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Glycobiology vol 7 no 1 pp. 67-77, 1997
© 1997

research-article

Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPA{alpha}1 expressed in Chinese hamster ovary cells

Martin Gohlke, Rolf Nuck2, Christoph Kannicht, Detlef Grunow, Günther Baude1, Peter Donner1 and Werner Reutter

Institut für Molekularbiologie und Biochemie der Freien Universität Berlin Berlin-Dahlem, Germany
1Research Laboratories of Schering AG Berlin, Germany

2To whom correspondence should be addressed at: Institut für Molekularbiologie und Biochemie der Freien Universität Berlin, Amimallee 22, D-14195 Berlin-Dahiem, Germany

Received on June 11, 1996; revised on July 24, 1996; accepted on August 19, 1996

The recombinant plasminogen activator (rDSPA{alpha}1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA{alpha}I expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-ß-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fiuorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was {alpha}2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA{alpha}1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA{alpha}1 is structurally closely related.

bat plasminogen activator oligosaccharide analysis rDSPA{alpha}1 recombinant glycoprotein site-specific N-glycosylation


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