myc expression vector pTW324, using primer pair #8629 (5′-ggtGAATTCcacggccaggaggagcagg-3′) and #8630 (5′- atgACCGGTatgcatattcaagtcttcttcagaaataagtttttgttcgc-3′). EcoRI and AgeI restriction sites included in the primers are indicated in capital letters. The PCR fragment was digested with EcoRI and AgeI and subcloned to generate construct pCMV3xFLAG-TEV-B4GAT1dTM-Myc6xHIS (pTW351), which expresses a mouse B4GAT1dTM fusion protein (amino acids 37–415) tagged with a N-terminal 3xFLAG and C-terminal Myc6xHis.</p></sec><sec id="s4-11"><title>Generation of cell lines stably expressing B4GAT1dTM proteins</title><p>HEK293T cells were transfected with constructs pTW351 (B4GAT1dTM) using FuGENE 6 (Roche Applied Science, Indianapolis, IN). The construct contains an IRES-puromycin resistance cassette and stable cell lines were selected in medium containing Puromycin (1 µg/ml, InvivoGen, San Diego, CA). Expression and secretion of B4GAT1dTM protein into the culture medium was confirmed by immunoblotting with anti-Myc antibody 4A6 (Millipore). The stable cell lines obtained in this way were adapted to serum-free medium 293SFMII (Invitrogen) and cultivated in CELLine bioreactors (CL1000, Argos Technologies, Elgin, IL).</p></sec><sec id="s4-12"><title>Purification of B4GAT1dTM and LARGEdTM</title><p>B4GAT1dTM and LARGEdTM secreted into the culture medium by HEK293T cells were purified using the Talon metal-affinity resin (Clontech, Mountain View, CA) according to the manufacturer's instructions. The purity of the protein was confirmed by SDS-PAGE and Coomassie Brilliant blue (CBB) staining (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1B</xref>). For the enzyme assay, the eluate was desalted and concentrated using an Amicon Ultra centrifugal filter unit (Millipore).</p></sec><sec id="s4-13"><title>DGFc340 in vitro LARGEdTM assay</title><p>To generate the DGFc340 and DGFc340-mut acceptor proteins we infected control and glycosylation-deficient MEFs and CHO-derived cell lines with Ad5-CMV DGFc340 adenoviral vectors at an MOI of 400. At 4 days post-infection the secreted proteins were isolated from the culture medium using Protein A-agarose beads (Santa Cruz, Dallas, TX). DGFc340 bound Protein A-agarose beads were washed three times with TBS and Protein A slurry prebound with ∼25 µg DGFc340 was added to the in vitro LARGEdTM assay. Enzyme reactions (50 µl) were carried out at 37°C, with 5 mM UDP-GlcA and 5 mM UDP-Xyl, in 0.1 M MES (2-(<italic>N</italic>-morpholino)ethanesulfonic acid) buffer (pH 6.0) supplemented with 10 mM MnCl<sub>2</sub>, 10 mM MgCl<sub>2</sub>, 0.2% Triton X-100 and 5 µg purified LARGEdTM protein. The reaction was terminated by adding 5× LSB and boiling for 5 min, The samples were subsequently analyzed by immunoblotting.</p></sec><sec id="s4-14"><title>DGFc340 [<sup>14</sup>C] radioactive sugar donor in vitro assay</title><p>DGFc340 (∼25 µg) and DGFc340-mut (∼25 µg) bound Protein A-agarose beads were washed with TBS and used in the in vitro LARGEdTM assay. 30 µl enzyme reactions were carried out at 37°C for 20 hr, with 0.05 µCi UDP-GlcA [GlcA-<sup>14</sup>C] (final conc. 5.5 µM) and 0.05 µCi UDP-Xyl [Xyl-<sup>14</sup>C] (final conc. 6.6 µM), in 0.1 M MES buffer (pH 6.0) supplemented with 10 mM MnCl<sub>2</sub>, 10 mM MgCl<sub>2</sub>, 0.2% Triton X-100 and 5 µg purified LARGEdTM protein. The reaction was terminated by adding 25 µl of 0.1 M EDTA. After three washes with TBS the Protein A-agarose-bound DGFc340 samples were analyzed by scintillation counting.</p><p>The reactions for B4GAT1dTM activity were carried out similarly. Again, 30 µl enzyme reactions were carried out at 37°C for 20 hr and with 0.05 µCi UDP-GlcA [GlcA-<sup>14</sup>C] (final conc. 5.5 µM), but in this case 0.1 M MOPS (3-(<italic>N</italic>-morpholino)propanesulfonic acid) buffer (pH 7.0) supplemented with 10 mM MnCl<sub>2</sub>, 10 mM MgCl<sub>2</sub>, 0.2% Triton X-100 was used, with 0.25 µg purified B4GAT1dTM protein.