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• W2953347000 abstract "•Human glycosyltransferases (170 GTf genes) organized in glycosylation pathway maps•The human glycome displayed in a natural context on the cell surface•Sustainable cell-based array resource to dissect biological functions of glycans•Microbial adhesins may bind to clustered patches of O-glycans The structural diversity of glycans on cells—the glycome—is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins. The structural diversity of glycans on cells—the glycome—is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins. The great structural diversity and complexity of the glycome of cells pose huge challenges for analytic and functional studies to extract and define specific biological roles and the underlying molecular basis. Glycan arrays have played a pivotal role in surveying and mapping the informational content of complex glycans. Different strategies have been undertaken to immobilize and display libraries of glycans in printed array formats, drawing parallels to DNA arrays (Rillahan and Paulson, 2011Rillahan C.D. Paulson J.C. Glycan microarrays for decoding the glycome.Annu. Rev. Biochem. 2011; 80: 797-823Crossref PubMed Scopus (348) Google Scholar), and approaches to produce comprehensive oligosaccharide libraries vary from chemical and chemoenzymatic synthesis to isolation of natural oligosaccharides and glycoconjugates. Different immobilization strategies have been used, with the most prevalent being coupling to N-hydroxysuccinimide (NHS)-activated slides and the neoglycolipid approach utilizing reductive amination to link released oligosaccharides to an amino-phospholipid (Blixt et al., 2004Blixt O. Head S. Mondala T. Scanlan C. Huflejt M.E. Alvarez R. Bryan M.C. Fazio F. Calarese D. Stevens J. et al.Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.Proc. Natl. Acad. Sci. USA. 2004; 101: 17033-17038Crossref PubMed Scopus (967) Google Scholar, Fukui et al., 2002Fukui S. Feizi T. Galustian C. Lawson A.M. Chai W. Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions.Nat. Biotechnol. 2002; 20: 1011-1017Crossref PubMed Scopus (527) Google Scholar, Palma et al., 2014Palma A.S. Feizi T. Childs R.A. Chai W. Liu Y. The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome.Curr. Opin. Chem. Biol. 2014; 18: 87-94Crossref PubMed Scopus (71) Google Scholar, Puvirajesinghe and Turnbull, 2016Puvirajesinghe T.M. Turnbull J.E. Glycoarray technologies: deciphering interactions from proteins to live cell responses.Microarrays (Basel). 2016; 5: E3Crossref PubMed Google Scholar). Two larger academic initiatives host these resources and offer services for the scientific community (www.functionalglycomics.org and www.imperial.ac.uk/glycosciences/). These glycan arrays are utilized with great success to probe glycan binding specificities of lectins and microbial adhesins; in particular, the Consortium for Functional Glycomics arrays and expanded sialoside microarrays have been instrumental in dissecting the subtle binding specificities of different influenza hemagglutinins (HAs) and their role in cross-species transmission (Padler-Karavani et al., 2012Padler-Karavani V. Song X. Yu H. Hurtado-Ziola N. Huang S. Muthana S. Chokhawala H.A. Cheng J. Verhagen A. Langereis M.A. et al.Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays.J. Biol. Chem. 2012; 287: 22593-22608Crossref PubMed Scopus (100) Google Scholar, Peng et al., 2017Peng W. de Vries R.P. Grant O.C. Thompson A.J. McBride R. Tsogtbaatar B. Lee P.S. Razi N. Wilson I.A. Woods R.J. Paulson J.C. Recent H3N2 viruses have evolved specificity for extended, branched human-type receptors, conferring potential for increased avidity.Cell Host Microbe. 2017; 21: 23-34Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, Rillahan and Paulson, 2011Rillahan C.D. Paulson J.C. Glycan microarrays for decoding the glycome.Annu. Rev. Biochem. 