A novel function - thermal protective properties of an antifreeze protein from the summer desert beetle Microdera punctipennis.

ABSTRACT

It is well known that antifreeze proteins play an important role in protecting poikilothermic organisms from freezing. However, the transcripts of antifreeze protein genes, Mpafps, were observed in the desert beetle Microdera punctipennis in summer. The mRNA levels of Mpafps increased significantly after heat shock at 50°C, which implies that a novel function may exist in the antifreeze proteins from M. punctipennis. Southern blot analysis suggested the presence of multiple copies of the Mpafps family in the genome. Transcripts of two cDNAs encoding antifreeze proteins (Mpafps77 and Mpafps52) were isolated from beetles collected in the summer. The deduced amino acid sequences of the MpAFPs expressed in the summer are shorter by one 12-residue repeat and have significantly different C-terminal end sequences relative to the AFPs expressed in winter. Mpafps77 was constructed and expressed in Escherichia coli as a fusion protein, maltose-binding protein (MBP)-MpAFPS77. An in vitro heat protection assay was done by measuring the survival of bacteria and yeast that were exposed to 50 and 42°C, respectively and showed that the fusion protein significantly increased the thermal tolerance of these cells. It also increased the thermotolerance of the lactate dehydrogenase (LDH) enzyme at 65°C. These studies are the first biochemical demonstration of a thermal protective function for MpAFP and suggest some novel protective mechanisms may be present in M. punctipennis.