Site-specific glycosylation of human recombinant erythropoietin: analysis of glycopeptides or peptides at each glycosylation site by fast atom bombardment-mass spectrometry

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Abstract
We have previously determined the carbohydrate structure of human recombinant erythropoietin [Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076]. The carbohydrate chains are distributed in three N-glycosylation sites and one O-glycosylation site. In order to examine the extent to which protein structure influences glycosylation, we have analyzed the saccharide structures at each glycosylation site (Asn24, Asn38, Asn83, and Ser126) of human recombinant erythropoietin. By high-performance liquid chromatography, we have succeeded in separation of glycopeptides containing different O-linked saccharides to the same peptide backbone. Fast atom bombardment mass spectrometry of the isolated glycopeptides combined with Edman degradation allowed us to elucidate the composition of glycopeptides and the amino acid attachment site. The analysis of glycopeptides and saccharides by fast atom bombardment mass spectrometry and high-performance liquid chromatography provided the following conclusions on N-glycans: (1) saccharides at Asn24 are heterogeneous and consist of biantennary, triantennary, and tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (2) saccharides at Asn38 mainly consist of well-processed saccharides such as tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (3) saccharides at Asn83, on the other hand, are homogeneous in the backbone structure and are composed mainly of tetraantennary without N-acetyllactosaminyl repeats. It was also noted that saccharides at Asn24 are much less sialylated than those at Asn38, although these two glycosylation sites are close to each other. These results clearly indicate that the protein structure and, possibly, the carbohydrate chain at the neighboring site greatly influence glycosylation of a given glycosylation site.