Skip to main content
SpringerLink
Log in

Inhibition Mechanism and the Effects of Structure on Activity of Male Reproduction-Related Peptidase Inhibitor Kazal-Type (MRPINK) of Macrobrachium rosenbergii

  • Original Article
  • Published:
Marine Biotechnology Aims and scope Submit manuscript

Abstract

In our previous reports, a Kazal family serine protease inhibitor, male reproduction-related peptidase inhibitor Kazal-type (MRPINK) has been identified from the prawn, Macrobrachium rosenbergii, and discovered having an inhibitory effect on the sperm gelatinolytic activity. MRPINK was predicated to inhibit chymotrypsin since it contains leucine and proline at P1 positions of the two domains, respectively. In this report, recombinant MRPINK was as expected 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. To elucidate the effects of structure on activity of MRPINK, the mutants (domain-1 only, domain-2 only, MRPINKP88I, MRPINKL37K, MRPINKL37A, and MRPINKL37G) were prepared and their inhibitory activities assayed. The results showed that domain-2 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 if it was replaced by Lys, Ala, or Gly, domain-1 was ineffective to the activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Asao T, Takahashi K, Tashiro M (1998) Interaction of second and third domains of Japanese quail ovomucoid with ten trypsins. BBA–Proteins Proteom 1387:415–421

    CAS  Google Scholar 

  • Bode W, Huber R (1992) Natural protein protease inhibitors and their interaction with proteases. Eur J Biochem 204:433–451

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quanitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Campos ITN, Silva MM, Azzolini SS, Souza AF, Sampaio CAM, Fritz H, Tanaka AS (2004) Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine protease specificities. Arch Biochem Biophys 425:87–94

    Article  PubMed  CAS  Google Scholar 

  • Cao JX, Dai JQ, Dai ZM, Yin GL, Yang WJ (2007) A male reproduction-related Kazal-type peptidase inhibitor gene in the prawn, Macrobrachium rosenbergii: molecular characterization and expression patterns. Mar Biotechnol 9:45–55

    Article  PubMed  CAS  Google Scholar 

  • Deraison C, Bonnart C, Lopez F, Besson C, Robinson R, Jayakumar A, Wagberg F, Brattsand M, Hachem JP, Leonardsson G, Hovnanian A (2007) LEKTI fragments specifically inhibit KLK5, KLK7, and KLK14 and control desquamation through a pH-dependent interaction. Mol Biol Cell 18:3607–3619

    Article  PubMed  CAS  Google Scholar 

  • Friedrich T, Kröger B, Bialojan S, Lemaire HG, Höffken HW, Reuschenbach P, Otte M, Dodt J (1993) A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus. J Biol Chem 268:16216–16222

    PubMed  CAS  Google Scholar 

  • Galliot B, Licina MM, Ghila L, Chera S (2007) RNAi gene silencing affects cell and developmental plasticity in hydra. C R Biologies 330:491–497

    Article  PubMed  CAS  Google Scholar 

  • Huang K, Lu W, Anderson S, Laskowski M Jr, James MNG (1995) Water molecules participate in proteinase-inhibitor interactions: crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B. Protein Sci 4:1985–1997

    Article  PubMed  CAS  Google Scholar 

  • Jarasrassamee B, Supungul P, Panyim S, Klinbunga S, Rimphanichayakit V, Tassanakajon A (2005) Recombinant expression and characterization of five-domain Kazal-type serine proteinase inhibitor of black tiger shrimp (Penaeus monodon). Mar Biotechnol 7:46–52

    Article  PubMed  CAS  Google Scholar 

  • Jayakumar A, Kang Y, Mitsudo K, Henderson Y, Frederick MJ, Wang M, El-Naggar AK, Marx UC, Briggs K, Clayman GL (2004) Expression of LEKTI domains 6–9¢ in the baculovirus expression system: recombinant LEKTI domains 6–9¢ inhibit trypsin and subtilisin A. Protein Expres Purif 35:93–101

    Article  CAS  Google Scholar 

  • Johansson MW, Keyser P, Söderhäll K (1994) Purification and cDNA cloning of a four-domain Kazal protease inhibitor from crayfish blood cells. Eur J Biochem 223:389–394

