Biochemical characterization of the O-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells.
Alterations in N- and O-linked glycosylation affect cell surface expression and antigenicity of recombinant glycophorin A expressed in transfected Chinese hamster ovary (CHO) cells. To understand these effects further, glycophorin A was purified by immunoaffinity chromatography from transfected wild type and glycosylation deficient CHO cells. The O-glycans were characterized both biochemically, using gel filtration and high performance anion exchange chromatography, and immunologically, using carbohydrate specific monoclonal antibodies to probe Western blots. The O-glycans of human erythrocyte glycophorin A consist mainly of short oligosaccharides with one, two, or three sialic acid residues linked to a common disaccharide core, Gal beta 1-3GalNAc alpha 1-Ser/Thr, with the disialylated structure being the most abundant. With the exception of the trisialylated derivative, the same structures were found on recombinant glycophorin A expressed by wild type CHO cells. However, in contrast to human erythrocyte glycophorin A, the monosialylated oligosaccharide was the most abundant structure on the recombinant protein. Furthermore, recombinant glycophorin A was shown to express a small amount of the Tn antigen (GalNAc alpha 1-Ser/Thr). Recombinant glycophorin A had the same O-glycan composition, whether purified from clones expressing high or moderate levels of the recombinant glycoprotein. This indicates that the level of expression of the transfected glycoprotein did not affect its O-glycan composition. Deletion of the N-linked glycosylation site at Asn26, by introducing the Mi.I mutation (Thr28-->Met) by site-directed mutagenesis, did not markedly affect the O-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Påhlsson
P
P
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104.
Blackall
D P
DP
Ugorski
M
M
Czerwinski
M
M
Spitalnik
S L
SL
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D006021:Glycophorins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry; D013045:Species Specificity
D006021:Glycophorins; D009844:Oligosaccharides; D011994:Recombinant Proteins