Kazal-type serine proteinase inhibitors from the black tiger shrimp Penaeus monodon and the inhibitory activities of SPIPm4 and 5

Serine proteinase inhibitors (SPIs) play important roles in physiological and immunological processes involving proteinases in all multicellular organisms. In black tiger shrimp Penaeus monodon, nine different Kazal-type SPIs, namely SPIPm1-9, were identified from the cDNA libraries of hemocyte, hepatopancreas, hematopoietic tissue, ovary and lymphoid organ. They are multi-domain SPIs containing 2–7 and possibly more Kazal domains. Two interesting cDNA clones, SPIPm4 and SPIPm5 coding for two-domain Kazal-type SPIs, were identified from the heat-treated hemocyte cDNA libraries. The SPIPm4 and SPIPm5 consist of open reading frames of 387 and 399 bp coding for polypeptides of 128 and 132 amino acids with putative signal peptides of 21 and 19 amino acid residues and mature SPIs of 107 and 113 amino acid residues, respectively. Recombinant expression in an Escherichia coli expression system yielded recombinant proteins, rSPIPm4 and rSPIPm5, with molecular masses of 12.862 and 13.433 kDa, respectively. The inhibitory activities of SPIPm4 and SPIPm5 were tested against trypsin, chymotrypsin, subtilisin and elastase. The SPIPm4 exhibited potent inhibitory activity against subtilisin and weakly against chymotrypsin whereas the SPIPm5 strongly inhibited subtilisin and elastase. The inhibition was a competitive type with inhibition constants (Ki) of 14.95 nM for SPIPm4 against subtilisin, 4.19 and 59.64 nM, respectively, for SPIPm5 against subtilisin and elastase. They had no bacteriostatic effect against Gram-positive bacteria: Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, and Gram-negative bacteria: Vibrio harveyi 639, E. coli JM109. Gene expression study revealed that the SPIPm5 gene was up-regulated in response to heat treatment suggesting the involvement of SPIs in stress responses.