N-glycosylation contributes to the intracellular stability of prothrombin precursors in the endoplasmic reticulum.
Rat prothrombin (rFII) and human prothrombin (hFII) are processed differently during their biosynthesis in a manner dependent upon gamma-glutamyl carboxylation, a vitamin K-dependent posttranslational modification of prothrombin precursors. The role of N-glycosylation in the cellular processing of prothrombin was examined in hepatoma (H-35 and HepG2) and transformed kidney (HEK293) cell lines. Aglyco-rFII obtained by tunicamycin treatment was degraded in warfarin-treated H-35 cells, but not in vitamin K-treated cells. Fully glycosylated rFII is also selectively retained and degraded in warfarin-treated H-35 cells. When rFII and hFII were transiently expressed in tunicamycin-treated HEK293 cells, rFII but not hFII was degraded to generate a 48-KD species. This degradation was independent of gamma-carboxylation, indicating that the sensitivity of aglyco-rFII and aglyco-hFII toward this specific proteolysis differs in HEK293 cells. By expressing chimeric rFII/hFII constructs in tunicamycin-treated HEK293 cells, it was shown that the kringle 2 structure of prothrombin was responsible for this difference. The 48-KD species was not observed in tunicamycin-treated H-35 cells, suggesting that this specific proteolytic processing of aglyco-rFII is also cell-type specific. Expression of rFII in other tunicamycin-treated nonhepatic cells suggests that this cell-specific difference in processing might be determined by a difference between hepatic and nonhepatic cells. The site-directed mutagenesis utilized in these studies also establishes that the N-glycosylation sites of rFII are at residues Asn77, 101, 378, and 518.
Wu
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Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, 53705-1569, USA.
Suttie
J W
JW
D000818:Animals; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology; D014859:Warfarin / Q000494:pharmacology
D011498:Protein Precursors; D011994:Recombinant Proteins; D014415:Tunicamycin; D014859:Warfarin; D011516:Prothrombin