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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Glycosylation Mutants of Cultured Mammalian Cells - Essentials of Glycobiology - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="Essentials of Glycobiology [Internet]. 3rd edition">
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<meta name="citation_title" content="Glycosylation Mutants of Cultured Mammalian Cells">
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<meta name="citation_publisher" content="Cold Spring Harbor Laboratory Press">
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<meta name="citation_date" content="2017">
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<meta name="citation_author" content="Jeffrey D. Esko">
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<meta name="citation_author" content="Pamela Stanley">
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<meta name="citation_pmid" content="28876848">
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<meta name="citation_doi" content="10.1101/glycobiology.3e.049">
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<meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK453088/">
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<meta name="DC.Title" content="Glycosylation Mutants of Cultured Mammalian Cells">
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<meta name="DC.Contributor" content="Jeffrey D. Esko">
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<meta name="DC.Contributor" content="Pamela Stanley">
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<meta name="description" content="Rapid progress in understanding glycosylation pathways of eukaryotes came with the application of genetic strategies to isolate mutants of mammalian cells and yeast with defects in glycan synthesis. This chapter reviews methods used to isolate mammalian cell glycosylation mutants and the diversity of mutants that may be obtained from selections and screens. The applications of glycosylation mutants to address functional roles of glycans and in glycosylation engineering are discussed briefly. Many of the cell lines described in this chapter are available through the American Type Culture Collection.">
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<meta name="og:description" content="Rapid progress in understanding glycosylation pathways of eukaryotes came with the application of genetic strategies to isolate mutants of mammalian cells and yeast with defects in glycan synthesis. This chapter reviews methods used to isolate mammalian cell glycosylation mutants and the diversity of mutants that may be obtained from selections and screens. The applications of glycosylation mutants to address functional roles of glycans and in glycosylation engineering are discussed briefly. Many of the cell lines described in this chapter are available through the American Type Culture Collection.">
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match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><p class="vip-notice"><strong><a href="/books/n/glyco4/?report=reader">A new version of this title is available</a></strong></p><p class="vip-notice"><strong><a href="/books/NBK579983/?report=reader">See the updated version of this chapter</a></strong></p><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK453088_"><span class="label">Chapter 49</span><span class="title" itemprop="name">Glycosylation Mutants of Cultured Mammalian Cells</span></h1><p class="contribs">Esko JD, Stanley P.</p><p class="fm-aai"><a href="#_NBK453088_pubdet_">Publication Details</a></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="_abs_rndgid_" itemprop="description"><p>Rapid progress in understanding glycosylation pathways of eukaryotes came with the application of genetic strategies to isolate mutants of mammalian cells and yeast with defects in glycan synthesis. This chapter reviews methods used to isolate mammalian cell glycosylation mutants and the diversity of mutants that may be obtained from selections and screens. The applications of glycosylation mutants to address functional roles of glycans and in glycosylation engineering are discussed briefly. Many of the cell lines described in this chapter are available through the American Type Culture Collection.</p></div><div id="Ch49_s1"><h2 id="_Ch49_s1_">HISTORY</h2><p>The success of bacterial and yeast genetics in isolating mutants and using them to define biochemical pathways led in the late 1960s to the development of somatic cell genetics using mammalian cells. Chinese hamster ovary (CHO) cells were selected by two independent groups for initial experiments to isolate stable mutants. Somatic cell genetic strategies were applied early to glycobiology, yielding numerous mutants in glycoprotein biosynthesis and later in proteoglycan, glycosylphosphatidylinositol (GPI) anchor, and glycolipid biosynthesis. The ability to isolate glycosylation mutants in mammalian cells made it possible to unravel pathways of glycan synthesis and degradation, and to identify, isolate, and map structural and regulatory genes. CHO cells thus became a focus for experiments to decipher glycosylation pathways, and importantly, provided mutant host cells for the production of viruses and glycoproteins with modified glycans. This proved to be extremely beneficial to the biotechnology industry because most recombinant therapeutics are glycoproteins. CHO cells and CHO glycosylation mutants are now the workhorse of the biotechnology industry. They are particularly useful because they produce only minor, if any, quantities of nonhuman glycans or glycan modifications that give rise to undesirable antibodies. Conserved glycosylation pathways in yeast were delineated by similar approaches (<a href="/books/n/glyco3/ch23/?report=reader">Chapter 23</a>).</p><p>Mutants in any cell type often accumulate the precursor immediately upstream of the block in a pathway and thereby reveal the structure of their substrate(s). Sequencing of mutant alleles reveals specific mutations that may give rise to a glycosylation phenotype. In most cases, mutations are loss-of-function and they reduce or abrogate the activity of an enzyme in a pathway; but there are also gain-of-function mutations that activate a silent glycosylation gene, elevate the expression of an existing activity, or inactivate a negative regulatory factor (<a class="figpopup" href="/books/NBK453088/figure/ch49.f1/?report=objectonly" target="object" rid-figpopup="figch49f1" rid-ob="figobch49f1">Figure 49.1</a>). In nearly all cases, glycosylation mutations lead to the presence of altered glycans on cell-surface glycoconjugates and changes in cell properties that link glycan structure to function. Although gene editing techniques using CRISPR/Cas9 or transcription activator–like effectors (TALENs) are now the method of choice for introducing a mutation that weakens or ablates a glycosylation gene (<a href="/books/n/glyco3/ch27/?report=reader">Chapters 27</a> and <a href="/books/n/glyco3/ch56/?report=reader">56</a>), such approaches do not allow for the serendipitous findings that often emerge from genetic screens. For example, a screen of HAP1 (haploid) human cells mutagenized by retroviral gene trap, led to the identification of multiple, previously unknown glycosylation genes required for the synthesis of the glycan ligand on α-dystroglycan that binds laminin (<a class="figpopup" href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" rid-figpopup="figch49f2" rid-ob="figobch49f2">Figure 49.2</a>).