2011; 80: 797-823Crossref PubMed Scopus (348) Google Scholar, Wang et al., 2013Wang Z. Chinoy Z.S. Ambre S.G. Peng W. McBride R. de Vries R.P. Glushka J. Paulson J.C. Boons G.J. A general strategy for the chemoenzymatic synthesis of asymmetrically branched N-glycans.Science. 2013; 341: 379-383Crossref PubMed Scopus (247) Google Scholar). Glycan array technology depends on continued synthesis and/or isolation of glycans and often requires larger community-supported efforts. There are limitations in the size and complexity of glycans that can be synthesized and in impact of the linkers used (Padler-Karavani et al., 2012Padler-Karavani V. Song X. Yu H. Hurtado-Ziola N. Huang S. Muthana S. Chokhawala H.A. Cheng J. Verhagen A. Langereis M.A. et al.Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays.J. Biol. Chem. 2012; 287: 22593-22608Crossref PubMed Scopus (100) Google Scholar, Rillahan and Paulson, 2011Rillahan C.D. Paulson J.C. Glycan microarrays for decoding the glycome.Annu. Rev. Biochem. 2011; 80: 797-823Crossref PubMed Scopus (348) Google Scholar). Perhaps the most important limitation is the unnatural context in which the oligosaccharides or neoglycolipids are displayed in high densities without context of the protein or lipid backbone to which the glycans are normally attached (Blixt et al., 2004Blixt O. Head S. Mondala T. Scanlan C. Huflejt M.E. Alvarez R. Bryan M.C. Fazio F. Calarese D. Stevens J. et al.Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.Proc. Natl. Acad. Sci. USA. 2004; 101: 17033-17038Crossref PubMed Scopus (967) Google Scholar), as well as interactions with adjacent glycans that have been suggested to generate “clustered saccharide patches” (Cohen and Varki, 2014Cohen M. Varki A. Modulation of glycan recognition by clustered saccharide patches.Int. Rev. Cell Mol. Biol. 2014; 308: 75-125Crossref PubMed Scopus (60) Google Scholar, Varki, 1994Varki A. Selectin ligands.Proc. Natl. Acad. Sci. USA. 1994; 91: 7390-7397Crossref PubMed Scopus (953) Google Scholar). Printed glycan arrays do not display glycans as they are found on the cell surface, and they most often report a complex collection of glycan hapten structures with limited guidance for further studies. While the human glycome is vast, the cellular glycosylation machinery producing this diversity is simpler and, arguably, better understood and approachable. The human genome contains over 200 distinct genes encoding glycosyltransferases, and the knowledge of their properties of these and their roles in the 15 known distinct glycosylation pathways in human cells is relatively advanced (Joshi et al., 2018aJoshi H.J. Hansen L. Narimatsu Y. Freeze H.H. Henrissat B. Bennett E. Wandall H.H. Clausen H. Schjoldager K.T. Glycosyltransferase genes that cause monogenic congenital disorders of glycosylation are distinct from glycosyltransferase genes associated with complex diseases.Glycobiology. 2018; 28: 284-294Crossref PubMed Scopus (32) Google Scholar). Clearly, orchestration of the human glycome involves a large number of additional enzymes, including those that modify glycans such as sulfotransferases and epimerases, transporters, and other proteins; and the glycosylation processes involve overlapping, competing, and intertwined reactions, in particular, by families of isoenzymes that may be difficult to predict. However, to a large extent, the general scaffolds and structures of the glycome may be predicted from the repertoire of only 153 glycosyltransferase genes, as proposed in Figures 1, S1, and S2. The glycosylation pathway maps organize glycosyltransferase genes into pathway-specific and pathway-nonspecific steps in the biosynthesis of distinct glycoconjugates, and illustrates potential redundancies for individual biosynthetic steps provided by isoenzymes and alternative competing biosynthetic steps. These maps provide predictions of structural glycan features affected by the presence or absence of individual genes and whether global or differential subtle outcomes are expected. Combined with the recent introduction of the facile nuclease-based gene-editing tools (Steentoft et al., 2014Steentoft C. Bennett E.P. Schjoldager K.T. Vakhrushev S.Y. Wandall H.H. Clausen H. Precision genome editing: a small revolution for glycobiology.Glycobiology. 