Molecular Biology Reports

, Volume 38, Issue 3, pp 1861–1868 | Cite as

Recombinant expression, characterization and expressional analysis of clam Meretrix meretrix cathepsin B, an enzyme involved in nutrient digestion

  • Xueliang Yao
  • Jiquan Zhang
  • Jinsheng Sun
  • Baozhong LiuEmail author


Cathepsin B is one of the most important proteolytic enzymes involved in the nutrient metabolism of clam Meretrix meretrix. The recombinant fusion protein GST-MmeCB (rGST-MmeCB) was obtained at a high level from Escherichia coli and identified using LC-ESI-MS/MS. The GST tag was cleaved from rGST-MmeCB, and the resulting recombinant MmeCB (rMmeCB) was able to degrade the selective substrate carbobenzoxy-l-arginyl-l-arginyl-7-amino-4-trifluoromethylcoumarin (Z-Arg-Arg-AFC) in vitro. The kinetic parameters of the rMmeCB were calculated as follows: K m, Vmax and k cat are 6.11 μM, 0.0174 μM min−1 and 277.57 s−1, respectively. Rabbit anti-rGST-MmeCB polyclonal antibodies was prepared and used to analyze the tissue distribution of MmeCB protein in M. meretrix. The results showed that the highest level of cathepsin B was found in the digestive gland and moderate levels were found in gill and mantle. Similar expression patterns were found at the mRNA level as detected by real time PCR. Further analysis showed that starvation caused a slight increase in MmeCB protein synthesis in the digestive gland, while refeeding after starvation caused an apparent increase in MmeCB synthesis in digestive gland, gill and mantle. Real time PCR analysis showed that MmeCB mRNA in digestive gland was significantly up-regulated by starvation and returned to normal level after the starved clams were refed. Together, these results indicated that cathepsin B is probably involved in the nutrient digestion of M. meretrix.


Cathepsin B Expression and purification Meretrix meretrix Nutrient digestion 



We thank Professor Li Sun for her advices and critical reading of the manuscript and also thank the reviewers for their suggestions in revising this work. This work was financially supported by NSFC (30871910), National Basic Research Program of China (2010CB126403) and the National High-Tech R&D Program (2006AA10A410).


