Protein glycosylation in rat fibroblast cells expressing deletion variants of the human hsp70 gene

Abstract

Elevated heat resistance is often associated with high cellular levels of hsp70. In the rat cell line, M21, increased heat resistance is associated with both the expression of an intact exogenous human hsp70 gene, and of an exogenous stress glycoprotein, GP62. In this study, we examined heat-stress induced alterations in protein glycosylation in two variant lines of M21 that contain specific deletions in the exogenous human hsp70 gene. The deletion mutant, MVHΔSm, lacks the nucleolar localization sequence for human hsp70 gene, whereas the other mutant, MVHΔBg, is characterized by deletion of the ATP binding domain in the human hsp70 gene. After heat induction, the MVHΔSm mutants exhibited constitutive and heat-induced endogenous hsp70 mRNA levels, protein glycosylation, as well as hsp70 and GP62 protein levels comparable to those in the parent cell line, Rat-1. Cellular heat sensitivity of MVHΔSm mutants was also similar to that of Rat-1 cells, although the mutant cells had a reduced capacity for thermotolerance development. On the other hand, MVHΔBg mutants before thermotolerance induction, exhibited constitutive endogenous and human exogenous hsp70 mRNA levels similar to those in MVHΔSm mutants. However, after thermotolerance induction, MVHΔBg mutants showed lower levels of heat-induced endogenous hsp70 mRNA than MVHΔSm mutants, an overall reduction in protein glycosylation, low hsp70 and GP62 levels, and increased heat sensitivity when compared to the parent Rat-1 cell line. Incorporation of D-[2-3H]mannose into oligosaccharide precursor pools and glycoproteins was consistent with protein glycosylation pattern for each cell line. Acute heat stress resulted in the selective glycosylation of the ‘prompt’ glycoprotein, P-SG64, in the two deletion mutants, similar to that in M21 cells expressing the intact human hsp70 gene. The data indicate that both protein glycosylation and hsp70 expression generally correlate with cellular heat resistance and thermotolerance expression. The presence of full or partially deleted copies of human hsp70 modulates thermotolerance and protein glycosylation in a complex manner that is not yet fully understood.