</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figch49f1" co-legend-rid="figlgndch49f1"><a href="/books/NBK453088/figure/ch49.f1/?report=objectonly" target="object" title="FIGURE 49.1." class="img_link icnblk_img figpopup" rid-figpopup="figch49f1" rid-ob="figobch49f1"><img class="small-thumb" src="/books/NBK453088/bin/ch49f01.gif" src-large="/books/NBK453088/bin/ch49f01.jpg" alt="FIGURE 49.1.. Alteration of cell-surface glycans by recessive and dominant glycosylation mutations." /></a><div class="icnblk_cntnt" id="figlgndch49f1"><h4 id="ch49.f1"><a href="/books/NBK453088/figure/ch49.f1/?report=objectonly" target="object" rid-ob="figobch49f1">FIGURE 49.1.</a></h4><p class="float-caption no_bottom_margin">Alteration of cell-surface glycans by recessive and dominant glycosylation mutations. </p></div></div><div class="iconblock whole_rhythm clearfix ten_col fig" id="figch49f2" co-legend-rid="figlgndch49f2"><a href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" title="FIGURE 49.2." class="img_link icnblk_img figpopup" rid-figpopup="figch49f2" rid-ob="figobch49f2"><img class="small-thumb" src="/books/NBK453088/bin/ch49f02.gif" src-large="/books/NBK453088/bin/ch49f02.jpg" alt="FIGURE 49.2.. Selections for glycosylation mutants." /></a><div class="icnblk_cntnt" id="figlgndch49f2"><h4 id="ch49.f2"><a href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" rid-ob="figobch49f2">FIGURE 49.2.</a></h4><p class="float-caption no_bottom_margin">Selections for glycosylation mutants. Cytotoxic lectins or agents that bind to specific sugar residues select for resistant cells (<i>left</i>). Screen for mutants using replica plating. Colonies on plastic are transferred to discs and screened for defects in <a href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" rid-ob="figobch49f2">(more...)</a></p></div></div></div><div id="Ch49_s2"><h2 id="_Ch49_s2_">ISOLATION OF GLYCOSYLATION MUTANTS</h2><p>Cells in culture mutate at a low rate (<10<sup>−6</sup> mutations per locus per generation). In CHO cells, many loci are functionally haploid (single copy), and in HAP1 human cells, essentially all loci are haploid, which means that a single hit may generate a recessive mutant. However, typical mammalian cells are diploid and immortalized cells are often hyperploid, so the frequency of finding recessive mutants is low. To greatly increase the probability of finding desirable mutants, mutations may be induced by treating cells with chemical (e.g., alkylating agents), physical (e.g., ionizing radiation), or biological (e.g., a virus) mutagens. Nevertheless, selection or enrichment is usually needed to find rare recessive or dominant mutants bearing a desired glycosylation phenotype (<a class="figpopup" href="/books/NBK453088/figure/ch49.f1/?report=objectonly" target="object" rid-figpopup="figch49f1" rid-ob="figobch49f1">Figure 49.1</a>). For example, direct selection for resistance to cytotoxic plant lectins (<a href="/books/n/glyco3/ch31/?report=reader">Chapters 31</a> and <a href="/books/n/glyco3/ch32/?report=reader">32</a>) that bind to cell-surface glycans gives a range of glycosylation mutants. Importantly, many mutants resistant to one or more lectins because of the loss of specific sugars, become supersensitive to a different group of lectins that recognize sugar residues exposed by the mutation (<a class="figpopup" href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" rid-figpopup="figch49f2" rid-ob="figobch49f2">Figure 49.2</a>). The latter may be used to select for revertants in the original mutant population. Nontoxic lectins are also useful for enriching lectin-binding mutants (e.g., by flow cytometry). Mutations that affect all stages of glycosylation reactions, including the generation and transport of nucleotide sugars, have been identified using lectins as selective agents.</p><p>In principle, any glycan-binding protein (GBP) or agent that recognizes cell-surface glycans or a glycoprotein can be used to isolate mutants with a glycosylation defect (<a class="figpopup" href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" rid-figpopup="figch49f2" rid-ob="figobch49f2">Figure 49.2</a>). Conjugation of a GBP or protein domain to a toxin that cannot enter the cell independently, but can kill the cell following entry, can be used to select mutants when cytotoxic lectins are not available. For example, basic fibroblast growth factor (FGF-2)–saporin complexes have been used for the selection of mutants deficient in heparan sulfate (HS). Lectins, antibodies, or ligands that are fluorescently tagged may be used to enrich for mutants that are either deficient in binding or have acquired a novel binding ability because of altered glycosylation or reduced expression of an antigen at the cell surface. Panning or immunodepletion are related techniques. For example, coating a plate with FGF-2 allows selection of mutant cells that fail to produce HS proteoglycans, and consequently fail to adhere to a FGF-2-coated plate. HAP1 cells that fail to glycosylate α-dystroglycan fail to bind a specific monoclonal antibody and may be enriched by immunodepletion (<a class="figpopup" href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" rid-figpopup="figch49f2" rid-ob="figobch49f2">Figure 49.2</a>). Radiation suicide is another direct selection method for obtaining glycosylation mutants. Incubation of cells with a radioactive sugar, sulfate, or other precursor of high radiospecific activity leads to labeled glycoproteins, glycolipids, or proteoglycans. After prolonged storage of the cells, radiation damage will kill wild-type cells, whereas mutants with reduced incorporation of the label survive. Animal cells can also be replica-plated, much like microbial colonies, using porous cloth made of polyester or nylon as the replica (<a class="figpopup" href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" rid-figpopup="figch49f2" rid-ob="figobch49f2">Figure 49.2</a>). Colonies of cells on the disc can be used to identify mutants with reduced incorporation of radioactive precursors, or to identify mutants that fail to bind to a lectin, an antibody, or a growth factor. An adaptation of this technique allows detection of mutants affecting a specific enzyme by direct assay for activity in colony lysates generated on a disc. Although this technique has great specificity, its limited capacity makes detection of rare mutants difficult, and mutagenesis before screening is usually a requirement.