    Article  PubMed  CAS  Google Scholar 

  • Kanost MR (1999) Serine protease inhibitors in arthropod immunity. Dev Comp Immunol 23:291–301

    Article  PubMed  CAS  Google Scholar 

  • Kato I, Schrode J, Kohr WJ, Laskowski M Jr (1987) Chicken ovomuciod: determination of its amino acid sequence, determination of the trypsin reactive site, and preparation of all three of its domains. Biochemistry 26:193–201

    Article  PubMed  CAS  Google Scholar 

  • Krowarsch D, Cierpicki T, Jelen F, Otlewski J (2003) Canonical protein inhibitors of serine proteases. Cell Mol Life Sci 60:2427–2444

    Article  PubMed  CAS  Google Scholar 

  • Laskowski M Jr, Kato I (1980) Protein inhibitors of proteinases. Ann Rev Biochem 49:593–626

    Article  PubMed  CAS  Google Scholar 

  • Li Y, Ma WM, Dai JQ, Feng CZ, Yang F, Ohira T, Nagasawa H, Yang WJ (2008) Inhibition of a novel sperm gelatinase in prawn sperm by the male reproduction-related Kazal-type peptidase inhibitor. Mol Reprod Dev 75:1327–1337

    Article  PubMed  CAS  Google Scholar 

  • Lu SM, Lu W, Qasim MA, Anderson S, Apostol I, Ardelt W, Bigler T, Chiang YW, Cook J, James MNG, Kato I, Kelly C, Kohr W, Komiyama T, Lin TY, Ogawa M, Otlewski J, Park SJ, Qasim S, Ranjbar M, Tashiro M, Warne N, Whatley H, Wieczorek A, Wieczorek M, Wilusz, i T, Wynn R, Zhang W, Laskowski M Jr (2001) Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteases. Proc Natl Acad Sci 98:1410–1415

    Article  PubMed  CAS  Google Scholar 

  • Mägert HJ, Ständker L, Kreutzmann P, Zucht HD, Reinecke M, Sommerhoff CP, Fritz H, Forssmann WG (1999) LEKTI, a novel 15-domain type of human serine protease inhibitor. J Biol Chem 274:21499–21502

    Article  PubMed  Google Scholar 

  • Morris MT, Coppin A, Tomavo S, Carruthers VB (2002) Functional analysis of Toxoplasma gondii protease inhibitor 1. J Biol Chem 277:45259–45266

    Article  PubMed  CAS  Google Scholar 

  • Moser M, Auerswald E, Mentele R, Eckerskorn C, Fink IHFE (1998) Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech (Hirudo medicinalis)—primary structure, expression in yeast, and characterisation of native and recombinant inhibitor. Eur J Biochem 253:212–220

    Article  PubMed  CAS  Google Scholar 

  • Ødum L, Bundgaard JR, Johnsen AH (1999) A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis. Eur J Biochem 259:872–876

    Article  PubMed  Google Scholar 

  • Qasim MA, Ganz PJ, Saunders CW, Bateman KS, James MNG, Laskowski M Jr (1997) Interscaffolding additivity. Association of P1 variants of eglin c and of turkey ovomucoid third domain with serine proteinases. Biochemistry 36:1598–1607

    Article  PubMed  CAS  Google Scholar 

  • Rawings ND, Tolle DP, Barrett AJ (2004) Evolutionary families of peptidase inhibitors. Biochem J 378:705–716

    Article  Google Scholar 

  • Réhault SM, Zechmeister-Machhart M, Fortenberry YM, Malleier J, Binz NM, Cooper ST, Geiger M, Church FC (2005) Characterization of recombinant human protein C inhibitor expressed in Escherichia coli. BBA–Proteins Proteom 1748:57–65

    Article  CAS  Google Scholar 

  • Saxena I, Tayyab S (1997) Protein protease inhibitors from avian egg whites. Cell Mol Life Sci 53:13–23

