N-glycosylation contributes to the intracellular stability of prothrombin precursors in the endoplasmic reticulum

Rat prothrombin (rFII) and human prothrombin (hFII) are processed different ly during their biosynthesis in a manner dependent upon gamma-glutamyl carb oxylation, a vitamin K-dependent posttranslational modification of prothrom bin precursors, The role of N-glycosylation in the cellular processing of p rothrombin was examined in hepatoma (H-35 and HepG2) and transformed kidney (HEK293) cell lines, Aglyco-rFII obtained by tunicamycin treatment was deg raded in warfarin-treated H-35 cells, but not in vitamin K-treated cells. F ully glycosylated rFII is also selectively retained and degraded in warfari n-treated H-35 cells. When rFII and hFII were transiently expressed in tuni camycin-treated HEK293 cells, rFII but not hFII was degraded to generate a 48-KD species. This degradation was independent of gamma-carboxylation, ind icating that the sensitivity of aglyco-rFII and aglyco-hFII toward this spe cific proteolysis differs in HEK293 cells, By expressing chimeric rFII/hFII constructs in tunicamycin-treated HEK293 cells, it was shown that the krin gle 2 structure of prothrombin was responsible for this difference. The 48- KD species was not observed in tunicamycin-treated H-35 cells, suggesting t hat this specific proteolytic processing of aglyco-rFII is also cell-type s pecific. Expression of rFII in other tunicamycin-treated nonhepatic cells s uggests that this cell-specific difference in processing might be determine d by a difference between hepatic and nonhepatic cells. The site-directed m utagenesis utilized in these studies also establishes that the N-glycosylat ion sites of rFII are at residues Asn77, 101, 378, and 518. (C) 1999 Elsevi er Science Ltd. All rights reserved.