Study Of Biological Function Of MRPINK (Male Reproduction-related Peptidase Inhibitor Kazal-type) In Macrobrachium Rosenbergii

Kazal-type protease inhibitors(KPIs)belong to the family of serine protease inhibitors and are extremely widespread.They are involved in regulating some physiological events,such as immune response,blood coagulation,development regulation and fertilization regulation.A number of Kazal-type protease inhibitors have been purified and identified.The typical Kazal-type domain is consisted of 50-60 amino acid residues with six conserved cysteines,which form three intra-domain disulfide bridges(1-5/2-4/3-6).The amino acid residue located at the second position in the C-terminal direction after the second cysteine residue is designated as P₁ residue.The segment forming a convex,extended,solvent-exposed loop on the surface of an inhibitor is called protease-binding loop.A general conclusion has emerged that the specificity of inhibition depends mainly on the P₁ amino acid residue and binding loop.Kazal-type inhibitors react with cognate enzymes according to a substrate-like standard mechanism.In the stable complex,the P₁ amino acid residue of inhibitor binds to the S₁ cavity of the cognate enzyme and the loop has a flat shape that fits into the active-site cleft of enzyme.In the previous study,a male reproduction-related Kazal-type protease inhibitor gene was identified and characterized from the prawn,Macrobrachium rosenbergii (MRPINK).The gene contained a 405 bp open reading frame(ORF)and the conceptually translated protein consisted of a 21 amino acid signal peptide and a 113 amino acid mature polypeptide with two Kazal-type domains.Significant accumulation of the mRNA was only observed in the male reproductive tract.In situ hybridization demonstrated the presence of the mRNA in the secretory epithelial cells of vas deferens and terminal ampullae.In this study,recombinant MRPINK and several mutants of it were prepared.The inhibitory activities of recombinant proteins against three serine protease were tested, respectively.The inhibitory mechanism and the effects of P₁ amino acid residue on the inhibitory activity of MRPINK were then assayed.Meanwhile,in order to clarify the physiological function of MRPINK,we have further studied the inhibitory effect of it on the prawn sperm.The results of our studies as follows:1.Recombinant MRPINK and all the mutants(MRPINK^L37K,MRPINK^L37A, MRPINK^L37G,MRPINK^P88I,domain1 and domain2)were successfully expressed in E. coli BL21(DE3).rMRPINK,rMRPINK^L37K,rMRPINK^L37A,rMRPINK^L37Gand rMRPINK^P88Iwere purified under denatured conditions,while domainl and domain2 were purified under native conditions.2.Recombinant MRPINK was found to specifically inhibit chymotrypsin,but no inhibition was detected against trypsin or thrombin.By the analysis of kinetic tests, the inhibition mechanism of MRPINK was determined to be typical competitive model with K_i of 354 nM.The inhibitory activities of mutants(domain1,domain2, rMRPINK^P88I,rMRPINK^L37K,rMRPINK^L37A,and rMRPINK^L37G)were then assayed. The results showed that domain2 was the key contributor to the inhibition of chymotrypsin(K_i of 416 nM)and P₁ Pro was crucial for the activity.Nevertheless, whether the P₁ amino acid residue was Leu,or even it was replaced by Lys,Ala or Gly, domain1 was ineffective to the activity.3.The proteolytic activity of sperm extracts to vitelline coat components was detected to be interfered by MRPINK and MRPINK was also discovered to have an inhibitory effect on the gelatinolytic activity of M.rosenbergii sperm. Immunofluorescence analysis revealed MRPINK bound specifically onto the base of sperm.4.A novel gelatinase on sperm was found to be specifically inhibited by MRPINK and was named M.rosenbergii sperm gelatinase(MSG).MSG was then isolated and purified by HPLC(reversed-phase high performance liquid chromatography)combining with gelatinolytic assay.By amino-terminal amino acid sequence analysis and molecular cloning,the primary structure of MSG was determined.Northern blot analysis indicated MSG is male reproductive tract specific. In situ hybridization demonstrated the presence of the mRNA in the secretory epithelial cells of vas deferens and terminal ampullae.Our results demonstrated that MRPINK competitively inhibited the activity of chymotrypsin,and domain2 was the key contributor to the inhibition,P₁ Pro was crucial for the activity.Meanwhile,a novel gelatinase on sperm(MSG)was found to be specifically inhibited by MRPINK.We assumed that MRPINK might be involved into the fertilization of M.rosenbergii.