title: The GalNAc-type O-Glycoproteome of CHO cells characterized by the SimpleCell strategy.
abstract: The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its "human-like" glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycan structures (glycostructures) on glycoproteins are well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O-glycans to proteins (glycosites) is minimal. This type of O-glycosylation is one of the most abundant forms of glycosylation, and it is differentially regulated in cells by expression of a subset of homologous polypeptide GalNAc-transferases. Here, we have genetically engineered CHO cells to produce homogeneous truncated O-glycans, so-called SimpleCells, which enabled lectin enrichment of O-glycoproteins and characterization of the O-glycoproteome. We identified 738 O-glycoproteins (1548 O-glycosites) in cell lysates and secretomes providing the first comprehensive insight into the O-glycosylation capacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/).
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark;
authors.forename: Zhang
authors.initials: Z
authors.lastname: Yang
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark;
authors.forename: Adnan
authors.initials: A
authors.lastname: Halim
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark;
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Narimatsu
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark;
authors.forename: Hiren
authors.initials: H
authors.lastname: Jitendra Joshi
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark;
authors.forename: Catharina
authors.initials: C
authors.lastname: Steentoft
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark;
authors.forename: Katrine Ter-Borch Gram
authors.initials: KT
authors.lastname: Schjoldager
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark;
authors.forename: Morten
authors.initials: M
authors.lastname: Alder Schulz
authors.affiliation: ¶Cell Sciences and Development, SAFC/Sigma-Aldrich, 2909 Laclede Avenue, St. Louis, Missouri 63103.
authors.forename: Natalie R
authors.initials: NR
authors.lastname: Sealover
authors.affiliation: ¶Cell Sciences and Development, SAFC/Sigma-Aldrich, 2909 Laclede Avenue, St. Louis, Missouri 63103.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Kayser
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark;
authors.forename: Eric
authors.initials: E
authors.lastname: Paul Bennett
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark;
authors.forename: Steven B
authors.initials: SB
authors.lastname: Levery
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; seva@sund.ku.dk hclau@sund.ku.dk.
authors.forename: Sergey Y
authors.initials: SY
authors.lastname: Vakhrushev
authors.affiliation: From the ‡Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §Novo Nordisk Foundation Center for Biosustainability, Danish Technical University, Lyngby, Denmark; seva@sund.ku.dk hclau@sund.ku.dk.
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D060846:Cell Engineering; D002460:Cell Line; D003412:Cricetulus; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D054794:Glycomics; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D016415:Sequence Alignment; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D000116:Acetylgalactosamine; D004921:Erythropoietin; D006023:Glycoproteins; D007527:Isoenzymes; D018832:Molecular Chaperones; D017350:N-Acetylgalactosaminyltransferases; D020543:Proteome; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C554270:EPO protein, human
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