    Article  PubMed  CAS  Google Scholar 

  • Schlott B, Wöhnert J, Icke C, Hartmann M, Ramachandran R, Gührs KH, Glusa E, Flemming J, Görlach M, Große F, Ohlenschläger O (2002) Interaction of Kazal-type inhibitor domains with serine proteases: Biochemical and structural studies. J Mol Biol 318:533–546

    Article  PubMed  CAS  Google Scholar 

  • Simonet G, Claeys I, Broeck JV (2002) Structural and functional properties of a novel serine protease inhibiting peptide family in arthropods. Comp Biochem Phys B 132:247–255

    Article  Google Scholar 

  • Somprasong N, Rimphanitchayakit V, Tassanakajon A (2006) A five-domain Kazal-type serine protease inhibitor from black tiger shrimp Penaeus monodon and its inhibitory activities. Dev Comp Immunol 30:998–1008

    Article  PubMed  CAS  Google Scholar 

  • Stubbs MT, Morenweiser R, Stürzebecheri J, Bauer M, Bode W, Huber Piechottka RGP, Matschiner G, Sommerhoff CP, Fritz H, Auerswald EA (1997) The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. J Biol Chem 272:19931–19937

    Article  PubMed  CAS  Google Scholar 

  • Tian MY, Kamoun S (2005) A two disulfide bridge Kazal domain from Phytophthora exhibits stable inhibitory activity against serine proteases of the subtilisin family. BMC Biochem 6:15

    Article  PubMed  CAS  Google Scholar 

  • Uhrin P, Dewerchin M, Hilpert M, Chrenek P, Schöfer C, Machhart MZ, Krönke G, Vales A, Carmeliet P, Binder BR, Geiger M (2000) Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility. J Clin Invest 106:1531–1539

    Article  PubMed  CAS  Google Scholar 

  • Vega FJ, Albores FV (2005) A four-Kazal domain protein in Litopenaeus vannamei hemocytes. Dev Comp Immunol 29:385–391

    Article  CAS  Google Scholar 

  • Yi Z, Vitek O, Qasim MA, Lu SM, Lu W, Ranjbar M, Li JT, Laskowski MC, Bailey-Kellogg C, Laskowski M Jr (2006) Functional evolution within a protein superfamily. Proteins 63:697–708

    Article  PubMed  CAS  Google Scholar 

  • Zaneveld LJD, Polakoski KL, Williams WL (1973) A protease and protease inhibitor of mammalian sperm acrosome. Biol Reprod 9:219–225

    PubMed  CAS  Google Scholar 

  • Zheng QL, Chen J, Nie ZM, Lv ZB, Wang D, Zhang YZ (2007) Expression, purification and characterization of a three-domain Kazal-type inhibitor from silkworm pupae (Bombyx mori). Comp Biochem Phys B 146:234–240

    Article  CAS  Google Scholar 

  • Zhu L, Song L, Chang Y, Xu W, Wu L (2006) Molecular cloning, characterization and expression of a novel serine protease inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819). Fish Shellfish Immunol 20:320–331

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Sciences Foundation of China (40730212) and the “863” program of China (2007AA09Z426).

Author information

Authors and Affiliations

  1. Institute of Cell Biology and Genetics, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, 310058, People’s Republic of China

    Ye Li, Ye-Qing Qian, Wen-Ming Ma & Wei-Jun Yang

  2. State Conservation Center for Gene Resources of Wildlife and the Key Laboratory of Conservation Genetics and Reproductive Biology for Wild Animals of the Ministry of Education, Hangzhou, Zhejiang, 310058, People’s Republic of China

    Ye Li & Wei-Jun Yang

Authors
  1. Ye Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ye-Qing Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wen-Ming Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wei-Jun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wei-Jun Yang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, Y., Qian, YQ., Ma, WM. et al. Inhibition Mechanism and the Effects of Structure on Activity of Male Reproduction-Related Peptidase Inhibitor Kazal-Type (MRPINK) of Macrobrachium rosenbergii . Mar Biotechnol 11, 252–259 (2009). https://doi.org/10.1007/s10126-008-9140-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10126-008-9140-7

Keywords

Search

Navigation