Abstract
Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest kcat/Km value of 20×103M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 μM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 μM.
References
Ahsan, M.N. and Watabe, S. (2001). Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus. J. Protein Chem.20, 49–58.10.1023/A:1011005104727Search in Google Scholar
Akita, H., Matsuyama, T., Iso, H., Sugita, M., and Yoshida, S. (1997). Effects of oxidative stress on the expression of limbic-specific protease neuropsin and avoidance learning in mice. Brain Res.769, 86–96.10.1016/S0006-8993(97)00674-4Search in Google Scholar
Basu, A., Dillon, R.D., Taylor, R., Davison, J.M., and Marshall, S.M. (2004). Is normalisation of serum potassium and magnesium always necessary in Gitelman syndrome for a successful obstetric outcome? BJOG111, 630–634.10.1111/j.1471-0528.2004.00148.xSearch in Google Scholar
Bernett, M.J., Blaber, S.I., Scarisbrick, I.A., Dhanarajan, P., Thompson, S.M., and Blaber, M. (2002). Crystal structure and biochemical characterization of human kallikrein 6 reveals that a trypsin-like kallikrein is expressed in the central nervous system. J. Biol. Chem.277, 24562–24570.10.1074/jbc.M202392200Search in Google Scholar
Bode, W. and Schwager, P. (1975). The single calcium-binding site of crystalline bovine β-trypsin. FEBS Lett.56, 139–143.10.1016/0014-5793(75)80128-1Search in Google Scholar
Borgono, C.A. and Diamandis, E.P. (2004). The emerging roles of human tissue kallikreins in cancer. Nat. Rev. Cancer4, 876–890.10.1038/nrc1474Search in Google Scholar PubMed
Borgono, C.A., Grass, L., Soosaipillai, A., Yousef, G.M., Petraki, C.D., Howarth, D.H., Fracchioli, S., Katsaros, D., and Diamandis, E.P. (2003). Human kallikrein 14: a new potential biomarker for ovarian and breast cancer. Cancer Res.63, 9032–9041.Search in Google Scholar
Brattsand, M., Stefansson, K., Lundh, C., Haasum, Y., and Egelrud, T. (2005). A proteolytic cascade of kallikreins in the stratum corneum. J. Invest. Dermatol.124, 198–203.10.1111/j.0022-202X.2004.23547.xSearch in Google Scholar PubMed
Campbell, R.C. (1989). Statistics for Biologists, 3rd ed. (Cambridge, UK: Cambridge University Press).10.1017/CBO9781139168076Search in Google Scholar
Cane, S., Bignotti, E., Bellone, S., Palmieri, M., De las Casas, L., Roman, J.J., Pecorelli, S., Cannon, M.J., O'Brien, T., and Santin, A.D. (2004). The novel serine protease tumor-associated differentially expressed gene-14 (KLK8/Neuropsin/Ovasin) is highly overexpressed in cervical cancer. Am. J. Obstet. Gynecol.190, 60–66.10.1016/j.ajog.2003.07.020Search in Google Scholar PubMed
Chen, Z.L., Yoshida, S., Kato, K., Momota, Y., Suzuki, J., Tanaka, T., Ito, J., Nishino, H., Aimoto, S., Kiyama, H., et al. (1995). Expression and activity-dependent changes of a novel limbic-serine protease gene in the hippocampus. J. Neurosci.15, 5088–5097.10.1523/JNEUROSCI.15-07-05088.1995Search in Google Scholar
Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J., and Deperthes, D. (2002). Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology. Eur. J. Biochem.269, 2747–2754.10.1046/j.1432-1033.2002.02960.xSearch in Google Scholar
Cloutier, S.M., Kundig, C., Felber, L.M., Fattah, O.M., Chagas, J.R., Gygi, C.M., Jichlinski, P., Leisinger, H.J., and Deperthes, D. (2004). Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substrates. Eur. J. Biochem.271, 607–613.10.1111/j.1432-1033.2003.03963.xSearch in Google Scholar
Davies, B.J., Pickard, B.S., Steel, M., Morris, R.G., and Lathe, R. (1998). Serine proteases in rodent hippocampus. J. Biol. Chem.273, 23004–23011.10.1074/jbc.273.36.23004Search in Google Scholar
Deperthes, D., Chapdelaine, P., Tremblay, R.R., Brunet, C., Berton, J., Hebert, J., Lazure, C., and Dube, J.Y. (1995). Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma. Biochim. Biophys. Acta1245, 311–316.10.1016/0304-4165(95)00118-2Search in Google Scholar
Diamandis, E.P., Yousef, G.M., Clements, J., Ashworth, L.K., Yoshida, S., Egelrud, T., Nelson, P.S., Shiosaka, S., Little, S., Lilja, H., et al. (2000a). New nomenclature for the human tissue kallikrein gene family. Clin. Chem.46, 1855–1858.10.1093/clinchem/46.11.1855Search in Google Scholar