</p><p>[<sup>14</sup>C] labeled sugar nucleotides were purchased from ARC (American Radiolabeled Chemicals, St. Louis, MO).</p></sec><sec id="s4-15"><title>Glycosidase digestion</title><p>Recombinant β-glucuronidase from <italic>E.coli</italic> was purchased from Sigma (G8295). Each digest was performed in a 100 µl volume at 37°C for 12 hr in 50 mM NaPO<sub>4</sub>, pH 7.0, 5 mM DTT, 1 mM EDTA, 0.1% Triton X-100 in the presence of 10 µg (100 units) β-glucuronidase.</p><p>Recombinant β-xylosidase from <italic>E.coli</italic> was purchased from Sigma (X3504). Each digest was performed in a 100 µl volume at 70°C for 60 min in 50 mM sodium acetate at pH 5.8 in the presence of 20 µg β-xylosidase.</p></sec><sec id="s4-16"><title>Analysis of enzymatic activities of B4GAT1 and LARGE</title><p>The HPLC-based enzyme assays for B4GAT1-Myc (100 µg cell lysates) and B4GAT1dTM (0.25 µg purified protein) were performed using Xyl-β-MU (0.1 mM) (Sigma) as the acceptor. The samples were incubated for 2 hr for analytical purposes and 24 hr for preparative purposes. 50 µl enzyme reactions were carried out at 37°C, with 5 mM UDP-GlcA, in 0.1 M MOPS buffer (pH 7.0) supplemented with 10 mM MnCl<sub>2</sub>, 10 mM MgCl<sub>2</sub>, and 0.2% Triton X-100. The reaction was terminated by adding 25 µl of 0.1 M EDTA and boiling for 5 min. The supernatant was analyzed using a <italic>LC</italic>18 column (4.6 × 250 mm Supelcosil LC-18 column (Supelco, Bellefonte, PA)) with Buffer A (50 mM ammonium formate pH 4.0) and Buffer B (80% acetonitrile in buffer A), using a 12% B isocratic run at 1 ml/min using Beckman Gold system (Beckman Coulter, Inc., Brea, CA). The elution of MU derivatives was monitored by fluorescence detection (325 nm for excitation, and 380 nm for emission). For the assessment of metal dependence, all ions were used at a concentration of 10 mM in 0.1 M MOPS pH 7.0. To test pH-dependent activity testing buffers ranging from pH 4.5–8.5 were used: 0.1 M sodium acetate (pH 4.5–5.5), 0.1 M MES (pH 5.5–6.5), 0.1 M MOPS (pH 6.5–7.5) and 0.1 M Tris (pH 7.5–8.5).</p><p>To assess endogenous B4GAT1 GlcA-T activity in MEFs, we solubilized the cells in TBS 1% TX-100. 100 µg total protein from crude lysates were added to each assay. 50 µl enzyme reactions were carried out for 18hr at 37°C, with 5 mM UDP-GlcA, in 0.1 M MOPS buffer (pH 7.0) supplemented with 10 mM MnCl<sub>2</sub>, 10 mM MgCl<sub>2</sub>, and 0.2% Triton X-100. For analysis of substrate specificity Xyl-α-MU (Sigma), Xyl-β-MU (Sigma) and Xyl-α1,3-GlcA-β-MU were added to the standard enzyme reaction at a concentration of 0.1 mM.</p><p>The HPLC-based enzymatic assay for LARGEdTM (5 µg purified protein) and endogenous LARGE was performed using GlcA-β-MU, GlcA-β1,3−Xyl-α-MU and GlcA-β1,4−Xyl-β−MU as the acceptor for Xyl-T activity and Xyl-α1,3-GlcA-β-MU for GlcA-T activity as described previously (<xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>, <xref ref-type="bibr" rid="bib26">2013</xref>, <xref ref-type="bibr" rid="bib27">2014</xref>). For the assessment of endogenous LARGE GlcA-T activity in MEF cells, we solubilized the cells in TBS 1% TX-100 and enriched glycoproteins from crude lysates (2 mg total protein) using WGA-agarose. N-Acetylglucosamine-eluted glycoproteins from WGA-bound glycoproteins were incubated in a volume of 50 μl for 18 hr at 37°C, with 0.1 mM MU-acceptor, 5 mM UDP-GlcA in 0.1 M MES buffer pH 6.0, 10 mM MnCl<sub>2</sub>, 10 mM MgCl<sub>2</sub> and 0.2% Triton X-100. The reaction was terminated by adding 25 μl of 0.1 M EDTA and boiling for 5 min, and the supernatant was analyzed with an LC-18 column using a 12% B isocratic run.</p></sec><sec id="s4-17"><title>Analysis of B4GAT1 GlcNAc-transferase enzyme activity</title><p>The test B4GAT1dTM for GlcNAc transferase activity Gal-β1,4-GlcNAc-β-MU (0.1 mM) was used as acceptor. The 50 µl enzyme reactions were carried out as described previously (<xref ref-type="bibr" rid="bib48">Sasaki et al., 1997</xref>) at 37°C, with 5 mM UDP-GlcNAc in 0.1 M cocodylate buffer (pH 7.0) supplemented with 20 mM MnCl<sub>2</sub>, 5 mM ATP and 0.25 µg B4GAT1dTM enzyme. The reaction was terminated by adding 25 µl of 0.1 M EDTA and boiling for 5 min. The supernatant was analyzed using a LC18 column (4.6 × 250 mm Supelcosil LC-18 column (Supelco)) with Buffer A (50 mM ammonium formate pH 4.0) and Buffer B (80% acetonitrile in buffer A), using a 16% B isocratic run at 1 ml/min using Beckman Gold system.