2014; 24: 663-680Crossref PubMed Scopus (47) Google Scholar), we posit that it is now timely and conceivable to take an entirely genetic approach to dissect and display the structural diversity of the glycome of a human cell. Clearly, the glycosylation pathway maps require continuous refinement with increased insight into nonredundant and competing functions of isoenzymes, which requires further studies in isogenic cell models with combinatorial engineering to evaluate outcome. This has been a fruitful strategy for the discovery of functions of the large family polypeptide GalNAc-transferase isoenzymes controlling O-glycosylation (Schjoldager et al., 2015Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation—GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO Rep. 2015; 16: 1713-1722Crossref PubMed Scopus (81) Google Scholar). To begin to facilitate such comprehensive gene targeting, we previously generated a validated CRISPR/Cas9 guide RNA (gRNA) library for highly efficient knockout (KO) targeting of all human glycosyltransferase genes (Narimatsu et al., 2018Narimatsu Y. Joshi H.J. Yang Z. Gomes C. Chen Y.H. Lorenzetti F.C. Furukawa S. Schjoldager K.T. Hansen L. Clausen H. et al.A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome.Glycobiology. 2018; 28: 295-305Crossref PubMed Scopus (55) Google Scholar) and strategies for stable, site-specific knockin (KI) of non-expressed genes (Yang et al., 2015bYang Z. Wang S. Halim A. Schulz M.A. Frodin M. Rahman S.H. Vester-Christensen M.B. Behrens C. Kristensen C. Vakhrushev S.Y. et al.Engineered CHO cells for production of diverse, homogeneous glycoproteins.Nat. Biotechnol. 2015; 33: 842-844Crossref PubMed Scopus (168) Google Scholar). Genetic approaches studying the biology of glycans have a long history. A large collection of Chinese hamster ovary (CHO) cell lines was originally developed through chemical mutagenesis and selection for loss of lectin binding or expression of cell-surface proteins (Conzelmann and Kornfeld, 1984Conzelmann A. Kornfeld S. Beta-linked N-acetylgalactosamine residues present at the nonreducing termini of O-linked oligosaccharides of a cloned murine cytotoxic T lymphocyte line are absent in a Vicia villosa lectin-resistant mutant cell line.J. Biol. Chem. 1984; 259: 12528-12535PubMed Google Scholar, Kingsley et al., 1986Kingsley D.M. Kozarsky K.F. Hobbie L. Krieger M. Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant.Cell. 1986; 44: 749-759Abstract Full Text PDF PubMed Scopus (240) Google Scholar, Patnaik and Stanley, 2006Patnaik S.K. Stanley P. Lectin-resistant CHO glycosylation mutants.Methods Enzymol. 2006; 416: 159-182Crossref PubMed Scopus (162) Google Scholar), and most of these cells have been characterized to have defects in a distinct glycosyltransferase gene, donor sugar nucleotide synthase gene, sugar nucleotide transporter, or sugar nucleotide epimerase gene, with resulting loss or gain of global glycan features such as complex N-glycans, complete loss of sialylation or fucosylation of all glycans, or complete loss of glycosaminoglycan structures. These mutant cells have been important tools for determining the requirement for global glycan features, but they do not enable deeper insight into glycan structures involved in biological interactions. Studies of deficiencies in glycosylation genes with rodents have also illustrated the wide biological importance of glycans and the glycosylation process (Lowe and Marth, 2003Lowe J.B. Marth J.D. A genetic approach to mammalian glycan function.Annu. Rev. Biochem. 