Molecular Cloning and Characterization of Anionic and Cationic Variants of Trypsin from Atlantic Salmon

• Published in: European Journal of Biochemistry
• Main Authors: Male, Rune, Lorens, James B., Smalås, Arne O., Torrissen, Krisna R.
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 1995
• Subjects: Atlantic salmon; Salmo salar
• Online Access: http://dx.doi.org/10.1111/j.1432-1033.1995.677zz.x; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1432-1033.1995.677zz.x; https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1995.677zz.x

Pancreatic cDNA libraries from Atlantic salmon ( Salmo salar ) were constructed and screened with salmon trypsin‐specific probes. Five clones containing near full‐length transcripts were selected for further characterization. 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 substrate‐binding pocket with aspartic acid at the bottom, and 12 cysteine residues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular mass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP‐I, SalTRP‐IA and SalTRP‐IB) possessed very similar sequences and may represent 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 trypsin clones (SalTRP‐II and SalTRP‐III) are probably encoded by separate gene loci. Analysis of genomic DNA by Southern blotting and hybridization to a trypsin probe showed a complex pattern, indicative of a large number of gene loci for trypsin in Atlantic salmon. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone represented a cationic variant. Multiple alignments of the Atlantic salmon trypsin sequences with trypsin, chymotrypsin and elastase from different species placed all Atlantic salmon sequences approximately equidistant from trypsins of other species. Interestingly, the distance between the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an early separation of these two types of trypsin, possibly prior to the derivation 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 ...