Prompt glycosylation of calreticulin is independent of Ca2+ homeostasis.
Selective glycosylation of "prompt" stress glycoproteins (P-SG), mainly P-SG67 and P-SG64 (M(r) of 64,000, pI = 5.1), occurs immediately during acute heat-stress. In the present study, P-SG64 was purified by sequential gel filtration, anion-exchange, affinity chromatography, and two-dimensional isoelectric focusing/SDS-PAGE. Purified P-SG64 was further characterized by microsequencing of a peptide fragment, PT-61, which showed a 100% sequence homology with calreticulin, suggesting that P-SG64 is identical to calreticulin. PT-61 also showed 55%, 58% and 63% sequence homologies with calnexin, HIV-1 gp120 and HIV-2 envelope polyprotein, respectively. 45Ca2+ overlay studies confirmed Ca(2+)-binding of P-SG64. P-SG67 was also recently identified as calreticulin (8), which suggests that CHO cells either have two isoforms of calreticulin or express variable states of calreticulin glycosylation during acute heat stress. The role of intracellular Ca2+ ([Ca2+]i) during heat-induced "prompt" glycosylation was also examined and indicated an 8-fold increase in [Ca2+]i. Chelation of this increased cytoplasmic Ca2+ by BAPTA reduced glycosylation of P-SG67/P-SG64/calreticulin only by approximately 20%. This observation suggests that altered [Ca2+]i homeostasis is not directly linked to calreticulin glycosylation, instead, heat-induced calreticulin glycosylation is a Ca(2+)-independent effect.
Jethmalani
S M
SM
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205.
Henle
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037282:Calreticulin; D006224:Cricetinae; D006031:Glycosylation; D006706:Homeostasis; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits; D012261:Ribonucleoproteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D001324:Autoantigens; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D012261:Ribonucleoproteins; D002118:Calcium