The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

The Chinese hamster ovary cell(CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its “human-like” glycosylationfeatures. CHO is used for production of several O- glycoproteintherapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O- glycoproteinsare not produced efficiently in CHO. We have previously shown that the capacity for O- glycosylationof proteins can be one limiting parameter for production of active proteins in CHO. Although the capacity of CHO for biosynthesis of glycanstructures (glycostructures) on glycoproteinsare well established, our knowledge of the capacity of CHO cells for attaching GalNAc-type O- glycansto proteins (glycosites) is minimal. This type of O- glycosylationis 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- glycoproteinsand characterization of the O- glycoproteome. We identified 738 O- glycoproteins(1548 O-glycosites) in cell lysates and secretomesproviding the first comprehensive insight into the O- glycosylationcapacity of CHO (http://glycomics.ku.dk/o-glycoproteome_db/).