  1. 1.
    Takio K, Towatari T, Katunuma N, Teller DC, Titani K (1983) Homology of amino acid sequences of liver cathepsin B and H with that of papain. Proc Natl Acad Sci USA 80:3666–3670CrossRefPubMedGoogle Scholar
  2. 2.
    Cygler M, Sivaraman J, Grochulski P, Coulombe R, Storer AC, Mort JS (1996) Structure of rat procathepsin B: model for inhibition of cysteine protease activity by proregion. Structure 4:405–416CrossRefPubMedGoogle Scholar
  3. 3.
    Stern I, Schaschke N, Moroder L, Turk D (2004) Crystal structure of NS-134 in complex with bovine cathepsin B: a two-headed epoxysuccinyl inhibitor extends along the entire active-site cleft. J Biochem 381:511–517CrossRefGoogle Scholar
  4. 4.
    Turk D, Podobnik M, Popovic T, Katunuma N, Bode W, Huber R, Turk V (1995) Crystal structure of cathepsin B inhibited with CA030 at 2 Å resolution: a basis for the design of specific epoxysuccinyl inhibitors. Biochemistry 34:4791–4797CrossRefPubMedGoogle Scholar
  5. 5.
    Chan VJ, McKerrow JH, Sakanari JA, Selzer PM (1999) Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease. J Biochem 340:113–117CrossRefGoogle Scholar
  6. 6.
    Hasnain S, Huber CP, Muir A, Rowan AD, Mort JS (1992) Investigation of structure function relationships in cathepsin B. Biol Chem Hoppe-Seyler 373:413–418PubMedGoogle Scholar
  7. 7.
    Hasnain S, Hirama T, Huber CP, Mason P, Mort JS (1993) Characterization of cathepsin B specificity by site-directed mutagenesis. Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization. J Biol Chem 268:235–240PubMedGoogle Scholar
  8. 8.
    Illy C, Quraishi O, Wang J, Purisima E, Vernet T, Mort JS (1997) Role of the occluding loop in cathepsin B activity. J Biol Chem 272:1197–1202CrossRefPubMedGoogle Scholar
  9. 9.
    Mishiro T, Nakano S, Takahara S, Miki M, Nakamura Y, Yasuoka S, Nikawa T, Yasui N (2004) Relationship between cathepsin B and thrombin in rheumatoid arthritis. J Rheumatol 31:1265–1273PubMedGoogle Scholar
  10. 10.
    Baici A, Hörler D, Lang A, Merlin C, Kissling R (1995) Cathepsin B in osteoarthritis: zonal variation of enzyme activity in human femoral head cartilage. Ann Rheum Dis 54:281–288CrossRefPubMedGoogle Scholar
  11. 11.
    Vasiljeva O, Papazoglou A, Krüger A, Brodoefel H, Korovin M, Deussing J, Augustin N, Nielsen BS, Almholt K, Bogyo M, Peters C, Reinheckel T (2006) Tumor cell-derived and macrophage-derived cathepsin B promotes progression and lung metastasis of mammary cancer. Cancer Res 66:5242–5250CrossRefPubMedGoogle Scholar
  12. 12.
    Aoki H, Ahsan MN, Watabe S (2003) Molecular cloning and characterization of cathepsin B from the hepatopancreas of northern shrimp Pandalus borealis. Comp Biochem Phys B 134:681–694CrossRefGoogle Scholar
  13. 13.
    Wang Y, Zhang S, Liu Z, Li H, Wang L (2004) Characterization and expression of AmphiCB encoding a cathepsin B proteinase from amphioxus Branchiostoma belcheri tsingtauense. Curr Sci 87:1717–1722Google Scholar
  14. 14.
    Cho WL, Tsao SM, Hays AR, Walter R, Chen JS, Snigirevskaya ES, Raikhel AS (1999) Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor. J Biol Chem 274:13311–13321CrossRefPubMedGoogle Scholar
  15. 15.
    Sajid M, McKerrow JH, Hansell E, Mathieu MA, Lucas KD, Hsieh I, Greenbaum D, Bogyo M, Salter JP, Lim KC, Franklin C, Kim JH, Caffrey CR (2003) Functional expression and characterization of Schistosoma mansoni cathepsin B and its transactivation by an endogenous asparaginyl endopeptidase. Mol Biochem Parasitol 131:65–75CrossRefPubMedGoogle Scholar
  16. 16.
    Xu YS, Kawasaki H (2001) Isolation and expression of cathepsin B cDNA in hemocytes during metamorphosis of Bombyx mori. Comp Biochem Phys B 130:393–399CrossRefGoogle Scholar
  17. 17.
    Liu B, Dong B, Tang B, Zhang T, Xiang J (2006) Effect of stocking density on growth, settlement and survival of clam larvae, Meretrix meretrix. Aquaculture 258:344–349CrossRefGoogle Scholar
  18. 18.
    Gricourt L, Mathieu M, Kellner K (2006) An insulin-like system involved in the control of Pacific oyster Crassostrea gigas reproduction: hrIGF-1 effect on germinal cell proliferation and maturation associated with expression of an homologous insulin receptor-related receptor. Aquaculture 25:85–98CrossRefGoogle Scholar
  19. 19.
    Grattarola M, Carloni M, Dondero F, Viarengo A, Vergani L (2006) Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20. Mol Biol Rep 33:265–272CrossRefPubMedGoogle Scholar
  20. 20.
    Tirape A, Bacque C, Brizard R, Vandenbulcke F, Boulo V (2007) Expression of immune-related genes in the oyster Crassostrea gigas during ontogenesis. Dev Comp Immunol 31:859–873CrossRefPubMedGoogle Scholar
  21. 21.
    Zhu L, Song L, Mao Y, Zhao J, Li C, Xu W (2008) A novel serine protease with clip domain from scallop Chlamys farreri. Mol Biol Rep 35:257–264CrossRefPubMedGoogle Scholar
  22. 22.
    Ladhar-Chaabouni R, Raja Mokdad-Gargouri R, Denis FO, Hamza-Chaffai A (2009) Cloning and characterization of cDNA probes for the analysis of metallothionein gene expression in the Mediterranean bivalves: Ruditapes decussatus and Cerastoderma glaucum. Mol Biol Rep 36:1007–1014CrossRefPubMedGoogle Scholar
