Alterations in the domain structure of tissue-type plasminogen activator change the nature of asparagine glycosylation.
The formation of N-linked oligosaccharides of eukaryotic glycoproteins starts with the attachment of a common precursor at the recognition site Asn-X-Ser/Thr. Subsequent processing, by yet unknown controlling factors, leads to the formation of three different glycans: the high mannose type, the complex type and the hybrid type. In order to gain insight into the processing mechanisms, we studied the glycan pattern of a panel of related molecules constructed by insertion, duplication or deletion of the domains encoded by the cDNA of a fibrinolytic glycoprotein, tissue-type plasminogen activator (t-PA). These variant molecules are identical in regard to the glycosylation sites originally situated in particular domains, but differ with respect to the sequential alignment of the domains. The variant and native t-PA genes were transfected into mouse C127 cells and their carbohydrate structures analyzed by the susceptibility to specific endoglycosidases and by reaction with sugar-specific lectins. We found that with one exception, all mutant activators lack the high mannose glycan found at asn 117 of native t-PA. The exception was a molecule that retains the original domain arrangement up to and through the glycosylation site at asn 117. These results demonstrate for the first time that structural alterations in the primary sequence distal to the actual glycosylation site can result in altered processing of N-linked oligosacharides.
Biotechnology and Microbiology Division, Wyeth-Ayerst Research, Inc., Radnor, PA 19087.
J
J
Wilhelm
S G
SG
Lee
N K
NK
Kalyan
S M
SM
Cheng
F
F
Wiener
W
W
Pierzchala
P P
PP
Hung
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002478:Cells, Cultured; D006031:Glycosylation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D009844:Oligosaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D010959:Tissue Plasminogen Activator