</p></sec><sec id="s4-18"><title>Separation and purification of the disaccharide generated by B4GAT1dTM</title><p>A large scale reaction was carried out using B4GAT1dTM purified using a metal-affinity resin as described previously for LARGEdTM (<xref ref-type="bibr" rid="bib25">Inamori et al., 2012</xref>). B4GAT1dTM was added to 10 mM of UDP-GlcA and Xylose-β−MU in 50 mM MOPS buffer pH 7.0, 10 mM MgCl<sub>2</sub>, 10 mM MnCl<sub>2</sub> and 0.5% TX-100 and incubated for 48 hr at 37°C with rotation. The sample was then run over a C18 column (4.6 × 250 mm Supelcosil LC-18 column (Supelco)) with Buffer A (50 mM ammonium formate pH 4.0) and Buffer B (80% acetonitrile in buffer A) using a 16% B isocratic run at 1 ml/min on a Beckman Gold system. The elution of MU derivatives was monitored by fluorescence detection (325 nm for excitation, and 380 nm for emission). The product in the peak fractions was collected and lyophilized. The dried sample was then brought up in Milli-Q water (500 µl) and lyophilized and this procedure was repeated three times, after which the sample was brought up in Milli-Q water. The product was quantitated based on the standard curve of GlcA-β-MU. This sample was used for NMR studies.</p></sec><sec id="s4-19"><title>Separation and purification of the tetrasaccharide generated by B4GAT1dTM and LARGEdTM</title><p>The GlcA-β1,4-xylose-β-MU disaccharide (B4GAT1 product) was added to 10 mM of UDP-Xyl in 50 mM sodium acetate buffer at pH 5.5 and with 10 mM MgCl<sub>2</sub>, 10 mM MnCl<sub>2</sub>, 0.5% TX-100 and LARGEdTM attached to metal-affinity resin and incubated for 48 hr at 37°C with rotation. The sample was then run over a LC18 column (4.6 × 250 mm Supelcosil LC-18 column (Supelco)) with Buffer A (50 mM ammonium formate pH 4.0) and Buffer B (80% acetonitrile in buffer A) using a 16% B isocratic run at 1 ml/min on a Beckman Gold system. The elution of MU derivatives was monitored by fluorescence detection (325 nm for excitation, and 380 nm for emission). The trisaccharide peak was collected and lyophilized. The lyophilized sample was then brought up in 10 mM UDP-GlcA in 50 mM MOPS buffer pH 6.0, 10 mM MgCl<sub>2</sub>, 10 mM MnCl<sub>2</sub> and 0.5% TX-100 and incubated for 48 hr at 37°C with rotation. It was again run on a C18 column with 16% B isocratic run. The product peak fraction was then collected and lyophilized. The dried sample was brought up in Milli-Q water (500 µl) and lyophilized. This procedure was repeated a total of three times. The last time the sample was brought up in Milli-Q water and the product was quantitated using a standard curve of GlcA-β-MU. This sample was used for NMR studies.</p></sec><sec id="s4-20"><title>Synthesis and purification of the disaccharide generated by B4GALT1</title><p>A large scale reaction was carried out using recombinant human B4GALT1 (purchased from R&D Systems cat# 3609-GT, Minneapolis, MN). B4GALT1 (1.5 µg) was added to 5 mM of UDP-Gal and 3 mM GlcNAc-β−MU in 50 mM Tris buffer pH 7.5, 10 mM MgCl<sub>2</sub> and 150 mM NaCl and incubated for 48 hr at 37°C with rotation. The sample was then run over a C18 column (4.6 × 250 mm Supelcosil LC-18 column (Supelco)) with Buffer A (50 mM ammonium formate pH 4.0) and Buffer B (80% acetonitrile in buffer A) using a 16% B isocratic run at 1 ml/min on a Beckman Gold system. The elution of MU derivatives was monitored by fluorescence detection (325 nm for excitation, and 380 nm for emission). Over time in the above reaction a peak was seen that ran about 1.5 min after the GlcNAc-β−MU peak at 21.5 min. This product peak was collected was and lyophilized. The dried sample was then brought up in Milli-Q water (500 µl) and lyophilized and this procedure was repeated three times, after which the sample was brought up in Milli-Q water. The product was quantitated based on the standard curve of GlcA-β-MU. This sample was used for NMR studies.