</p><p>Regardless of the technique used to isolate mutants, the resulting strains must be cloned and carefully characterized for stability and the biochemical and molecular basis of mutation. Additional genetic analyses include somatic cell hybridization for dominance/recessive testing and assigning mutants to different genetic complementation groups. Biochemical analysis involves the characterization of glycan structures produced by mutant cells (<a href="/books/n/glyco3/ch50/?report=reader">Chapter 50</a>), the quantitation and analysis of intermediates, and assays for activities thought to be missing or acquired based on the properties of the mutant. Identifying the molecular basis of mutation requires isolation of a complementing cDNA that reverts the mutant phenotype and determining whether the mutation arose from defective transcription, translation, or stability of the gene product, or from a missense or nonsense mutation in the coding region of the gene. Targeted gene mutation (<a href="/books/n/glyco3/ch56/?report=reader">Chapter 56</a>) can also be used to validate a phenotype after a gene has been identified in a selected mutant.</p></div><div id="Ch49_s3"><h2 id="_Ch49_s3_">CELL LINES FROM MICE OR HUMANS WITH A GLYCOSYLATION MUTATION</h2><p>Transgenic mice that overexpress a glycosylation gene, or mutant mice that lack a glycosylation activity because of targeted gene inactivation (<a href="/books/n/glyco3/ch56/?report=reader">Chapter 56</a>), are a source of mutant cells that may be used for glycobiology research. Fibroblasts or lymphoblasts can be obtained readily from humans with a disorder of glycosylation. Cells may be grown as primary cultures or immortalized by viral transformation. By crossing mutant mice with the Immortomouse, which carries a temperature-sensitive SV40 T antigen in every cell, immortalized mutant cell lines can be derived from essentially any cell type. For mutations that cause embryos to die during gestation, mutant embryonic stem (ES) cells can be derived from blastocysts, provided the mutation does not result in cell-autonomous lethality. The resulting mutant ES cell lines can be used to investigate functions for specific glycans during differentiation in embryoid cell culture, or in vivo in mouse chimeras. A chimera is obtained by injecting wild-type or mutant ES cells into the inner cell mass of a mouse blastocyst. If the ES cells survive, the resulting mouse is a mosaic of cells derived from the ES cells and cells derived from the blastocyst. Mutant ES cells may not contribute equally well to all tissues. For example, ES cells lacking MGAT1 are unable to make complex or hybrid N-glycans (<a href="/books/n/glyco3/ch9/?report=reader">Chapter 9</a>), but they differentiate normally into many cell types in cultured embryoid bodies. However, following introduction into blastocysts, ES cells lacking MGAT1 do not contribute to the organized layer of bronchial epithelium in chimeric embryos.</p><p>Immortalized fibroblasts from patients with defects in glycosylation can be used to study the underlying defect (<a href="/books/n/glyco3/ch45/?report=reader">Chapter 45</a>). Immortalized human cell lines bearing defects in lysosomal degradation are also available. Induced pluripotent stem cells derived from fibroblasts from patients with glycosylation disorders provide another approach for obtaining various differentiated cell lines for further study.</p></div><div id="Ch49_s4"><h2 id="_Ch49_s4_">RECESSIVE GLYCOSYLATION MUTANTS</h2><p>Selection schemes based on isolating rare mutants resistant to cytotoxic plant lectins have yielded a large number of glycosylation mutants affected in diverse aspects of glycan synthesis (<a class="figpopup" href="/books/NBK453088/table/CH49TB1/?report=objectonly" target="object" rid-figpopup="figCH49TB1" rid-ob="figobCH49TB1">Table 49.1</a>). Some mutations affect several types of glycans, such as mutants with reduced nucleotide sugar formation or transport into the Golgi. For example, the UDP-Gal transporter defect in Lec8 mutant cells affects transfer of galactose to O- and N-glycans on glycoproteins, as well as to glycosaminoglycans (GAGs) and glycolipids. The ldlD mutant is particularly interesting in this regard, because it lacks the epimerase responsible for converting UDP-Glc to UDP-Gal and UDP-GlcNAc to UDP-GalNAc (<a class="figpopup" href="/books/NBK453088/figure/ch49.f3/?report=objectonly" target="object" rid-figpopup="figch49f3" rid-ob="figobch49f3">Figure 49.3</a>). Because there are salvage pathways for importing Gal and GalNAc into cells (<a href="/books/n/glyco3/ch5/?report=reader">Chapter 5</a>), the composition of different classes of glycans can be controlled in ldlD cells by nutritional supplementation with either of these two sugars. Mutations in glycosyltransferase genes may ablate activity or affect the kinetic properties of an enzyme (e.g., Lec1A, <a class="figpopup" href="/books/NBK453088/table/CH49TB1/?report=objectonly" target="object" rid-figpopup="figCH49TB1" rid-ob="figobCH49TB1">Table 49.1</a>), or its subcellular localization (e.g., Lec4A, <a class="figpopup" href="/books/NBK453088/table/CH49TB1/?report=objectonly" target="object" rid-figpopup="figCH49TB1" rid-ob="figobCH49TB1">Table 49.1</a>). Sequencing mutant alleles provides leads for further site-directed mutagenesis of the gene in order to define important functional domains of the protein required for catalysis or compartmentalization.</p><div class="iconblock whole_rhythm clearfix ten_col fig" id="figch49f3" co-legend-rid="figlgndch49f3"><a href="/books/NBK453088/figure/ch49.f3/?report=objectonly" target="object" title="FIGURE 49.3." class="img_link icnblk_img figpopup" rid-figpopup="figch49f3" rid-ob="figobch49f3"><img class="small-thumb" src="/books/NBK453088/bin/ch49f03.gif" src-large="/books/NBK453088/bin/ch49f03.jpg" alt="FIGURE 49.3.. Mutation of UDP-Gal-4-epimerase in ldlD mutant Chinese hamster ovary (CHO) cells prevents the generation of UDP-Gal and UDP-GalNAc preventing addition of Gal and GalNAc to all glycans." /></a><div class="icnblk_cntnt" id="figlgndch49f3"><h4 id="ch49.f3"><a href="/books/NBK453088/figure/ch49.f3/?report=objectonly" target="object" rid-ob="figobch49f3">FIGURE 49.3.</a></h4><p class="float-caption no_bottom_margin">Mutation of UDP-Gal-4-epimerase in ldlD mutant Chinese hamster ovary (CHO) cells prevents the generation of UDP-Gal and UDP-GalNAc preventing addition of Gal and GalNAc to all glycans. Salvage reactions that generate UDP-Gal or UDP-GalNAc by an alternate <a href="/books/NBK453088/figure/ch49.f3/?report=objectonly" target="object" rid-ob="figobch49f3">(more...)