2003; 72: 643-691Crossref PubMed Scopus (528) Google Scholar), while in humans, a large number of congenital disorders of glycosylation (CDGs) have contributed substantially to our understanding of the importance of many different types of glycosylation (Ng and Freeze, 2018Ng B.G. Freeze H.H. Perspectives on glycosylation and Its congenital disorders.Trends Genet. 2018; 34: 466-476Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In many cases, the complexities of whole-organism-level analyses have precluded identification of specific structure-function relationships. Past studies with mutant cell lines and organisms clearly demonstrate the power of genetic approaches to probe the glycome, and perhaps one of the best illustrations hereof is the complete unraveling of the many genes necessary for biosynthesis of the extraordinarily complex O-mannose glycan structure required for laminin binding to α-dystroglycan (Jae et al., 2013Jae L.T. Raaben M. Riemersma M. van Beusekom E. Blomen V.A. Velds A. Kerkhoven R.M. Carette J.E. Topaloglu H. Meinecke P. et al.Deciphering the glycosylome of dystroglycanopathies using haploid screens for lassa virus entry.Science. 2013; 340: 479-483Crossref PubMed Scopus (222) Google Scholar). This and more recent studies using precise gene engineering to delineate and reprogram glycosylation pathways in mammalian cell lines demonstrate the ability to dissect biological roles of glycosylation genes and their contribution to the glycome (Lavrsen et al., 2018Lavrsen K. Dabelsteen S. Vakhrushev S.Y. Levann A.M.R. Haue A.D. Dylander A. Mandel U. Hansen L. Frödin M. Bennett E.P. Wandall H.H. De novo expression of human polypeptide N-acetylgalactosaminyltransferase 6 (GalNAc-T6) in colon adenocarcinoma inhibits the differentiation of colonic epithelium.J. Biol. Chem. 2018; 293: 1298-1314Crossref PubMed Scopus (48) Google Scholar, Schulz et al., 2018Schulz M.A. Tian W. Mao Y. Van Coillie J. Sun L. Larsen J.S. Chen Y.H. Kristensen C. Vakhrushev S.Y. Clausen H. Yang Z. Glycoengineering design options for IgG1 in CHO cells using precise gene editing.Glycobiology. 2018; 28: 542-549Crossref PubMed Scopus (22) Google Scholar, Stolfa et al., 2016Stolfa G. Mondal N. Zhu Y. Yu X. Buffone Jr., A. Neelamegham S. Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and glycosphingolipids to human leukocyte-endothelium adhesion.Sci. Rep. 2016; 6: 30392Crossref PubMed Scopus (39) Google Scholar, Yang et al., 2015bYang Z. Wang S. Halim A. Schulz M.A. Frodin M. Rahman S.H. Vester-Christensen M.B. Behrens C. Kristensen C. Vakhrushev S.Y. et al.Engineered CHO cells for production of diverse, homogeneous glycoproteins.Nat. Biotechnol. 2015; 33: 842-844Crossref PubMed Scopus (168) Google Scholar). These studies also show that genetic reprogramming of glycosylation can be performed with a high degree of predictability. Here, we apply a fairly comprehensive genetic “tree-pruning” approach—glycotopiary—to engineer and reprogram the glycosylation capacities of a human cell line in order to construct a cell-based glycan array that covers a large part of the structural diversity of the human glycome. We used a rational combinatorial approach to eliminate and/or introduce de novo glycosylation capacities to develop sublibraries of stably engineered HEK293 isogenic cells that individually display the loss or gain of distinct features of the human glycome. Importantly, combinatorial engineering of isoenzyme families with poorly understood functions enabled dissection and display of uniquely regulated glycan features. We demonstrate performance of the array with a series of plant, microbial, and human lectins. We confirmed the hypothesis that the glycoconjugate and cellular context of glycans provide additional and necessary diversity in structural permutations of the human glycome. Cell-based array analysis of avian and human influenza virus HAs fully recapitulated the known selective binding to α2-3 or α2-6-linked sialic acids (SAs) (Rillahan and Paulson, 2011Rillahan C.D. Paulson J.C. Glycan microarrays for decoding the glycome.Annu. Rev. Biochem. 