Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis

Abstract

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X₃-Ser-X₅-X₆-Cys-X₈-X₉-Ala-X₁₁-Thr-X₁₃, where X₃ and X₁₁ tend toward charged residues, X₅ tends toward threonine or serine, X₆ toward asparagine or aspartate, X₉ toward asparagine or lysine, and X₁₃ toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor.