Kazal-type serine proteinase inhibitors from the black tiger shrimp Penaeus monodon and the inhibitory activities of SPI Pm4 and 5

Abstract

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 SPI Pm1-9, 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, SPI Pm4 and SPI Pm5 coding for two-domain Kazal-type SPIs, were identified from the heat-treated hemocyte cDNA libraries. The SPI Pm4 and SPI Pm5 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, rSPI Pm4 and rSPI Pm5, with molecular masses of 12.862 and 13.433 kDa, respectively. The inhibitory activities of SPI Pm4 and SPI Pm5 were tested against trypsin, chymotrypsin, subtilisin and elastase. The SPI Pm4 exhibited potent inhibitory activity against subtilisin and weakly against chymotrypsin whereas the SPI Pm5 strongly inhibited subtilisin and elastase. The inhibition was a competitive type with inhibition constants ( K i) of 14.95 nM for SPI Pm4 against subtilisin, 4.19 and 59.64 nM, respectively, for SPI Pm5 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 SPI Pm5 gene was up-regulated in response to heat treatment suggesting the involvement of SPIs in stress responses.