  23. 23.
    Song J, Li L, Liu Z, Li Q, Ran P (2009) Sequence analysis and expression of a cDNA clone encoding tropomysin in Sinonovacula constricta. Mol Biol Rep 36:315–321CrossRefPubMedGoogle Scholar
  24. 24.
    Xu K, Kanno M, Yu H, Li Q, Kijima A (2010) Complete mitochondrial DNA sequence and phylogenetic analysis of Zhikong scallop Chlamys farreri (Bivalvia: Pectinidae). Mol Biol Rep. doi: 10.1007/s11033-010-9974-8
  25. 25.
    Wang X, Liu B, Wang G, Tang B, Xiang J (2008) Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix. Aquaculture 282:41–46CrossRefGoogle Scholar
  26. 26.
    Kuhelj R, Dolinar M, Pungercar J, Turk V (1995) The preparation of catalytically active human cathepsin B from its precursor expressed in Escherichia coli in the form of inclusion bodies. Eur J Biochem 229:533–539CrossRefPubMedGoogle Scholar
  27. 27.
    Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M (1996) Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two-dimensional gels. Proc Natl Acad Sci USA 93:14440–14445CrossRefPubMedGoogle Scholar
  28. 28.
    Barrett AJ, Kirschke H (1981) Cathepsin B, Cathepsin H, and Cathepsin L. Methods Enzymol 80:535–561CrossRefPubMedGoogle Scholar
  29. 29.
    Hu KJ, Leung PC (2004) Shrimp cathepsin L encoded by an intronless gene has predominant expression in hepatopancreas, and occurs in the nucleus of oocyte. Comp Biochem Phys B 137:21–33CrossRefGoogle Scholar
  30. 30.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  31. 31.
    Bos OG, Philippart CJM, van der Meer J (2007) Effects of temporary food limitation on development and mortality of Macoma balthica larvae. Mar Ecol Prog Ser 330:155–162CrossRefGoogle Scholar
  32. 32.
    Moran AL, Manahan DT (2004) Physiological recovery from prolonged ‘starvation’ in larvae of the Pacific oyster Crassostrea gigas. J Exp Mar Biol Ecol 306:17–36CrossRefGoogle Scholar
  33. 33.
    Pechenik JA, Hammer K, Weise C (1996) The effect of starvation on acquisition of competence and post-metamorphic performance in the marine prosobranch gastropod Crepidula fornicata (L.). J Exp Mar Biol Ecol 199:137–152CrossRefGoogle Scholar
  34. 34.
    Tang B, Liu B, Wang G, Zhang T, Xiang J (2006) Effects of various algal diets and starvation on larval growth and survival of Meretrix meretrix. Aquaculture 254:526–533CrossRefGoogle Scholar
  35. 35.
    Song X, Wang L, Song L, Zhao J, Zhang H, Zheng P, Qiu L, Liu XL, Wu L (2009) A cyclophilin A inducible expressed in gonad of Zhikong scallop Chlamys farreri. Mol Biol Rep 36:1637–1645CrossRefPubMedGoogle Scholar
  36. 36.
    Zhu L, Song L, Zhang H, Zhao J, Li C, Xu W (2008) Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops Argopecten irradians. Mol Biol Rep 35:125–132CrossRefPubMedGoogle Scholar
  37. 37.
    Cong M, Ni D, Song L, Wang L, Zhao J, Qiu L, Li L (2009) Molecular cloning, characterization and mRNA expression of peroxiredoxin in Zhikong scallop Chlamys farreri. Mol Biol Rep 36:1451–1459CrossRefPubMedGoogle Scholar
  38. 38.
    Zhu L, Song L, Xu W, Qian P (2009) Identification of a C-type lectin from the bay scallop Argopecten irradians. Mol Biol Rep 36:1167–1173CrossRefPubMedGoogle Scholar
  39. 39.
    Hegedus D, Grady MO, Chamankhah M, Baldwin D, Gleddie S, Braun L, Erlandson M (2002) Changes in cysteine protease activity and localization during midgut metamorphosis in the crucifer root maggot (Delia radicum). Insect Biochem Mol Biol 32:1585–1596CrossRefPubMedGoogle Scholar
  40. 40.
    Fox T, de Miguel E, Mort JS, Storer AC (1992) Potent slow-binding inhibition of cathepsin B by its propeptide. Biochemistry 31:12571–12576CrossRefPubMedGoogle Scholar
  41. 41.
    Laycock MV, MacKay RM, Fruscio MD, Gallant JW (1991) Molecular cloning of three cDNAs that encode cysteine proteinases in the digestive gland of the American lobster (Homarus americanus). FEBS Lett 292:115–120CrossRefPubMedGoogle Scholar
  42. 42.
    Manahan DT (1989) Amino acid fluxes to and from seawater in axenic veliger larvae of a bivalve (Crassostrea gigas). Mar Ecol Prog Ser 53:247–255CrossRefGoogle Scholar
  43. 43.
    Gomme J (2001) Transport of exogenous organic substances by invertebrate integuments: the field revisited. J Exp Zool 289:254–265CrossRefPubMedGoogle Scholar
  44. 44.
    Stephens GC (1988) Epidermal amino acid transport in marine invertebrates. Biochim Biophys Acta 947:113–138PubMedGoogle Scholar
  45. 45.
    Saleem MA, Wilkins RM, Mantle D, Shakoori AR (2003) Effect of starvation on proteases in insecticide-resistant and susceptible strains of Tribolium castaneum. Pak J Zool 35:197–204Google Scholar
  46. 46.
    Saleem MA, Wilkins RM, Mantle D, Sohail A, Shakoori AR (2003) Effect of starvation on proteases in insecticide-resistant and susceptible strains of Musca domestica. Pak J Zool 35:311–318Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Xueliang Yao
    • 1
    • 2
  • Jiquan Zhang
    • 2
  • Jinsheng Sun
    • 1
  • Baozhong Liu
    • 2
    Email author
  1. 1.Tianjin Normal UniversityTianjinChina
  2. 2.Institute of OceanologyChinese Academy of SciencesQingdaoChina

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