</a></p></div></div><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figCH49TB1"><a href="/books/NBK453088/table/CH49TB1/?report=objectonly" target="object" title="Table 49.1." class="img_link icnblk_img figpopup" rid-figpopup="figCH49TB1" rid-ob="figobCH49TB1"><img class="small-thumb" src="/books/NBK453088/table/CH49TB1/?report=thumb" src-large="/books/NBK453088/table/CH49TB1/?report=previmg" alt="Table 49.1." /></a><div class="icnblk_cntnt"><h4 id="CH49TB1"><a href="/books/NBK453088/table/CH49TB1/?report=objectonly" target="object" rid-ob="figobCH49TB1">Table 49.1.</a></h4><p class="float-caption no_bottom_margin">Examples of recessive glycosylation mutants </p></div></div><p>Some lectin-resistant mutants are defective in the formation of dolichol-P-oligosaccharides or in the processing reactions that remove Glc or Man after transfer of the glycan chain to a glycoprotein (<a href="/books/n/glyco3/ch9/?report=reader">Chapter 9</a>). The latter mutants revealed the identity and importance of α-mannosidases in the formation of N-glycans. However, when the α-mannosidase II gene <i>Man2a1</i> was ablated in mice, no effect was seen in certain tissues because another previously unknown α-mannosidase gene (<i>Man2a2</i>) allowed N-glycans to be synthesized. This finding emphasizes a limitation of somatic cells in that they may not express glycosylation genes that are developmentally regulated in a tissue-specific manner, thereby precluding the isolation of mutants affected in those genes from that cell line.</p></div><div id="Ch49_s5"><h2 id="_Ch49_s5_">DOMINANT GLYCOSYLATION MUTANTS</h2><p>The recessive mutants in <a class="figpopup" href="/books/NBK453088/table/CH49TB1/?report=objectonly" target="object" rid-figpopup="figCH49TB1" rid-ob="figobCH49TB1">Table 49.1</a> lack a glycosylation activity or fail to make a precursor. Dominant mutations that activate a silent gene reveal activities that may normally be expressed only in a few, very specialized cells in the body. Therefore, dominant mutants are important in glycosylation gene discovery, for identifying mechanisms of glycosylation gene regulation, and for defining pathways of glycan biosynthesis. The mutants in <a class="figpopup" href="/books/NBK453088/table/CH49TB2/?report=objectonly" target="object" rid-figpopup="figCH49TB2" rid-ob="figobCH49TB2">Table 49.2</a> show a gain-of-function, dominant, lectin-resistant phenotype caused by the increased expression of a glycosyltransferase that is normally silent or expressed at very low levels. The activation of a glycosyltransferase gene may reflect a mutation in a regulatory region of the gene or in a <i>trans</i>-acting factor. The genetic bases of the mutants in <a class="figpopup" href="/books/NBK453088/table/CH49TB2/?report=objectonly" target="object" rid-figpopup="figCH49TB2" rid-ob="figobCH49TB2">Table 49.2</a> are not known, but their characterization may reveal novel genes or regulatory factors that may not have been previously known to exist.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figCH49TB2"><a href="/books/NBK453088/table/CH49TB2/?report=objectonly" target="object" title="Table 49.2." class="img_link icnblk_img figpopup" rid-figpopup="figCH49TB2" rid-ob="figobCH49TB2"><img class="small-thumb" src="/books/NBK453088/table/CH49TB2/?report=thumb" src-large="/books/NBK453088/table/CH49TB2/?report=previmg" alt="Table 49.2." /></a><div class="icnblk_cntnt"><h4 id="CH49TB2"><a href="/books/NBK453088/table/CH49TB2/?report=objectonly" target="object" rid-ob="figobCH49TB2">Table 49.2.</a></h4><p class="float-caption no_bottom_margin">Examples of dominant mutants expressing a new activity </p></div></div></div><div id="Ch49_s6"><h2 id="_Ch49_s6_">MUTANTS IN GPI-ANCHOR BIOSYNTHESIS</h2><p>Glycosylation defects in GPI-anchor biosynthesis reduce expression of GPI-anchored proteins at the cell surface (<a href="/books/n/glyco3/ch12/?report=reader">Chapter 12</a>). Originally, many GPI-anchor mutants were isolated by strategies that took advantage of antibodies to a GPI-anchored glycoprotein. For example, lymphoma cells expressing Thy-1 on their surface were incubated with an antibody to Thy-1 and serum-containing complement, which lysed cells expressing the Thy-1 antigen. Loss of GPI-anchor biosynthesis reduced the expression of Thy-1 on the surface and conferred resistance to the cytolytic effect. Other mutants have been obtained by sorting cells that do not bind to a fluorescent antibody or with bacterial toxins that bind GPI glycans. The GPI-anchor mutants obtained to date fall into many genetic complementation groups, each having a different lesion in GPI-anchor biosynthesis (<a href="/books/n/glyco3/ch12/?report=reader">Chapter 12</a>). These mutants reveal the complexity of GPI-anchor biosynthesis: multiple gene products are involved in forming the <i>N</i>-acetylglucosamine linkage to phosphatidylinositol, the first committed intermediate in the pathway; dolichol-P-Man is used as the donor of Man; at least three enzymes are involved in the attachment of ethanolamine phosphate residues; and five genes are required for the transfer of the GPI anchor to protein. The available strains show the importance of genetic approaches for identifying genes that might not be obvious from measuring biosynthetic reactions in vitro.</p></div><div id="Ch49_s7"><h2 id="_Ch49_s7_">MUTANTS IN PROTEOGLYCAN ASSEMBLY</h2><p>A large collection of mutants defective in GAG/proteoglycan biosynthesis has been isolated (<a class="figpopup" href="/books/NBK453088/table/CH49TB3/?report=objectonly" target="object" rid-figpopup="figCH49TB3" rid-ob="figobCH49TB3">Table 49.3</a>). Many of these mutants were obtained by replica plating methods using sulfate incorporation to monitor GAG production in colonies (<a class="figpopup" href="/books/NBK453088/figure/ch49.f2/?report=objectonly" target="object" rid-figpopup="figch49f2" rid-ob="figobch49f2">Figure 49.2</a>). Mutants in the early steps of GAG biosynthesis (complementation groups A, B, and G) lack both CS and HS chains, and enzymatic assays showed that they lack enzymes responsible for the assembly of the core protein linkage tetrasaccharide shared by both these types of GAGs (<a href="/books/n/glyco3/ch17/?report=reader">Chapter 17</a>). Another class of mutants (group D) is defective only in HS biosynthesis. This mutation defines a bifunctional enzyme (EXT1) that catalyzes the alternating addition of GlcNAc and glucuronic acid (GlcA) residues to growing HS chains. Some of the mutant alleles depress both enzyme activities, whereas others only affect the GlcA transfer activity. Thus, the mutants define different functional domains of the protein, which have been mapped by sequencing various mutant alleles. Mutants in another bifunctional enzyme, <i>N</i>-acetylglucosamine N-deacetylase/N-sulfotransferase (NDST1), have only a partial deficiency in N-sulfation of HS chains. Further analysis of the mutant showed that more than one isozyme is present in CHO cells and that the defect affects only one locus. Thus, the mutants revealed early on that the assembly of HS is much more complex than had been appreciated on the basis of known structures, enzymatic reactions measured in cell extracts, or intermediates observed in pulse-labeling experiments.