</p></sec><sec id="s4-21"><title>NMR analysis</title><p>Samples were prepared for NMR by fractionation (using gel filtration and/or LC-18 chromatography) as described above, followed by the exchange of hydroxyl hydrogens by lyophilization and dissolution in 10 mM sodium phosphate buffer pH 6.5, in 100% D<sub>2</sub>O. <sup>1</sup>H homonuclear two-dimensional DQF-COSY (<xref ref-type="bibr" rid="bib47">Rance et al., 1983</xref>), TOCSY (<xref ref-type="bibr" rid="bib8">Braunschweiler and Ernst, 1983</xref>), and ROESY (<xref ref-type="bibr" rid="bib14">Davis and Bax, 1985</xref>) experiments, and <sup>1</sup>H/<sup>13</sup>C heteronuclear two-dimensional HMQC, HMBC, and H2BC experiments (<xref ref-type="bibr" rid="bib44">Nyberg et al., 2005</xref>) were collected using a Bruker Avance II 800 MHz NMR spectrometer equipped with a sensitive cryoprobe. All NMR spectra were recorded at 25°C. The <sup>1</sup>H chemical shifts were referenced to 2,2-dimethyl- 2-silapentane-5-sulfonate (DSS). NMR spectra were processed using the NMRPipe software package (<xref ref-type="bibr" rid="bib17">Delaglio et al., 1995</xref>) and analyzed using NMRView software (<xref ref-type="bibr" rid="bib29">Johnson and Blevins, 1994</xref>).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank the Gene Transfer Vector Core (UI, supported by NIH/NIDDK P30 DK 54759) for generating adenoviruses; We thank Pamela Stanley, Monty Krieger and Jeff Esko for providing us with CHO mutant cells, David Ginty for providing us with <italic>B4gat1</italic> (<italic>B3gnt1</italic><italic>)</italic>-deficient mice, Hans v. Bokhoven for providing us with patient fibroblasts, members of the Campbell laboratory for fruitful discussions; Andrew Crimmins for technical support; Christine Blaumueller for critical reading of the manuscript. This work was supported in part by a Paul D. Wellstone Muscular Dystrophy Cooperative Research Center Grant (1U54NS053672, KPC and TW), a MDA grant (238219, KPC and TW) and an ARRA Go Grant (1 RC2 NS069521-01, KPC and TW). KPC is an investigator of the Howard Hughes Medical Institute.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>TW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>K-I, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>DV, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con4"><p>CH, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con5"><p>GM, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con6"><p>YH, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con7"><p>DBVB, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con8"><p>LY, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con9"><p>KMW, Analysis and interpretation of data, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con10"><p>KPC, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: Animal care, ethical usage and procedures were approved and performed in accordance with the standards set forth by the National Institutes of Health and the Animal Care Use and Review Committee at the University of Iowa (protocol #4081122). At the University of Iowa all mice are socially housed (unless single housing is required) under specific pathogen-free conditions in an AAALAC accredited animal facility. Housing conditions are as specified in the Guide for the Care and Use of Laboratory Animals (NRC). Mice are housed on Thoren brand, HEPA filtered ventilated racks, in solid bottom cages with mixed paper bedding. A standard 12/12-h light/dark cycle was used. 