Diamandis, E.P., Yousef, G.M., Soosaipillai, A.R., Grass, L., Porter, A., Little, S., and Sotiropoulou, G. (2000b). Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications. Clin. Biochem.33, 369–375.10.1016/S0009-9120(00)00145-4Search in Google Scholar
Diamandis, E.P., Okui, A., Mitsui, S., Luo, L.Y., Soosaipillai, A., Grass, L., Nakamura, T., Howarth, D.J., and Yamaguchi, N. (2002). Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma. Cancer Res.62, 295–300.Search in Google Scholar
Egelrud, T. (1993). Purification and preliminary characterization of stratum corneum chymotryptic enzyme: a proteinase that may be involved in desquamation. J. Invest. Dermatol.101, 200–204.10.1111/1523-1747.ep12363804Search in Google Scholar
Elmoujahed, A., Gutman, N., Brillard, M., and Gauthier, F. (1990). Substrate specificity of two kallikrein family gene products isolated from the rat submaxillary gland. FEBS Lett.265, 137–140.10.1016/0014-5793(90)80903-VSearch in Google Scholar
Felber, L.M., Borgono, C.A., Cloutier, S.M., Kundig, C., Kishi, T., Ribeiro Chagas, J., Jichlinski, P., Gygi, C.M., Leisinger, H.J., Diamandis, E.P., and Deperthes, D. (2005). Enzymatic profiling of human kallikrein 14 using phage-display substrate technology. Biol. Chem.386, 291–298.10.1515/BC.2005.035Search in Google Scholar PubMed
Fiedler, F. and Leysath, G. (1979). Substrate specificity of porcine pancreatic kallikrein. Adv. Exp. Med. Biol.120A, 261–271.10.1007/978-1-4757-0926-1_26Search in Google Scholar PubMed
Franzke, C.W., Baici, A., Bartels, J., Christophers, E., and Wiedow, O. (1996). Antileukoprotease inhibits stratum corneum chymotryptic enzyme. Evidence for a regulative function in desquamation. J. Biol. Chem.271, 21886–21890.10.1074/jbc.271.36.21886Search in Google Scholar PubMed
Gomis-Ruth, F.X., Bayes, A., Sotiropoulou, G., Pampalakis, G., Tsetsenis, T., Villegas, V., Aviles, F.X., and Coll, M. (2002). The structure of human prokallikrein 6 reveals a novel activation mechanism for the kallikrein family. J. Biol. Chem.277, 27273–27281.10.1074/jbc.M201534200Search in Google Scholar PubMed
Hansson, L., Stromqvist, M., Backman, A., Wallbrandt, P., Carlstein, A., and Egelrud, T. (1994). Cloning, expression, and characterization of stratum corneum chymotryptic enzyme. A skin-specific human serine proteinase. J. Biol. Chem.269, 19420–19426.Search in Google Scholar
Huntington, J.A., Read, R.J., and Carrell, R.W. (2000). Structure of a serpin-protease complex shows inhibition by deformation. Nature407, 923–926.10.1038/35038119Search in Google Scholar PubMed
Jeohn, G.H., Serizawa, S., Iwamatsu, A., and Takahashi, K. (1995). Isolation and characterization of gastric trypsin from the microsomal fraction of porcine gastric antral mucosa. J. Biol. Chem.270, 14748–14755.10.1074/jbc.270.24.14748Search in Google Scholar PubMed
Kapadia, C., Chang, A., Sotiropoulou, G., Yousef, G.M., Grass, L., Soosaipillai, A., Xing, X., Howarth, D.H., and Diamandis, E.P. (2003). Human kallikrein 13: production and purification of recombinant protein and monoclonal and polyclonal antibodies, and development of a sensitive and specific immunofluorometric assay. Clin. Chem.49, 77–86.10.1373/49.1.77Search in Google Scholar PubMed
Kapadia, C., Yousef, G.M., Mellati, A.A., Magklara, A., Wasney, G.A., and Diamandis, E.P. (2004). Complex formation between human kallikrein 13 and serum protease inhibitors. Clin. Chim. Acta339, 157–167.10.1016/j.cccn.2003.10.009Search in Google Scholar PubMed
Kishi, T., Kato, M., Shimizu, T., Kato, K., Matsumoto, K., Yoshida, S., Shiosaka, S., and Hakoshima, T. (1999). Crystal structure of neuropsin, a hippocampal protease involved in kindling epileptogenesis. J. Biol. Chem.274, 4220–4224.10.1074/jbc.274.7.4220Search in Google Scholar PubMed
Kishi, T., Grass, L., Soosaipillai, A., Scorilas, A., Harbeck, N., Schmalfeldt, B., Dorn, J., Mysliwiec, M., Schmitt, M., and Diamandis, E.P. (2003a). Human kallikrein 8, a novel biomarker for ovarian carcinoma. Cancer Res.63, 2771–2774.Search in Google Scholar
Kishi, T., Grass, L., Soosaipillai, A., Shimizu-Okabe, C., and Diamandis, E.P. (2003b). Human kallikrein 8: immunoassay development and identification in tissue extracts and biological fluids. Clin. Chem.49, 87–96.10.1373/49.1.87Search in Google Scholar PubMed
