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 (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhi bits a superior pharmacological profile if compared to tissue-type pla sminogen activator (t-PA) or streptokinase. In the present study the s tructures of the carbohydrate moieties at the two N-glycosylation site s (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ova ry cells were determined, N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N-4-(N-acetyl-beta-glu cosaminyl)asparagine amidase F digestion and separated by two-dimensio nal HPLC, Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by se quence analysis in which the fluorescently labeled glycans were cleave d with an array of specific exoglycosidases, More than 30 different ol igosaccharides were identified, The results revealed that Asn-117 carr ied a mixture of one high-mannose structure (17% of site-specific glyc osylation), three hybrid glycans (26%) and predominantly biantennary c omplex N-glycans (54%), Glycosylation site Asn-362 was found to compri se complex glycans with biantennary (50%), 2,4- and 2,6-branched trian tennary (21%, 11%), and tetraantennary structures (10%), which were fu cosylated at the innermost residue of N-acetylglucosamine, Mainly neut ral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites, Sialic acid was a lpha 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 durin g biosynthesis than the high-mannose structures linked to Asn-117 of t -PA, to which the polypeptide backbone of rDSPA alpha 1 is structurall y closely related.