2011; 80: 797-823Crossref PubMed Scopus (348) Google Scholar), and the added context of the cell provided evidence for binding selectivities beyond the simple SA linkage. Analysis of streptococcal serine-rich repeat adhesins produced refinement of the recognized O-glycan structures, compared to information derived from printed glycan arrays, providing evidence for the recognition of clusters or patterns of O-glycans created by the protein carrier. Thus, the cell-based glycan array fully complements the traditional printed glycan arrays and further provides insight into the genetic and biosynthetic regulation of glycan recognition events, with broader context of glycoconjugate nature and higher order presentation. We organized current knowledge of 169 glycosyltransferase genes directing the human glycome into a rainbow diagram that organizes these genes into the 15 distinct glycosylation pathways symbolized by the color used for the first monosaccharide (Figure 1; Joshi et al., 2018aJoshi H.J. Hansen L. Narimatsu Y. Freeze H.H. Henrissat B. Bennett E. Wandall H.H. Clausen H. Schjoldager K.T. Glycosyltransferase genes that cause monogenic congenital disorders of glycosylation are distinct from glycosyltransferase genes associated with complex diseases.Glycobiology. 2018; 28: 284-294Crossref PubMed Scopus (32) Google Scholar, Joshi et al., 2018bJoshi H.J. Narimatsu Y. Schjoldager K.T. Tytgat H.L.P. Aebi M. Clausen H. Halim A. SnapShot: O-Glycosylation Pathways across Kingdoms.Cell. 2018; 172: 632-632.e632Abstract Full Text PDF PubMed Scopus (53) Google Scholar, Narimatsu et al., 2018Narimatsu Y. Joshi H.J. Yang Z. Gomes C. Chen Y.H. Lorenzetti F.C. Furukawa S. Schjoldager K.T. Hansen L. Clausen H. et al.A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome.Glycobiology. 2018; 28: 295-305Crossref PubMed Scopus (55) Google Scholar), with the predicted functions in biosynthetic steps and pathways as shown in Figure S2. 44 genes can be assigned to pathway-specific functions in the initiation of glycosylation of different types of glycoconjugates, 16 genes can be assigned to assembly of the lipid-linked oligosaccharide precursor and oligosaccharyltransferase dedicated to N-glycosylation, and 57 genes can be assigned to pathway-specific functions in immediate core extension and branching steps. Thus, 117 of the 169 genes are assignable to distinct glycosylation pathways, and several of these predictions were previously validated with CHO mutant cells (Patnaik and Stanley, 2006Patnaik S.K. Stanley P. Lectin-resistant CHO glycosylation mutants.Methods Enzymol. 2006; 416: 159-182Crossref PubMed Scopus (162) Google Scholar), targeted CHO KO cells (Yang et al., 2015bYang Z. Wang S. Halim A. Schulz M.A. Frodin M. Rahman S.H. Vester-Christensen M.B. Behrens C. Kristensen C. Vakhrushev S.Y. et al.Engineered CHO cells for production of diverse, homogeneous glycoproteins.Nat. Biotechnol. 2015; 33: 842-844Crossref PubMed Scopus (168) Google Scholar), and other mammalian cell lines (Stolfa et al., 2016Stolfa G. Mondal N. Zhu Y. Yu X. Buffone Jr., A. Neelamegham S. Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and glycosphingolipids to human leukocyte-endothelium adhesion.Sci. Rep. 2016; 6: 30392Crossref PubMed Scopus (39) Google Scholar). We classified 17 genes to pathway-nonspecific elongation or branching and another 35 genes to pathway-nonspecific capping, including sialylation and fucosylation. While it is possible to reliably assign most of the glycosyltransferases that belong to the large isoenzyme families to general biosynthetic steps, it is important to note that, for most of these isoenzymes, our understanding of their specific non-redundant functions is still very limited. We previously demonstrated how genetic KO and/or KI dissection of isoenzyme genes can be used to identify non-redundant functions