</p><div class="iconblock whole_rhythm clearfix ten_col table-wrap" id="figCH49TB3"><a href="/books/NBK453088/table/CH49TB3/?report=objectonly" target="object" title="Table 49.3." class="img_link icnblk_img figpopup" rid-figpopup="figCH49TB3" rid-ob="figobCH49TB3"><img class="small-thumb" src="/books/NBK453088/table/CH49TB3/?report=thumb" src-large="/books/NBK453088/table/CH49TB3/?report=previmg" alt="Table 49.3." /></a><div class="icnblk_cntnt"><h4 id="CH49TB3"><a href="/books/NBK453088/table/CH49TB3/?report=objectonly" target="object" rid-ob="figobCH49TB3">Table 49.3.</a></h4><p class="float-caption no_bottom_margin">Examples of mutants defective in proteoglycan assembly </p></div></div></div><div id="Ch49_s8"><h2 id="_Ch49_s8_">MUTANTS DEFECTIVE IN GLYCOLIPID OR O-GLYCAN SYNTHESIS</h2><p>Glycolipids and glycans linked by O-GalNAc are often relatively simple in cultured cells. For example, CHO cells synthesize mainly gangliosides GM3 and lactosylceramide with a small amount of glucosylceramide. O-GalNAc glycans contain up to only four sugars in glycoproteins from CHO cells. O-Fuc, O-Glc, and O-Man glycans are expressed on only a small subset of glycoproteins and are generally not detected by glycomic profiling methods (<a href="/books/n/glyco3/ch50/?report=reader">Chapter 50</a>). All of these glycans are affected in the mutants described in <a class="figpopup" href="/books/NBK453088/table/CH49TB1/?report=objectonly" target="object" rid-figpopup="figCH49TB1" rid-ob="figobCH49TB1">Table 49.1</a> in which CMP-Neu5Ac, UDP-Gal, UDP-GalNAc, or GDP-Fuc are reduced in the Golgi. Similarly, a defective sialyltransferase or galactosyltransferase may cause these glycans to be truncated. A mutant of B16 melanoma cells that is defective in ceramide glucosyltransferase (glucosylceramide synthase) lacks all glycolipids because this enzyme catalyzes the first step in the synthetic pathway (<a href="/books/n/glyco3/ch11/?report=reader">Chapter 11</a>). However, cultured cell mutants defective in polypeptide O-GalNAc transferases (GALNTs) or protein O-fucosyltransferase (POFUT1) have not been isolated. This may reflect the paucity of cytotoxic lectins or toxins that bind to O-glycans and glycolipids or, in some cases, because of redundancy of enzymes (<a href="/books/n/glyco3/ch10/?report=reader">Chapters 10</a> and <a href="/books/n/glyco3/ch11/?report=reader">11</a>). Mice lacking specific glycolipid biosynthetic enzymes and glycosyltransferases that transfer GlcNAc or Fuc to protein have been generated and provide a source of mutant cells that may be studied in culture. Interestingly, cells lacking the O-GlcNAc transferase (OGT) that acts in the cytoplasm to transfer GlcNAc to protein have not been obtained, and mouse mutants defective in this transferase become arrested in development at the two-cell-stage embryo, showing that this O-GlcNAc addition is essential for cell viability.</p></div><div id="Ch49_s9"><h2 id="_Ch49_s9_">USES OF MAMMALIAN GLYCOSYLATION MUTANTS</h2><p>Fortunately for glycobiologists, the vast majority of glycosylation mutations still allow single cell viability in vitro under ideal culture conditions. Glycosylation mutants of mammalian cells have thus been used to address many questions in glycobiology and for glycosylation engineering of recombinant glycoproteins (<a href="/books/n/glyco3/ch56/?report=reader">Chapter 56</a>). Because mutant selections are broad and often not intentionally biased, they generate mutants defective in both known and novel reactions. Thus, glycosylation mutants play an important role in research to define the pathways and regulation of glycosylation in mammals. In this regard, they are more useful tools than mutant mice because cells in culture are viable in the absence of glycolipids, GPI anchors, proteoglycans, O-GalNAc, O-Fuc, O-Glc, O-Man glycans, and complex or hybrid N-glycans. Glycosylation mutants make truncated or altered glycans and thus provide an opportunity to study functional roles for cell-surface glycans in the context of a living cell. Important insights have been gained into specific sugars required for viral, bacterial, or parasite adhesion and infection, and for leukocyte cell adhesion and motility. In addition, functional roles for glycans in the intracellular sorting and secretion of glycoproteins, in growth factor binding and activation, and in receptor functions have been identified using glycosylation mutants. For example, a panel of CHO glycosylation mutants was used in a coculture assay to show that ligand-induced Notch signaling is reduced when GDP-Fuc levels are low, but is unaffected by reductions in Sia. Similarly, one of the first demonstrations for coreceptor functions for HS used mutant CHO cells defective in HS synthesis and engineered to express the FGF receptor.</p><p>Although glycosylation is in many cases dispensable for survival of isolated cells in a culture dish, it is often crucial in vivo. Gene ablation studies in mice have identified several instances in which an intact glycosylation pathway is essential for embryogenesis. Examples include mutants that lack complex and hybrid N-glycans and proteoglycan mutants defective in HS, whereas the corresponding mutants in CHO cells do not cause an obvious growth phenotype. Thus, one theme that emerges from the study of mutants is that glycosylation is critical in the context of a multicellular organism but dispensable in isolated cells. This conclusion has been driven home in recent years by the discovery of human genetic diseases, which arise from mutations in genes involved in glycosylation (<a href="/books/n/glyco3/ch45/?report=reader">Chapter 45</a>).</p><p>CHO cells have become the cells of choice for the biotechnology industry in the production of recombinant therapeutic glycoproteins and in glycosylation engineering (see <a href="/books/n/glyco3/ch56/?report=reader">Chapter 56</a>). For example, CHO cells lacking FUT8, which adds fucose to the core GlcNAc of complex N-glycans are used to produce cytotoxic therapeutic antibodies that have a greatly enhanced ability to kill their target cells. In another example, CHO cells with multiple mutations that simplify N- and O-glycans are being used by X-ray crystallographers to produce homogeneous preparations of membrane glycoproteins with highly truncated N- and O-glycans, greatly facilitating their crystallization.