Heat shock-induced prompt glycosylation. Identification of P-SG67 as calreticulin.
Acute heat shock initiates the phenomenon of "prompt" glycosylation, which is characterized by selective glycosylation of specific cellular proteins called the prompt stress glycoproteins (P-SG). Prompt glycosylation rapidly occurs even during short heating periods, e.g. 10 min at 45 degrees C, and is not affected by the presence of cycloheximide (Henle, K. J., Kaushal, G. P., Nagle, W. A., and Nolen, G. T. (1993) Exp. Cell Res. 207, 245-251). The major P-SG in Chinese hamster ovary cells, P-SG67, was characterized by an M(r) of 67,000 and a pI = 5.1. In the present study, we purified P-SG67 by sequential gel filtration, anion exchange, affinity chromatography with concanavalin A-Sepharose, and two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis. The purified protein was digested and partially characterized by microsequencing of three major peptide fragments. The fragments, comprising a total of 46 amino acid residues, had an almost 100% sequence homology with calreticulin and partial homology with calnexin. Calcium binding studies with 45Ca2+ overlay confirmed that P-SG67 is a Ca(2+)-binding protein. These observations support the notion that P-SG67 is identical to calreticulin and that the glycosylation status of calreticulin can respond to environmental stress conditions.
Jethmalani
S M
SM
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205-5484.
Henle
K J
KJ
Kaushal
G P
GP
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D037282:Calreticulin; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013312:Stress, Physiological / Q000378:metabolism*; D013997:Time Factors
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D006023:Glycoproteins; D010446:Peptide Fragments; D012261:Ribonucleoproteins