Cloning and expression of murine high molecular mass heat shock proteins, HSP105

Abstract

We have shown that the 105-kDa heat shock protein (HSP105) and the 42°C- specific heat shock protein (42°C-HSP) constitute high molecular mass heat shock proteins. To elucidate the structure of these heat shock proteins, we have screened a cDNA library constructed with poly(A)+ RNA derived from mouse FM3A cells preheated at 42 °C for 2 h using an antibody against murine HSP105. Two full-length cDNA clones were obtained: the pB105-1 insert encoded an 858-amino acid protein, and the pB105-2 insert encoded an 814-amino acid protein and lacked 44 amino acids found in pB105-1. The two clones contained the amino acid sequence found in the 17-kDa polypeptide fragments from HSP105 and 42 °C-HSP by lysylendopeptidase digestion. In vitro translation products of the RNA transcripts from pB105-1 and pB105-2 migrated to the same positions of HSP105 and 42 °C-HSP, respectively, on SDS-polyacrylamide gel electrophoresis. Northern blot analysis showed that the transcript was 4 kilobases in murine FM3A cells and was strongly induced by heat shock and by treatment with arsenite or an amino acid analog. By reverse transcription- polymerase chain reaction analysis using primers by which deletion of 132 nucleotides in pB105-2 could be detected, the polymerase chain reaction product corresponding to pB105-2 was increased only after heat shock at 42 °C, whereas the product corresponding to pB105-1 was induced by heat shock at either 42 or 45 °C and also by other stresses. Thus, the cDNA clones pB105-1 and pB105-2 encode HSP105 and 42 °C-HSP, respectively, and HSP105 and 42 °C-HSP (a short form of HSP105) are suggested to be produced by alternative splicing. Here, HSP105 and 42 °C-HSP are renamed HSP105α and HSP105β, respectively. A protein sequence homology search revealed that HSP105 shares 54, 34, and 25% amino acid identity with human HSP70RY, the sea urchin egg receptor for sperm, and murine inducible HSP70, respectively. Furthermore, by Northern blot analysis, HSP105 mRNA was revealed to be present in most murine tissues and to be highly expressed in the brain.