</p><p>Somatic cell genetics arose from the desire to manipulate the genome of cultured cells in vitro. Today, the availability of genomic sequences from multiple organisms has shifted the emphasis in genetics toward the generation of mutant organisms using the techniques of transgenesis, homologous recombination for gene replacement, conditional gene inactivation and gene editing. However, the study of somatic cell mutants still plays an important role in glycobiology research because it provides a less-expensive and faster method for studying the effects of deleting or expressing particular glycosylation gene products in a cell. Gain-of-function mutants may of course be generated by transfection of cDNAs encoding glycosylation genes, and reduced expression of any gene can be achieved by the use of RNA interference (RNAi), antisense cDNA strategies, or gene editing. Although extremely valuable, the latter approaches generally target only known genes, whereas cell-based selections or screens make it possible to discover new genes by screening for phenotypic changes directly related to glycosylation changes. Additionally, cells and mutants with well-characterized glycosylation pathways are ideal hosts for investigating the activity encoded by a putative glycosylation gene identified in genome sequence databases. These mutant cells also provide a platform to test the severity of human mutations in a complementation test: the normal human gene rescues defective glycosylation when transfected into the mutant cell, but the same gene with a pathological mutation does not. Thus, somatic cell mutants provide access to novel genes involved in glycosylation, which in turn guide strategies for sophisticated gene-manipulation experiments in animals. By combining the two approaches, the biological function of a particular glycosyltransferase, sugar residue, or lectin can be defined. Coupled with powerful mass spectrometry techniques for determining glycan structures (<a href="/books/n/glyco3/ch50/?report=reader">Chapter 50</a>) from small samples of tissue or cells, glycosylation mutants of cells and animals provide complementary material for structure/function analyses and identifying mechanistic bases of glycan functions in mammals.</p></div><div id="ack46"><h2 id="_ack46_">ACKNOWLEDGMENTS</h2><p>The authors acknowledge contributions to previous versions of this chapter by Carolyn R. Bertozzi and appreciate helpful comments and suggestions from Michelle Dookwah, Chengcheng Huang, and Krithika Vaidyanathan.</p></div><div id="rl49"><h2 id="_rl49_">FURTHER READING</h2><ul class="simple-list"><li class="half_rhythm"><p><div class="bk_ref" id="CH49C1">Stanley P. 1984. Glycosylation mutants of animal cells. Annu Rev Genet
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18:
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525–552. [<a href="https://pubmed.ncbi.nlm.nih.gov/6241454" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 6241454</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C2">Esko JD. 1989. Replica plating of animal cells. Methods Cell Biol
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32:
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387–422. [<a href="https://pubmed.ncbi.nlm.nih.gov/2691858" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 2691858</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C3">Esko JD. 1991. Genetic analysis of proteoglycan structure, function and metabolism. Curr Opin Cell Biol
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3:
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805–816. [<a href="https://pubmed.ncbi.nlm.nih.gov/1931081" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1931081</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C4">Stanley P. 1992. Glycosylation engineering. Glycobiology
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2:
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99–107. [<a href="https://pubmed.ncbi.nlm.nih.gov/1606361" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 1606361</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C5">Stanley P, Raju TS, Bhaumik M. 1996. CHO cells provide access to novel N-glycans and developmentally regulated glycosyltransferases. Glycobiology
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695–699. [<a href="https://pubmed.ncbi.nlm.nih.gov/8953280" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8953280</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C6">Esko JD, Selleck SB. 2002. Order out of chaos: Assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem
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435–471. [<a href="https://pubmed.ncbi.nlm.nih.gov/12045103" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 12045103</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C7">Maeda Y, Ashida H, Kinoshita T. 2006. CHO glycosylation mutants: GPI anchor. Methods Enzymol
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182–205. [<a href="https://pubmed.ncbi.nlm.nih.gov/17113867" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17113867</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C8">Patnaik SK, Stanley P. 2006. Lectin-resistant CHO glycosylation mutants. Methods Enzymol
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159–182. [<a href="https://pubmed.ncbi.nlm.nih.gov/17113866" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17113866</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C9">Zhang L, Lawrence R, Frazier BA, Esko JD. 2006. CHO glycosylation mutants: Proteoglycans. Methods Enzymol
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205–221. [<a href="https://pubmed.ncbi.nlm.nih.gov/17113868" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17113868</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C10">Jae LT, Raaben M, Riemersma M, van Beusekom E, Blomen VA, Velds A, Kerkhoven RM, Carette JE, Topaloglu H, Meinecke P, et al.
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2013. Deciphering the glycosylome of dystroglycanopathies using haploid screens for Lassa virus entry. Science
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479–483. [<a href="/pmc/articles/PMC3919138/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3919138</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/23519211" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 23519211</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C11">Steentoft C, Bennett EP, Schjoldager KT, Vakhrushev SY, Wandall HH, Clausen H. 2014. Precision genome editing: A small revolution for glycobiology. Glycobiology
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663–680. [<a href="https://pubmed.ncbi.nlm.nih.gov/24861053" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24861053</span></a>]</div></p></li><li class="half_rhythm"><p><div class="bk_ref" id="CH49C12">Yang Z, Wang S, Halim A, Schulz MA, Frodin M, Rahman SH, Vester-Christensen MB, Behrens C, Kristensen C, Vakhrushev SY, et al.
