Thorpe, M.; Akula, S.; Fu, Z.; Hellman, L. The Extended Cleavage Specificity of Channel Catfish Granzyme-like II, A Highly Specific Elastase, Expressed by Natural Killer-like Cells. Int. J. Mol. Sci.2024, 25, 356.
Thorpe, M.; Akula, S.; Fu, Z.; Hellman, L. The Extended Cleavage Specificity of Channel Catfish Granzyme-like II, A Highly Specific Elastase, Expressed by Natural Killer-like Cells. Int. J. Mol. Sci. 2024, 25, 356.
Thorpe, M.; Akula, S.; Fu, Z.; Hellman, L. The Extended Cleavage Specificity of Channel Catfish Granzyme-like II, A Highly Specific Elastase, Expressed by Natural Killer-like Cells. Int. J. Mol. Sci.2024, 25, 356.
Thorpe, M.; Akula, S.; Fu, Z.; Hellman, L. The Extended Cleavage Specificity of Channel Catfish Granzyme-like II, A Highly Specific Elastase, Expressed by Natural Killer-like Cells. Int. J. Mol. Sci. 2024, 25, 356.
Abstract
The extended cleavage specificity of catfish granzyme-like II has been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated by using a panel of recombinant substrates. This serine protease, which has previously been isolated as cDNA from a catfish natural killer-like cell line showed a preference for alanine in the P1 position of the substrate, and for multiple basic amino acids N-terminally of the cleavage site. Using a set of recombinant substrates revealed the enzyme was most active on substrates with an alanine preceded by 3 or 4 basic amino acids in a row, but also showed activity against substrates with two basic amino acids, as well as with two basic amino acids separated by one amino acid just N-terminally of the Ala. The characteristics of this separating amino acid seemed less important, as many different amino acids were accepted in this position, except for proline. The amino acids following the basic amino acids and the P1 Ala residue also appeared to be of major importance for efficient cleavage. The most preferred P1 residue was Ala, and replacing Ala with Gly or Leu resulted in a clear reduction in the cleavage rate. A closely-related zebrafish serine protease (Zebrafish esterase-like) showed a very similar cleavage specificity, indicating an evolutionary conservation of this protease specificity among various fish species.
Two catfish serine proteases, originating from NK-like cells, have now been isolated and characterized. One of them is highly specific met-ase with similar characteristics as the mammalian granzyme M. This enzyme may be involved in the induction of apoptosis in virus infected cells, with a potential target in (catfish) caspase 6. In contrast to catfish granzyme -like I, the second enzyme analysed here, doesn’t seem to have a direct counterpart in mammalian NK-cells, and its role in the immune function of catfish NK cells is therefore still not known. However, this enzyme seems to be able to cleave a number of cytoskeletal proteins indicating a separate strategy to induce apoptosis in target cells. Both of these enzymes are very interesting targets for further studies of their roles in catfish immunity, as enzymes with similar specificities have been identified also in zebrafish.
Keywords
fish; serine protease; cleavage specificity; tryptase; NK cells; evolution
Subject
Biology and Life Sciences, Immunology and Microbiology
Copyright:
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