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An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “B4GAT1 synthesizes a glucuronyl-xylosyl acceptor required for initiation of LARGE-mediated α-dystroglycan glycosylation” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Vivek Malhotra (Senior editor) and 4 reviewers, one of whom, Suzanne Pfeffer, is a member of our Board of Reviewing Editors. The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The dystroglycanopathies are a subset of muscular dystrophies. Many result from genetic defects in the formation of a novel GlcA-Xyl glycosaminoglycan (GAG), assembled by the LARGE glycosyltransferase on a trisaccahride-Man-PO4 containing core. Knowledge of this interesting and complex receptor glycan has been driven by the identification of a number of disease genes predicted to encode novel glycosyltransferases. However, the biochemical contributions of many of the gene products, and the nature of the linker between the GAG and the Man-PO4 core, remain unknown. This manuscript 1) identifies the final sugar of the linker as a beta-GlcA, 2) shows that the beta-GlcA becomes modified by the alpha-3-XylT activity of LARGE, 3) shows that Xyl contributes to the linker, and 4) provides strong evidence that the previously identified B3GNT1 disease gene is directly responsible for addition of beta-GlcA to the underlying core, possibly forming a GlcA(beta)1-4Xyl(beta)-linkage. Overall, this is a thorough and excellently documented piece of detective work to characterize the LARGE primer on alpha-DG, in which a role for B3GNT1/B4GAT1 is systematically built up first using small molecules, then an alpha-DG surrogate and alpha-DG itself. In addition, the new activity assigned to this gene will induce reinterpretation of the basis for its interesting cellular functions in cancer. Other interesting information regarding the roles of other GT-like genes, the biochemical and cellular effects of 3 B3GNT1/B4GAT1 disease mutations, and expression of B3GNT1/B4GAT1 RNA, is also presented. The paper would, however, be strengthened if the following issues are addressed.</p><p>1) The authors put forward a stronger conclusion, that the terminal primer for LARGE is a GlcA(beta)1-4Xyl(beta)-moiety, based on detection of the corresponding activity using recombinantly prepared B3GNT1. However, there is a problem, because they do not directly demonstrate the presence of the disaccharide on native alpha-DG or DGFc340. Although they show that B3GNT1 is capable of catalyzing the transfer of GlcA from UDP-GlcA in beta-linkage to the 4 position of a beta-linked Xyl residue, this was carried out at high enzyme and substrate concentrations (ratio unspecified) that might drive alternative reactions that are not relevant at native concentrations in vivo (e.g.,: Lairson et al. (2006) Nat Chem Biol 2:724-8). While they convincingly showed that the enzyme has high selectivity toward UDP-GlcA relative to other potential donor substrates, they did not screen other possible acceptors. However, they did show that the B3GNT1 acceptor activity of DGFc340 from B3GNT1-deficient cells was inhibited by pretreatment with a commercial preparation of beta-xylosidase, but the enzyme concentration was high, release of Xyl was not verified, and alternative glycosidase activities were not excluded. Thus the conclusion that the sugar underlying GlcA is Xyl(beta) is based on converging lines of evidence that alone are rather soft. This conclusion will not be incontrovertible until the presence of the disaccharide on DGFc340 or alpha-DG is directly demonstrated, such as might be supported by mass spectrometry. The authors could quite easily label DGFc340 produced in B4GAT1 mutant MEFs with 3H-xylose, show that treatment of the captured DGFc340 substrate by beta-xylosidase releases 3H-xylose and destroys it as a substrate of recombinant B4GAT, and show that, once B4GALT1 has acted on untreated DGFc340, 3H-xylose can no longer be released by treatment with beta-xylosidase. If these data are not forthcoming (and this may well be), a qualification of their conclusions is warranted.