PROMPT GLYCOSYLATION OF CALRETICULIN IS INDEPENDENT OF CA2+ HOMEOSTASIS

Selective glycosylation of ''prompt'' stress glycoproteins (P-SG), mai nly P-SG67 and P-SG64 (M(r) of 64,000, pl=5.1),occurs immediately duri ng acute heat-stress. In the present study, P-SG64 was purified by seq uential gel filtration, anion-exchange, affinity chromatography, and t wo-dimensional isoelectric focusing/ SOS-PAGE. Purified P-SG64 was fur ther characterized by microsequencing of a peptide fragment, PT-61, wh ich showed a 100% sequence homology with calreticulin, suggesting that PSG64 is identical to calreticulin. PT-Si also showed 55%, 58% and 63 % sequence homologies with calnexin, HIV-1 gp120 and HIV-2 envelope po lyprotein, respectively. Ca-45(2+) overlay studies confirmed Ca2+-bind ing of P-SG64. P-SG67 was also recently identified as calreticulin (8) , which suggests that CHO cells either have two isoforms of calreticul in or express variable states of calreticulin glycosylation during acu te heat stress. The role of intracellular Ca2+ ([Ca2+]]) during heat-i nduced ''promp'' glycosylation was also examined and indicated an 8-fo ld increase in [Ca2+](i). Chelation of this increased cytoplasmic Ca2 by BAPTA reduced glycosylation of P-SG67/P-SG64/calreticulin only by similar to 20%. This observation suggests that altered [Ca2+](i) homeo stasis is not directly linked to calreticulin glycosylation, instead, heat-induced calreticulin glycosylation is a Ca2+-independent effect. (C) 1994 Academic Press, Inc.