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

The recombinant plasminogen activator (rDSPA1) from the vampirebat Desmodus rotundus is a promising new thrombolytic agentthat exhibits a superior pharmacological profile if comparedto tissue-type plasminogen activator (t-PA) or streptokinase.In the present study the structures of the carbohydrate moietiesat the two N-glycosylation sites (Asn-117, Asn-362) of rDSPAIexpressed in Chinese hamster ovary cells were determined. N-Linkedglycans were enzymatically released from isolated tryptic glycopeptidesby peptide-N⁴-(N-acetyl-ß-glucosaminyl)asparagineamidase F digestion and separated by two-dimensional HPLC. Oligosaccharidestructures were characterized by analysis of carbohydrate compositionand linkage, by mass spectrometry, and by sequence analysisin which the fiuorescently labeled glycans were cleaved withan array of specific exoglycosidases. More than 30 differentoligosaccharides were identified. The results revealed thatAsn-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%). Glycosylationsite Asn-362 was found to comprise complex glycans with biantennary(50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennarystructures (10%), which were fucosylated at the innermost residueof N-acetylglucosamine. Mainly neutral and monosialylated glycans,and smaller quantities of disialylated glycans, were detectedat both glycosylation sites. Sialic acid was 2-3 linked to galactoseexclusively. As shown in this study the N-glycans attached toAsn-117 of rDSPA1 are more processed during biosynthesis thanthe high-mannose structures linked to Asn-117 of t-PA, to whichthe polypeptide backbone of rDSPA1 is structurally closely related. bat plasminogen activator oligosaccharide analysis rDSPA1 recombinant glycoprotein site-specific N-glycosylation