</p><p>2) The authors also rename B3GNT1 as B4GAT1. However, they do not show that it lacks the beta-3-GlcNAc-transferase (i-antigen forming) activity originally assigned to it. While it can be argued that the latter activity was also assigned based on indirect evidence, the authors did not adequately address the potential for beta-3-GlcNAc-transferase activity because they only tested Gal(beta)-MU as an acceptor, which is not a substrate, and did not test the preferred Gal(beta)1-4GlcNAc-. If it is not too difficult, they should try to clarify this directly. The authors should explain clearly at the beginning that their data will show that the gene called B3GNT1 in the literature, in fact encodes a glucuronisyltransferase and not an N-acetylglucosaminyltransferase as previously thought.</p><p>3) While many experiments add up to strong support for the authors' conclusions re B4GAT1, their interpretation of the data in <xref ref-type="fig" rid="fig1">Figure 1B</xref> may or may not be the case. The data in <xref ref-type="fig" rid="fig1">Figure 1B</xref> show that LARGE cannot override a defect in FKRP, FKTN, B4GAT1 or TMEM5. However, the latter does not mean that all these activities act prior to LARGE in cells. The evidence that B4GAT1 acts prior to LARGE is strong and described in the manuscript. It is also clear as shown later that LARGE acting in the absence of these activities in vitro can generate laminin binding glycans. However, one or more of the remaining three activities could potentially act after LARGE and be required to modify LARGE-generated glycans such that they become capable of binding to laminin or a different binding partner when LARGE is expressed at endogenous levels in vivo. Determining whether these activities act prior to or post LARGE is beyond the scope of this manuscript. However, the authors should qualify their conclusions with respect to <xref ref-type="fig" rid="fig1">Figure 1</xref> and the model in Figure 18.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03941.022</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The authors put forward a stronger conclusion, that the terminal primer for LARGE is a GlcA(beta)1-4Xyl(beta)-moiety, based on detection of the corresponding activity using recombinantly prepared</italic> B3GNT1<italic>. However, there is a problem, because they do not directly demonstrate the presence of the disaccharide on native alpha-DG or DGFc340. Although they show that</italic> B3GNT1 <italic>is capable of catalyzing the transfer of GlcA from UDP-GlcA in beta-linkage to the 4 position of a beta-linked Xyl residue, this was carried out at high enzyme and substrate concentrations (ratio unspecified) that might drive alternative reactions that are not relevant at native concentrations</italic> in vivo <italic>(e.g.,: Lairson et al (2006) Nat Chem Biol 2:724-8). While they convincingly showed that the enzyme has high selectivity toward UDP-GlcA relative to other potential donor substrates, they did not screen other possible acceptors. However, they did show that the</italic> B3GNT1 <italic>acceptor activity of DGFc340 from</italic> B3GNT1<italic>-deficient cells was inhibited by pretreatment with a commercial preparation of beta-xylosidase, but the enzyme concentration was high, release of Xyl was not verified, and alternative glycosidase activities were not excluded. Thus the conclusion that the sugar underlying GlcA is Xyl(beta) is based on converging lines of evidence that alone are rather soft. This conclusion will not be incontrovertible until the presence of the disaccharide on DGFc340 or alpha-DG is directly demonstrated, such as might be supported by mass spectrometry. The authors could quite easily label DGFc340 produced in</italic> B4GAT1 <italic>mutant MEFs with 3H-xylose, show that treatment of the captured DGFc340 substrate by beta-xylosidase releases 3H-xylose and destroys it as a substrate of recombinant</italic> B4GAT<italic>, and show that, once</italic> B4GALT1 <italic>has acted on untreated DGFc340, 3H-xylose</italic> can <italic>no longer be released by treatment with beta-xylosidase. If these data are not forthcoming (and this may well be), a qualification of their conclusions is warranted</italic>.