of isoenzymes (Schjoldager et al., 2015Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation—GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO Rep. 2015; 16: 1713-1722Crossref PubMed Scopus (81) Google Scholar), and this is clearly the strategy needed to dissect the large β3/4Gal-transferase, β3GlcNAc-transferase, and α2-3 or α2-6 sialyltransferase isoenzyme families. We previously also classified human glycosyltransferase genes grossly into regulated and non-regulated based on organ transcriptome data (Joshi et al., 2018aJoshi H.J. Hansen L. Narimatsu Y. Freeze H.H. Henrissat B. Bennett E. Wandall H.H. Clausen H. Schjoldager K.T. Glycosyltransferase genes that cause monogenic congenital disorders of glycosylation are distinct from glycosyltransferase genes associated with complex diseases.Glycobiology. 2018; 28: 284-294Crossref PubMed Scopus (32) Google Scholar), and this provides indications of differentially regulated glycosylation steps and pathways that contribute to the diversity of the glycome. We selected the HEK293 cell line as the platform for construction of the cell-based glycan display because structural analyses of different types of glycans suggest a high degree of complexity in glycosylation (Fujitani et al., 2013Fujitani N. Furukawa J. Araki K. Fujioka T. Takegawa Y. Piao J. Nishioka T. Tamura T. Nikaido T. Ito M. et al.Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers.Proc. Natl. Acad. Sci. USA. 2013; 110: 2105-2110Crossref PubMed Scopus (117) Google Scholar, Termini et al., 2017Termini J.M. Silver Z.A. Connor B. Antonopoulos A. Haslam S.M. Dell A. Desrosiers R.C. HEK293T cell lines defective for O-linked glycosylation.PLoS ONE. 2017; 12: e0179949Crossref PubMed Scopus (14) Google Scholar, Yang et al., 2012Yang X. Tao S. Orlando R. Brockhausen I. Kan F.W. Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells.Carbohydr. Res. 2012; 358: 47-55Crossref PubMed Scopus (11) Google Scholar), and this cell line is widely used for recombinant expression and characterization of glycoproteins (Thomas and Smart, 2005Thomas P. Smart T.G. HEK293 cell line: a vehicle for the expression of recombinant proteins.J. Pharmacol. Toxicol. Methods. 2005; 51: 187-200Crossref PubMed Scopus (467) Google Scholar). We used RNA-sequencing (RNA-seq) transcriptomics as a rough prediction of the glycosylation capacity of HEK293 cells, and 121 of the 169 glycosyltransferase genes had detectable transcripts (fragments per kilobase million [FPKM] ≥ 1), while 47 were not or were poorly detectable (FPKM < 1) (Figure S1). Figure 1 illustrates the glycosyltransferase genes predicted to be expressed and their proposed functions, and the interpretation largely correlates with reported structural analysis (Yang et al., 2012Yang X. Tao S. Orlando R. Brockhausen I. Kan F.W. Structures and biosynthesis of the N- and O-glycans of recombinant human oviduct-specific glycoprotein expressed in human embryonic kidney cells.Carbohydr. Res. 2012; 358: 47-55Crossref PubMed Scopus (11) Google Scholar). Thus, HEK293 cells are predicted to have the capacity for all types of lipid and protein glycosylation, comprehensive elaboration of pathway-specific elongation and branching features, type 2 chain LacNAc and GalNAcβ1-4GlcNAc (LacDiNAc) core chains, and both α2-3 and α2-6SA capping. SA in HEK293 is primarily Neu5Ac, unless cells are cultured in bovine serum from where Neu5Gc can be scavenged, and acetylation has been reported (Wasik et al., 2017Wasik B.R. Barnard K.N. Ossiboff R.J. Khedri Z. Feng K.H. Yu H. Chen X. Perez D.R. Varki A. Parrish C.R. Distribution of O-acetylated sialic acids among target host tissues for influenza virus.mSphere. 