Prompt protein glycosylation during acute heat stress.
Constitutive patterns of protein synthesis and protein glycosylation are severely disrupted by acute heat stress. Stressed cells respond by preferential synthesis of specific proteins, e.g., the well-known family of heat shock proteins. We observed another response that rapidly occurs during heating periods as short as 10 min at 45 degrees C. During that period, CHO cells began to glycosylate specific proteins, designated as "prompt" stress glycoproteins (P-SG), while constitutive protein glycosylation ceased. Labeling of P-SGs showed a dose response with time and with temperature and appeared regardless of the label used (D-[3H]mannose or D-[3H]glucose). On SDS-PAGE, the major P-SG was characterized by M(r) approximately 67 kDa (P-SG67) and pI = 5.1. Other less prominent P-SGs appeared at M(r) 160, 100, 64, 60, and 47 kDa; incorporated label showed little turnover during 24 h at 37 degrees C. Prompt glycosylation was inhibited by tunicamycin, and label incorporated into P-SGs was sensitive to N-glycosidase F, but not to O-glycosidase. Analysis of enzymatically digested P-SG67 indicated that label had been incorporated into both high-mannose (Man9GlcNAc) and complex-type oligosaccharides. Brefeldin A did not eliminate P-SG67 labeling, but caused the further appearance of novel, Brefeldin-associated P-SGs. Labeling of P-SG67 oligosaccharides occurred without significant concomitant protein synthesis, suggesting that addition of labeled oligosaccharides largely occurred on mature, rather than nascent proteins. The functional significance of prompt glycosylation remains to be defined, but we propose that this novel phenomenon is an integral part of the cellular heat stress response.
Henle
K J
KJ
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205-5484.
Kaushal
G P
GP
Nagle
W A
WA
Nolen
G T
GT
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D020126:Brefeldin A; D016466:CHO Cells; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation; D006360:Heat-Shock Proteins / Q000378:metabolism; D006358:Hot Temperature / Q000009:adverse effects*; D008358:Mannose / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D013312:Stress, Physiological; D013997:Time Factors; D014316:Tritium; D014415:Tunicamycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D003517:Cyclopentanes; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D009844:Oligosaccharides; D014316:Tritium; D014415:Tunicamycin; D020126:Brefeldin A; D005947:Glucose; D008358:Mannose