</p><p>We thank the reviewers for raising an important point and suggesting some interesting additional experiments. We followed up with [3H] xylose labeling of DGFc340 in cells as suggested by the reviewers. As metabolic radioactive labeling of DGFc340 with [3H] xylose sounds very feasible on paper it proved to be technically challenging in reality. When we performed [3H] xylose metabolic cell labeling only ∼0.01% of the total radioactivity was incorporated into the secreted DGFc340 fusion protein. We validated the presence of the DGFc340 and DGFc340mut fusion protein by Western Blot, however the radioactive labeling for DGFc340 and DGFc340mut expressed in B3gnt1-deficient MEFs were indistinguishable and not significant. As stated by Snider M (Metabolic Labeling of Glycoproteins with Radioactive Sugars, Current protocols in cell biology, 2002) [3H] xylose is poorly taken up into cells creating a bottleneck that made this experimental approach not feasible.</p><p>Since the metabolic labeling of DGFc340 with [3H]-Xylose was not successful we did not include this data in the revised manuscript at this point. However, we present the data as <xref ref-type="fig" rid="fig9">Author response image 1</xref>. If the reviewers feel this data exhibits value for the manuscript we will include the data in the final manuscript.<fig id="fig9" position="float"><label>Author response image 1.</label><caption><p>[3H] Xylose metabolic labeling of DGFc340. DGFc340 and DGFc340mut fusion proteins were enriched by Protein A-agarose from culture medium of [3H] Xylose labeled control and B4gat-deficient MEF cells. (<bold>A</bold>) Expression and functional glycosylation analysis of Fc fusion proteins by Coomassie brilliant blue (CBB) stain and by immunoblot. The SDS-PAGE was stained with CBB and the immunoblot was incubated with antibodies against glyco α-DG (IIH6) and anti-Fc. (<bold>B</bold>) The figure represents the incorporation of radiolabeled [3H] Xylose into DGFc340/DGFc340mut fusion proteins (n = 3). Error bars represent s.d. Statistical analyses were performed by two-tail Student's <italic>t</italic> test. ns: not significant (p > 0.05).</p></caption><graphic xlink:href="elife03941f009"/></fig></p><p>Alternatively the reviewers suggested validating the Xylose sugar on DGFc340 by mass spectrometry. Given the fact that only a very minor portion of the secreted DGFc340 fusion protein gets modified with the functional glycan this approach is not feasible either.</p><p>We feel that the sum of our data and especially the in vitro synthesis of a GlcA-β1,3-Xyl-α1,3-GlcA-β1,4-Xyl-β-MU -tetrasaccharide by the sequential action of B4GAT1 and LARGE (Figure 13) provide strong and convincing evidence that B4GAT1 contributes to the primer/acceptor disaccharide (GlcA-β1,4-Xyl) that can be extended by LARGE to form the heteropolymer known to bind α-DG ligands.</p><p><italic>2) The authors also rename</italic> B3GNT1 <italic>as</italic> B4GAT1<italic>. However, they do not show that it lacks the beta-3-GlcNAc-transferase (i-antigen forming) activity originally assigned to it. While it</italic> can <italic>be argued that the latter activity was also assigned based on indirect evidence, the authors did not adequately address the potential for beta-3-GlcNAc-transferase activity because they only tested Gal(beta)-MU as an acceptor, which is not a substrate, and did not test the preferred Gal(beta)1-4GlcNAc-. If it is not too difficult, they should try to clarify this directly. The authors should explain clearly at the beginning that their data will show that the gene called</italic> B3GNT1 <italic>in the literature, in fact encodes a glucuronisyltransferase and not an N-acetylglucosaminyltransferase as previously thought</italic>.