A complex family of highly heterogeneous and internally repetitive hyperactive antifreeze proteins from the beetle Tenebrio molitor

We have previously identified a Thr- and Cys-rich thermal hysteresis (antif reeze) protein (THP) in the beetle Tenebrio molitor that has 10-100 times t he freezing point depression activity of fish antifreeze proteins. Because this 8.4 kDa protein is significantly different in its properties from THP preparations previously reported from this insect, a thorough search was un dertaken for other antifreeze types. Many active proteins were observed, bu t all appeared to be isoforms of the THP that differed in their number of 1 2-amino acid repeats (consensus sequence CTxSxxCxxAxT), amino acid substitu tions, and N-linked glycosylation, Mass spectral analysis has matched most of these isoforms with cDNA sequences of 17 different clones from a larval fat body library that encode eight different mature THPs containing 84, 96, or 120 amino acids. Genomic Southern blots suggest there may be 30-50 tigh tly linked copies of the gene, which is a signature consistently seen with unrelated fish antifreeze protein genes, and one that has been associated w ith the need to rapidly increase gene product in response to climate change . A three-dimensional model is proposed for the fully disulfide-bonded stru cture of T. molitor THP, which can accommodate addition or deletion of 12-a mino acid repeats. The structure is a beta-helix that places most of the Th r in a regular array on one side of the protein to form a putative ice-bind ing surface.