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2015. Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat Biotechnol
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842–844. [<a href="https://pubmed.ncbi.nlm.nih.gov/26192319" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 26192319</span></a>]</div></p></li></ul></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK453088_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Jeffrey D. Esko</span> and <span itemprop="author">Pamela Stanley</span>.</p><h3>Publication History</h3><p class="small">Published online: 2017.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright</a> 2015-2017 by The Consortium of Glycobiology Editors, La Jolla, California. All rights reserved.<p class="small">PDF files are not available for download.</p></div></div><h3>Publisher</h3><p><a href="http://www.cshlpress.com/default.tpl?action=full&cart=12210755385880789&--eqskudatarq=666" ref="pagearea=page-banner&targetsite=external&targetcat=link&targettype=publisher">Cold Spring Harbor Laboratory Press</a>, Cold Spring Harbor (NY)</p><h3>NLM Citation</h3><p>Esko JD, Stanley P. Glycosylation Mutants of Cultured Mammalian Cells. 2017. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 3rd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017. Chapter 49.<span class="bk_cite_avail"></span> doi: 10.1101/glycobiology.3e.049</p></div><div class="small-screen-prev"><a href="/books/n/glyco3/ch48/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/glyco3/ch50/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article><article data-type="fig" id="figobch49f1"><div id="ch49.f1" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK453088/bin/ch49f01.jpg" alt="FIGURE 49.1.. Alteration of cell-surface glycans by recessive and dominant glycosylation mutations." /></div><h3><span class="label">FIGURE 49.1.</span></h3><div class="caption"><p>Alteration of cell-surface glycans by recessive and dominant glycosylation mutations.</p></div><p><a href="/books/NBK453088/bin/ch49f01.pptx">Download Teaching Slide</a><span class="small"> (PPTX, 1.8M)</span></p></div></article><article data-type="fig" id="figobch49f2"><div id="ch49.f2" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK453088/bin/ch49f02.jpg" alt="FIGURE 49.2.. Selections for glycosylation mutants." /></div><h3><span class="label">FIGURE 49.2.</span></h3><div class="caption"><p>Selections for glycosylation mutants. Cytotoxic lectins or agents that bind to specific sugar residues select for resistant cells (<i>left</i>). Screen for mutants using replica plating. Colonies on plastic are transferred to discs and screened for defects in incorporation of radioactive precursors, binding to lectins and antibodies, or direct enzymatic assay. Mutants are colonies lacking a strong signal (<i>middle</i>). HAP1 human haploid cells mutagenized by infection with a gene trap retrovirus and selected for resistance to a Lassa pseudovirus (which needs glycosylated α-DG to infect cells) or immunodepleted for glycosylated α-DG (<i>right</i>).</p></div><p><a href="/books/NBK453088/bin/ch49f02.pptx">Download Teaching Slide</a><span class="small"> (PPTX, 2.1M)</span></p></div></article><article data-type="fig" id="figobch49f3"><div id="ch49.f3" class="figure bk_fig"><div class="graphic"><img data-src="/books/NBK453088/bin/ch49f03.jpg" alt="FIGURE 49.3.. Mutation of UDP-Gal-4-epimerase in ldlD mutant Chinese hamster ovary (CHO) cells prevents the generation of UDP-Gal and UDP-GalNAc preventing addition of Gal and GalNAc to all glycans." /></div><h3><span class="label">FIGURE 49.3.</span></h3><div class="caption"><p>Mutation of UDP-Gal-4-epimerase in ldlD mutant Chinese hamster ovary (CHO) cells prevents the generation of UDP-Gal and UDP-GalNAc preventing addition of Gal and GalNAc to all glycans. Salvage reactions that generate UDP-Gal or UDP-GalNAc by an alternate pathway may be used to differentially rescue the generation of UDP-Gal or UDP-GalNAc.</p></div><p><a href="/books/NBK453088/bin/ch49f03.pptx">Download Teaching Slide</a><span class="small"> (PPTX, 1.7M)</span></p></div></article><article data-type="table-wrap" id="figobCH49TB1"><div id="CH49TB1" class="table"><h3><span class="label">Table 49.1.</span></h3><div class="caption"><p>Examples of recessive glycosylation mutants</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK453088/table/CH49TB1/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__CH49TB1_lrgtbl__"><table class="no_bottom_margin"><colgroup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:ali="http://www.niso.org/schemas/ali/1.0/" xmlns:xi="http://www.w3.org/2001/XInclude" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:pmc="http://www.pubmedcentral.gov/pmc" xmlns:xlink="http://www.w3.org/1999/xlink" span="1"><col align="left" span="1" /><col align="left" span="1" /><col align="left" span="1" /><col align="left" span="1" /></colgroup><thead><tr><th id="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Mutant</th><th id="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Biochemical defect</th><th id="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Mutated gene</th><th id="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Glycosylation phenotype</th></tr></thead><tbody><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec32 (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">CMP-NeuAc synthetase</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Cmah</i>
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</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">reduced CMP-Neu5Ac synthesis; glycans lack terminal Sia; terminate in Gal</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec2 (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">CMP-NeuAc transporter</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">Slc35a1</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">reduced CMP-Neu5Ac transport into Golgi; glycans lack terminal Sia; terminate in Gal</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec8 (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">UDP-Gal transporter</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Slc35a2</i>
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</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">reduced UDP-Gal transport into Golgi; N-glycans terminate in GlcNAc; O-glycans terminate in GalNAc</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec13 (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">GDP-Man-4,6-dehydratase</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Gmds</i>
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</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">reduced synthesis GDP-Fuc; glycans lack fucose</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">ldlD (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">UDP-Gal-4-epimerase</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Gale</i>
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</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">reduced UDP-Gal and UDP-GalNAc synthesis; N-glycans lack Gal; O-GalNAc glycans and chondroitin sulfate not synthesized</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec1 (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">GlcNAc-TI</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;"><i>Mgat1</i>-null</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">no complex or hybrid N-glycans; replaced by Man<sub>5</sub>GlcNAc<sub>2</sub>Asn</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec1A (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">GlcNAc-TI</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;"><i>Mgat1</i>-defective</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">kinetic MGAT1 mutant; partial defect in complex and hybrid N-glycan synthesis</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec4A (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">GlcNAc-TV mislocalized</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Mgat5</i>
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</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">complex N-glycans lack the β1-6 GlcNAc branch</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec4 (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">GlcNAc-TV</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Mgat5</i>
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</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">MGAT5 inactivated; defect as above</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec20 (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">β1–4Gal-TI</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>B4galt1</i>
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</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">many glycans low in β1-4 Gal</td></tr><tr><td headers="hd_h_CH49TB1_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">2A10 (CHO)</td><td headers="hd_h_CH49TB1_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">ST8SiaIV</td><td headers="hd_h_CH49TB1_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>St8sia4</i>