</p><p>We appreciate the reviewers concerns regarding B4GAT1 having beta 1,3 GlcNAc -T enzyme activity as originally described by Sasaki et al. (1997). Indeed the tested Gal-b-MU is not the preferred acceptor substrate for the reported iGnT enzyme activity. To further validate our statement that B4GAT1 has no GlcNAc-transferase activity we synthesized and purified the hypothesized preferred substrate Gal-b1,4-GlcNAc-MU in vitro, confirmed it by NMR and tested it with B4GAT1dTM as acceptor for GlcNAc transferase activity. Using the same iGnT acceptor and enzyme conditions as described in Sasaki et al. (1997) we were not able to detect any enzyme product, further confirming that B4GAT1 does not have GlcNAc transferase activity and that the enzyme originally described as iGnT / B3GNT1 had been falsely assigned.</p><p>We hope that our additional experiments included in the manuscript as Figure 15 will satisfy the reviewers and grant the renaming of the enzyme previously known as iGnT/B3GNT1 to B4GAT1.</p><p>For further clarification we also added this sentence to the Introduction: “We present experimental evidence that this enzyme B4GAT1, which was previously described in the literature as B3GNT1 (<xref ref-type="bibr" rid="bib48">Sasaki et al., 1997</xref>) in fact encodes for a β1,4 glucuronyltransferase and not a β1,3 N-acetylglucosaminyltransferase as previously thought.”</p><p><italic>3) While many experiments add up to strong support for the authors' conclusions re</italic> B4GAT1<italic>, their interpretation of the data in</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1</italic>B</xref> <italic>may or may not be the case. The data in</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1</italic>B</xref> <italic>show that LARGE cannot override a defect in FKRP, FKTN,</italic> B4GAT1 <italic>or TMEM5. However, the latter does not mean that all these activities act prior to LARGE in cells. The evidence that</italic> B4GAT1 <italic>acts prior to LARGE is strong and described in the manuscript. It is also clear as shown later that LARGE acting in the absence of these activities</italic> in vitro <italic>can generate laminin binding glycans. However, one or more of the remaining three activities could potentially act after LARGE and be required to modify LARGE-generated glycans such that they become capable of binding to laminin or a different binding partner when LARGE is expressed at endogenous levels</italic> in vivo<italic>. Determining whether these activities act prior to or post LARGE is beyond the scope of this manuscript. However, the authors should qualify their conclusions with respect to</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref> <italic>and the model in Figure 18</italic>.</p><p>The reviewers bring up an interesting hypothesis that FKRP, FKTN, B4GAT1 and/or TMEM5 possibly do not act prior to LARGE glycan synthesis but rather modify the LARGE glycan itself. Patient studies have shown that all loss-of-function mutations in FKRP, FKTN, B4GAT1 and TMEM5 result in loss of aDG functional glycosylation and ligand binding. Furthermore it was demonstrated by Goddeeris et al., 2013 that the GlcA-Xyl heteropolymer synthesized by LARGE is sufficient for ligand binding. If any of the proteins in question would be involved in the post LARGE modification, mutations should not result in loss of ligand binding as the basic ligand binding LARGE glycan scaffold would still be preserved and present. Based on our current knowledge of the aDG functional glycosylation we feel that an involvement of FKRP, FKTN, B4GAT1 and/or TMEM5 in post LARGE glycan modification can be considered highly unlikely. We included a new <xref ref-type="fig" rid="fig1">Figure 1</xref> and added the following statement to the figure legend: “The putative glycosyltransferases B4GAT1 (B3GNT1), FKTN, FKRP and TMEM5 are proposed to act prior to LARGE, which adds a [GlcA-Xyl] heteropolymer that is responsible for ligand binding. However based on current knowledge it cannot be completely ruled out that they are involved in the modification of the LARGE glycan repeat itself to modulate ligand binding.”</p></body></sub-article></article> |