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 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 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-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 fluorescently 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.
Gohlke
M
M
Institut für Molekularbiologie und Biochemie der Freicn Universität Berlin, Berlin-Dahlem, Germany.
Nuck
R
R
Kannicht
C
C
Grunow
D
D
Baude
G
G
Donner
P
P
Reutter
W
W
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002685:Chiroptera; D006224:Cricetinae; D003412:Cricetulus; D006020:Glycopeptides / Q000032:analysis; D006031:Glycosylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D010449:Peptide Mapping; D010960:Plasminogen Activators / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012463:Saliva / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006020:Glycopeptides; D009844:Oligosaccharides; D011994:Recombinant Proteins; C069588:salivary plasminogen activator alpha 1, Desmodus rotundus; D010960:Plasminogen Activators