Identification and characterization of kazal-type serine proteinase inhibitors, SPIpm4 and SPIpm5, from haemocytes of black tiger shrimp penaeus monodon

Serine proteinase inhibitors (SPIs) play important roles in physiological and immunological processes involving proteinases in all multicellular organisms. In black tiger shrimp P. monodon, nine different Kazal-type SPIs, namely SPIPm1-9, were identified from the cDNA libraries. The semi-quantitative RT-PCR of seven Kazal-type SPI genes after V. harveyi 639 challenge showed that the expression of SPIPm1 was significantly increased for 1.92 fold at 6 hpi. Other SPI genes: SPIPm2, 6, 7 and 9 showed hardly any differences in mRNA expression level. RT-PCR study of the SPIPm5 gene revealed that it was up-regulated in response to heat treatment. 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 E. coli expression system yielded recombinant proteins, rSPIPm4 and rSPIPm5, with molecular masses of 12.862 and 13.433 kDa, respectively. The SPIPm4 inhibitor exhibited potent inhibitory activity against subtilisin and SPIPm5 against 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 B. subtilis, B. megaterium, S. aureus, V. harveyi 639 and E. coli JM109.