MOLECULAR CLOTTING AND CHARACTERIZATION OF ANIONIC AND CATIONIC VARIANTS OF TRYPSIN FROM ATLANTIC SALMON

Pancreatic cDNA libraries from Atlantic salmon (Salmo salar) were cons tructed and screened with salmon trypsin-specific probes. Five clones containing near full-length transcripts were selected for further char acterization. Comparison of deduced amino acid sequences revealed that all variants possessed the canonical serine protease catalytic triad, consisting of histidine, aspartic acid and serine residues, a substra te-binding pocket with aspartic acid at the bottom, and 12 cysteine re sidues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular m ass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP-I, SalT RP-IA and SalTRP-IB) possessed very similar sequences and may represen t allelic variants encoded by the same gene locus; however, existence as tetraploid loci or isoloci where disomic inheritance is incomplete may also exist in Atlantic salmon and cannot be excluded. Two other tr ypsin clones (SalTRP-II and SalTRP-III) are probably en coded by separ ate gene loci. Analysis of genomic DNA by Southern blotting and hybrid ization to a trypsin probe showed a complex pattern, indicative of a l arge number of gene loci for trypsin in Atlantic salmon. The charged a mino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone rep resented a cationic variant. Multiple alignments of the Atlantic salmo n trypsin sequences with trypsin, chymotrypsin and elastase from diffe rent species placed all Atlantic salmon sequences approximately equidi stant from trypsins of other species. Interestingly, the distance betw een the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an e arly separation of these two types of trypsin, possibly prior to the d erivation of fish during evolution. A structural model based on X-ray diffraction studies of the salmon trypsin protein was very similar to that of the mammalian enzyme. All residues which differ in charge betw een anionic and cationic trypsins were located at exposed regions of t he proteins.