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</td><td headers="hd_h_CH49TB1_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">reduced poly-Sia on N-glycans</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_margin">Note on nomenclature: capital first letter and lowercase is used for loss-of-function recessive mutants (e.g., Lec32).</p></div></dd></dl><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_margin">CHO, Chinese hamster ovary.</p></div></dd></dl></dl></div></div></div></article><article data-type="table-wrap" id="figobCH49TB2"><div id="CH49TB2" class="table"><h3><span class="label">Table 49.2.</span></h3><div class="caption"><p>Examples of dominant mutants expressing a new activity</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK453088/table/CH49TB2/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__CH49TB2_lrgtbl__"><table class="no_bottom_margin"><colgroup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:ali="http://www.niso.org/schemas/ali/1.0/" xmlns:xi="http://www.w3.org/2001/XInclude" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:pmc="http://www.pubmedcentral.gov/pmc" xmlns:xlink="http://www.w3.org/1999/xlink" span="1"><col align="left" span="1" /><col align="left" span="1" /><col align="left" span="1" /><col align="left" span="1" /></colgroup><thead><tr><th id="hd_h_CH49TB2_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Mutant</th><th id="hd_h_CH49TB2_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Biochemical change</th><th id="hd_h_CH49TB2_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Affected gene</th><th id="hd_h_CH49TB2_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Glycosylation phenotype</th></tr></thead><tbody><tr><td headers="hd_h_CH49TB2_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">LEC10 (CHO)</td><td headers="hd_h_CH49TB2_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">GlcNAc-TIII-expressed</td><td headers="hd_h_CH49TB2_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Mgat3</i>
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</td><td headers="hd_h_CH49TB2_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">complex N-glycans have the bisecting <i>N</i>-acetylglucosamine residue</td></tr><tr><td headers="hd_h_CH49TB2_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">LEC11 (CHO)</td><td headers="hd_h_CH49TB2_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">α3Fuc-TVI-expressed</td><td headers="hd_h_CH49TB2_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Fut6A, Fut6B</i>
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</td><td headers="hd_h_CH49TB2_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">fucose on poly-<i>N</i>-acetyllactosamine generates Le<sup>X</sup>, SLe<sup>X</sup>, and VIM-2 determinants</td></tr><tr><td headers="hd_h_CH49TB2_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">LEC12 (CHO)</td><td headers="hd_h_CH49TB2_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">α3Fuc-TIX-expressed</td><td headers="hd_h_CH49TB2_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Fut9</i>
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</td><td headers="hd_h_CH49TB2_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">fucose on poly-<i>N</i>-acetyllactosamine generates Le<sup>X</sup> and VIM-2 determinants</td></tr><tr><td headers="hd_h_CH49TB2_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">LEC29 (CHO)</td><td headers="hd_h_CH49TB2_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">α3Fuc-TIX-expressed</td><td headers="hd_h_CH49TB2_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Fut9</i>
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</td><td headers="hd_h_CH49TB2_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">fucose on poly-<i>N</i>-acetyllactosamine generates Le<sup>X</sup> but not VIM-2</td></tr><tr><td headers="hd_h_CH49TB2_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">LEC30 (CHO)</td><td headers="hd_h_CH49TB2_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">α3Fuc-TIX-expressed</td><td headers="hd_h_CH49TB2_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Fut4, Fut9</i>
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</td><td headers="hd_h_CH49TB2_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">fucose on poly-<i>N</i>-acetyllactosamine generates Le<sup>X</sup> and VIM-2 determinants</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_margin">Note on nomenclature: uppercase is used for gain-of-function dominant mutants (e.g., LEC10).</p></div></dd></dl><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_margin">CHO, Chinese hamster ovary.</p></div></dd></dl></dl></div></div></div></article><article data-type="table-wrap" id="figobCH49TB3"><div id="CH49TB3" class="table"><h3><span class="label">Table 49.3.</span></h3><div class="caption"><p>Examples of mutants defective in proteoglycan assembly</p></div><p class="large-table-link" style="display:none"><span class="right"><a href="/books/NBK453088/table/CH49TB3/?report=objectonly" target="object">View in own window</a></span></p><div class="large_tbl" id="__CH49TB3_lrgtbl__"><table class="no_bottom_margin"><colgroup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:ali="http://www.niso.org/schemas/ali/1.0/" xmlns:xi="http://www.w3.org/2001/XInclude" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:pmc="http://www.pubmedcentral.gov/pmc" xmlns:xlink="http://www.w3.org/1999/xlink" span="1"><col align="left" span="1" /><col align="left" span="1" /><col align="left" span="1" /><col align="left" span="1" /></colgroup><thead><tr><th id="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Strain</th><th id="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Biochemical defect</th><th id="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Mutated gene</th><th id="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="text-align:left;vertical-align:top;">Glycosylation phenotype</th></tr></thead><tbody><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">pgsA (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">xylosyltransferase 2</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Xylt2</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">lack of HS and CS</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">pgsI (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">UDP-xylose synthase</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Uxs1</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">lack of HS and CS</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">pgsB (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">galactosyltransferase I</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>B4galt7</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">lack of HS and CS</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">pgsG (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">glucuronyltransferase I</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>B3gat1</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">lack of HS and CS</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">pgsD (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">GlcA and GlcNAc transferase</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Ext1</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">HS deficient and accumulates CS</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">ldlD (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">UDP-Gal/UDP-GalNAc-4-epimerase</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>GalE</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">lack of CS when starved for <i>N</i>-acetyl- galactosamine and fed galactose; lack of all GAG chains when starved for galactose</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Lec8 (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">UDP-Gal transporter</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Slc35a2</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">reduced KS</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">pgsC (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">sulfate transporter</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Slc26a2</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">normal GAG biosynthesis due to salvage of sulfate from oxidation of sulfur-containing amino acids</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">pgsE (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">GlcNAc <i>N</i>-deacetylase/<i>N</i>-sulfotransferase</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Ndst1</i>
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</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">undersulfated HS</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">pgsF (CHO)</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;">HS uronyl 2-O-sulfotransferase</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Hs2st</i>
|
|
</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">defective 2-O-sulfation of uronic acids in HS; defective FGF-2 binding</td></tr><tr><td headers="hd_h_CH49TB3_1_1_1_1" rowspan="1" colspan="1" style="vertical-align:top;">Mouse LTA cells</td><td headers="hd_h_CH49TB3_1_1_1_2" rowspan="1" colspan="1" style="vertical-align:top;"><i>N</i>-sulfoglucosamine 3<i>-</i>O-sulfotransferase</td><td headers="hd_h_CH49TB3_1_1_1_3" rowspan="1" colspan="1" style="vertical-align:top;">
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<i>Hs3st1</i>
|
|
</td><td headers="hd_h_CH49TB3_1_1_1_4" rowspan="1" colspan="1" style="vertical-align:top;">defective 3-O-sulfation of <i>N</i>-sulfoglucosamine units; defective antithrombin binding</td></tr></tbody></table></div><div class="tblwrap-foot"><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_margin">CHO, Chinese hamster ovary; HS, heparan sulfate; CS, chondroitin sulfate; KS, keratan sulfate.</p></div></dd></dl></dl></div></div></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script></div></div>
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