Molecular Cloning of Horse Hsp90 cDNA and Its Comparative Analysis with Other Vertebrate Hsp90 Sequences

Abstract

Heat shock protein 90 (Hsp90), a molecular chaperone, is ubiquitous and involved in numerous cellular processes. To contribute to the relatively small collection of vertebrate Hsp90 sequences in the gene data bank, we cloned and sequenced horse (Equus caballus) Hsp90 a and β cDNAs. This enabled identification of horse-specific primers for development of a convenient PCR-based method that could monitor horse stress tolerance. We analyzed the sequence data comparatively and phylogenetically with other Hsp90 cDNA sequences, and identified vertebrate-specific and isoform-specific conserved regions to facilitate future molecular investigations of Hsp90 functions. We found 4 highly conserved regions to vertebrate Hsp90 exclusively and 27 amino acids conserved among but differing between Hsp90 a and Hsp90 β sequences. Protein-based phylogenetic trees revealed high conservation between mammal species within Hsp90 a and β clusters. Comparison of nucleotide and amino acid substitution levels suggests that horse Hsp90 β has undergone strong purifying selection, while rat Hsp90 β and hamster Hsp90 a have been positively selected. Surprisingly, fish Hsp90 a genes clearly clustered with Hsp90 β genes, and no distinct placement of fish Hsp90 a protein was found. The Hsp90 a isoform is apparently the result of β gene duplication. Our results highlight the importance of organism-and isoform-specific Hsp90 functional analyses in describing the role of Hsp90 in cells.