Characterization of the expression and function of Rana catesbeiana HSP30 and Xenopus laevis HSP27

ii I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. Abstract Exposure of cells to environmental or chemical stressors will initiate the heat shock response, which is mediated by heat shock proteins. Heat shock proteins are molecular chaperones which are classified by size into six main families: HSP100, HSP90, HSP70, HSP60, HSP40 and the small heat shock proteins (sHsps). The sHsp family members bind to denatured proteins and maintain them in a folding competent state such that they may be refolded by other molecular chaperones. The present study examined the expression and function of two amphibian sHsps, namely, Rana catesbeiana HSP30 and Xenopus laevis HSP27. Initially, an antisense riboprobe was produced to study the mRNA accumulation of Rana hsp30 in cultured tongue fibroblast (FT) cells. Results showed that Rana hsp30 mRNA was optimally induced when maintained at 35 o C for 2 h. An antibody to the recombinant Rana HSP30 protein was also produced in order to study HSP30 protein accumulation in Rana FT cells. Analysis showed that Rana HSP30 was heat-inducible and accumulated maximally at 4 h when maintained at 35 o C and then allowed to recover at 22 o C for 2 h. Immunocytochemical analysis indicated that Rana HSP30 protein was present primarily in the nucleus, with diffuse localization in the cytoplasm. Additional immunocytochemical analysis showed that Rana HSP30 remained in the nucleus following heat stress and extended periods of recovery. The molecular chaperone function of Rana HSP30 was also studied. Recombinant Rana HSP30 was found to inhibit the heat induced aggregation of various target proteins including citrate synthase, luciferase and malate dehydrogenase. Also, no major difference iv was detected between Rana HSP30 and Xenopus HSP30C in the inhibition of heat-induced aggregation of target proteins. This study also examined the expression and function of Xenopus laevis HSP27. Analysis of the putative amino acid sequence of the Xenopus hsp27 cDNA revealed that it had an identity of 71% with chicken, 65% with zebrafish, 63% with human and 53% with topminnow. Most of the identity was located within the α-crystallin domain of the protein. Interestingly, Xenopus HSP27 shared only a 19% identity with 2 other Xenopus sHsps, HSP30C and HSP30D. Western blot analysis using an anti-Xenopus …