PARTIAL HOMOLOGY OF STRESS GLYCOPROTEIN GP62 WITH HSP70

Thermotolerance and heat resistance are often associated with elevated levels of heat shock proteins (HSPs) and a selective increase in prot ein glycosylation, In the present study, we have characterized heat st ress-induced protein glycosylation in M21 cells, derived from the rat fibroblast line, Rat-1. M21 cells are characterized by constitutive ov erexpression of human HSP70 gene and show increased heat resistance wi thout loss of its normal capacity for thermotolerance development afte r heat conditioning (Li et al., 1991, Proc. Natl. Acad. Sci. USA 88, 1 681-1685). The data presented here show that the elevated heat resista nce in these cells is associated not only with the constitutive overex pression of human HSP70, but also with increased glycosylation of a ma jor stress glycoprotein, GP62 (M-r of 62,000). We further purified GP6 2 by sequential preparative isoelectric focusing and two dimensional i soelectric focusing/SDS-polyacrylamide gel electrophoresis. The purifi ed protein was digested and partially characterized by microsequencing of two peptide fragments, comprising of 14-15 amino acids each. These fragments had a 100% sequence homology with HSP70 and a 71-100% seque nce homology with HSC70 from various species. Western blotting using b oth HSP70 and HSC70 antibodies showed positive reactivity of GP62 with HSP70. Affinity characterizations showed strong binding of GP62 to wh eat germ agglutinin and concanavalin A, consistent with the presence o f both alpha-D-mannosyl/glucosyl and N-acetyl-beta-D-glucosylaminyl/gl ucosamine oligomer residues in GP62. These data confirm the glycosylat ed status of GP62 and indicate that GP62 is a heat stress-induced glyc oprotein with partial homology to HSP70. (C) 1997 Academic Press.