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Biochemical characterization of theO-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells

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Abstract

Alterations inN- andO-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. TheO-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. TheO-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, Galβ1-3GalNAcα1-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 crythrocyte 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 (GalNAcα1-Ser/Thr). Recombinant glycophorin A had the sameO-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 itsO-glycan composition. Deletion of theN-linked glycosylation site at Asn26, by introducing the Mi.I mutation (Thr28 → Met) by site-directed mutagenesis, did not markedly affect theO-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells. This demonstrates that the presence or absence of theN-glycan did not influenceO-linked glycosylation of the recombinant glycoprotein. Finally, theO-glycans on recombinant glycophorin A expressed in the Lec 2 and Lec 8 glycosylation deficient CHO cells were characterized. TheO-glycans on Lec 2 cell glycophorin A were predominantly Galβ1-3GalNAcα1-Ser/Thr (T antigen), while those on Lec 8 glycophorin A were exclusively GalNAcα1-Ser/Thr (Tn antigen). These results will lead to a better understanding of the cell biology and immunology of this important human erythrocyte glycoprotein.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of Pennsylvania, 19104, Philadelphia, PA, USA

    Peter Påhlsson, Douglas P. Blackall, Maciej Ugorski, Marcin Czerwinski & Steven L. Spitalnik

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  1. Peter Påhlsson
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  2. Douglas P. Blackall
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  3. Maciej Ugorski
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  4. Marcin Czerwinski
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  5. Steven L. Spitalnik
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Påhlsson, P., Blackall, D.P., Ugorski, M. et al. Biochemical characterization of theO-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells. Glycoconjugate J 11, 43–50 (1994). https://doi.org/10.1007/BF00732431

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