2017; 2 (e00379-16)Crossref PubMed Scopus (42) Google Scholar). The limited glycan features not predicted to be produced in HEK293 cells are globoseries glycosphingolipids (A4GALT); core1 extended (B3GNT3) and core3/4 branched GalNAc-type O-glycans (B3GNT6, GCNT3, and GCNT4); type 1 chain N-acetyl-lactosamine (LacNAc) structures (B3GALT1, T2, T4, and T5); and capping by blood group ABH, Sda, and Lewis antigens (ABO, B4GALNT2, FUT1, FUT2, and FUT3). Moreover, the capacity for α3 fucosylation and α2-8 sialylation is predicted to be low or absent, due to the limited expression of members of the large isoenzyme FUT and ST8SIA families. The basic glycotopiary concept to add and remove branches of glycan complexity by genetic KO and/or KI of glycosyltransferase genes, in order to generate isogenic cells displaying loss or gain of particular glycosylation features, is presented in Figure 2A. This illustrates how combinatorial CRISPR/Cas9 KO targeting of the genes controlling the earliest essential steps in elongation or elaboration of glycans found on glycosphingolipids (B4GALT5/6), N-glycoproteins (MGAT1), and GalNAc-type O-glycoproteins (C1GALT1/COSMC) results in isogenic cells differentially displaying glycan features on one or more of these glycoconjugates. The performed KO and/or KI targeting is indicated by red and green dots, respectively, with lines between dots representing combinatorial gene engineering. We designed sublibraries with the capacity to differentially display defined glycan features grouped according to the rainbow biosynthetic scheme (Figure 2B). The sublibraries consist of groups of isogenic cells with reprogrammed glycosylation capacities for the major steps in glycosylation. Sublibrary 1 was designed to differentially display the major types of glycoconjugates by eliminating the earliest pathway-specific elongation steps for one or more glycoconjugates, resulting in the display of N-glycans (KO MGAT1), GalNAc-type O-glycans (KO COSMC), and/or glycosphingolipids (KO B4GALT5/6), as well as independently Man-type O-glycans (KO POMGNT1, POMGNT2, and TMTC1-4) and glycosaminoglycans (GAGs) (KO B4GALT7). Sublibraries 2–5 differentially display most pathway-specific glycan features separately for glycosphingolipids, N-glycan branching, GalNAc-type O-glycan branching, and GAG core structures, respectively. Sublibrary 6 differentially displays pathway-nonspecific elongation by type 2 chain LacNAc and/or LacDiNAc and poly-LacNAc. Sublibraries 7 and 8 differentially display pathway-nonspecific Gal capping by α2-3 and/or α2-6SA, as well as GalNAc capping by α2-6SA. Importantly, the individual and combinatorial targeting of isoenzyme genes enables the display of the contribution of individual isoenzymes to the glycome and the dissection of their unique non-redundant functions and interpretation of the underlying structural glycan features (Schjoldager et al., 2015Schjoldager K.T. Joshi H.J. Kong Y. Goth C.K. King S.L. Wandall H.H. Bennett E.P. Vakhrushev S.Y. Clausen H. Deconstruction of O-glycosylation—GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.EMBO Rep. 2015; 16: 1713-1722Crossref PubMed Scopus (81) Google Scholar). We used site-directed KI integration of human glycosyltransferase genes to introduce glycan features not endogenously expressed in HEK293 cells. These cells do not express the complex GalNAc-type core3/4 O-glycans, which are generally poorly expressed in cancer cell lines. The core3 pathway competes with core1, so we generated a stable KI of the core3 synthase (B3GNT6) in HEK293 cells with a KO of the core1 (COSMC). The important cancer-associated STn O-glycan is not endogenously displayed in HEK293 cells, even after KO of COSMC (Steentoft et al., 2011Steentoft C. Vakhrushev S.Y. Vester-Christensen M." @default.
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• W2953347000 title "An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells" @default.
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• W2953347000 doi "https://doi.org/10.1016/j.molcel.2019.05.017" @default.
• W2953347000 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6660356" @default.
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