A three-domain Kazal-type serine proteinase inhibitor exhibiting domain inhibitory and bacteriostatic activities from freshwater crayfish Procambarus clarkii.
In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this study, we report a Kazal-type serine proteinase inhibitor, named hcPcSPI1, from freshwater crayfish (Procambarus clarkii). We found that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099) or became decreased after a white spot syndrome virus (WSSV) challenge. In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not alpha-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPI1 was also found to possess bacteriostatic activity against the B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.
Li
Xin-Cang
XC
School of Life Sciences, Shandong University, Jinan, Shandong 250100, China.
Wang
Xian-Wei
XW
Wang
Zong-Heng
ZH
Zhao
Xiao-Fan
XF
Wang
Jin-Xing
JX
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D003400:Astacoidea / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000254:growth & development / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000254:growth & development / Q000378:metabolism*; D007113:Immunity, Innate; D007700:Kinetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D011485:Protein Binding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D029941:Fish Proteins; D015842:Serine Proteinase Inhibitors