Kishi, T., Soosaipillai, A., Grass, L., Little, S.P., Johnstone, E.M., and Diamandis, E.P. (2004). Development of an immunofluorometric assay and quantification of human kallikrein 7 in tissue extracts and biological fluids. Clin. Chem.50, 709–716.10.1373/clinchem.2003.029538Search in Google Scholar PubMed
Laxmikanthan, G., Blaber, S.I., Bernett, M.J., Scarisbrick, I.A., Juliano, M.A., and Blaber, M. (2005). 1.70 Å X-ray structure of human apo kallikrein 1: structural changes upon peptide inhibitor/substrate binding. Proteins58, 802–814.Search in Google Scholar
Lovgren, J., Airas, K., and Lilja, H. (1999). Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors. Eur. J. Biochem.262, 781–789.Search in Google Scholar
Luo, L.Y., Grass, L., Howarth, D.J., Thibault, P., Ong, H., and Diamandis, E.P. (2001). Immunofluorometric assay of human kallikrein 10 and its identification in biological fluids and tissues. Clin. Chem.47, 237–246.10.1093/clinchem/47.2.237Search in Google Scholar
Luo, L.Y., Shan, S.J., Elliott, M.B., Soosaipillai, A., and Diamandis, E.P. (2006). Purification and characterization of human kallikrein 11, a candidate prostate and ovarian cancer biomarker, from seminal plasma. Clin. Cancer Res.12, 742–750.10.1158/1078-0432.CCR-05-1696Search in Google Scholar
Maeda, S. (1989). Expression of foreign genes in insects using baculovirus vectors. Annu. Rev. Entomol.34, 351–372.10.1146/annurev.en.34.010189.002031Search in Google Scholar
Magklara, A., Mellati, A.A., Wasney, G.A., Little, S.P., Sotiropoulou, G., Becker, G.W., and Diamandis, E.P. (2003). Characterization of the enzymatic activity of human kallikrein 6: autoactivation, substrate specificity, and regulation by inhibitors. Biochem. Biophys. Res. Commun.307, 948–955.10.1016/S0006-291X(03)01271-3Search in Google Scholar
Michael, I.P., Sotiropoulou, G., Pampalakis, G., Magklara, A., Ghosh, M., Wasney, G., and Diamandis, E.P. (2005). Biochemical and enzymatic characterization of human kallikrein 5 (hK5), a novel serine protease potentially involved in cancer progression. J. Biol. Chem.280, 14628–14635.10.1074/jbc.M408132200Search in Google Scholar
Momota, Y., Yoshida, S., Ito, J., Shibata, M., Kato, K., Sakurai, K., Matsumoto, K., and Shiosaka, S. (1998). Blockade of neuropsin, a serine protease, ameliorates kindling epilepsy. Eur. J. Neurosci.10, 760–764.10.1046/j.1460-9568.1998.00068.xSearch in Google Scholar
Obiezu, C.V., Shan, S.J., Soosaipillai, A., Luo, L.Y., Grass, L., Sotiropoulou, G., Petraki, C.D., Papanastasiou, P.A., Levesque, M.A., and Diamandis, E.P. (2005). Human kallikrein 4: quantitative study in tissues and evidence for its secretion into biological fluids. Clin. Chem.51, 1432–1442.10.1373/clinchem.2005.049692Search in Google Scholar
Okabe, A., Momota, Y., Yoshida, S., Hirata, A., Ito, J., Nishino, H., and Shiosaka, S. (1996). Kindling induces neuropsin mRNA in the mouse brain. Brain Res.728, 116–120.10.1016/0006-8993(96)00473-8Search in Google Scholar
Rajapakse, S., Ogiwara, K., Takano, N., Moriyama, A., and Takahashi, T. (2005). Biochemical characterization of human kallikrein 8 and its possible involvement in the degradation of extracellular matrix proteins. FEBS Lett.579, 6879–6884.10.1016/j.febslet.2005.11.039Search in Google Scholar
Russin, D.J., Floyd, B.F., Toomey, T.P., Brady, A.H., and Awad, W.M. Jr. (1974). The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (pronase). Stabilization of the trypsin component by calcium and guanidine. J. Biol. Chem.249, 6144–6148.10.1016/S0021-9258(19)42232-1Search in Google Scholar
Shimizu, C., Yoshida, S., Shibata, M., Kato, K., Momota, Y., Matsumoto, K., Shiosaka, T., Midorikawa, R., Kamachi, T., Kawabe, A., and Shiosaka, S. (1998). Characterization of recombinant and brain neuropsin, a plasticity-related serine protease. J. Biol. Chem.273, 11189–11196.10.1074/jbc.273.18.11189Search in Google Scholar PubMed
Shimizu-Okabe, C., Yousef, G.M., Diamandis, E.P., Yoshida, S., Shiosaka, S., and Fahnestock, M. (2001). Expression of the kallikrein gene family in normal and Alzheimer's disease brain. Neuroreport12, 2747–2751.10.1097/00001756-200108280-00031Search in Google Scholar
Stamey, T.A., Yang, N., Hay, A.R., McNeal, J.E., Freiha, F.S., and Redwine, E. (1987). Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N. Engl. J. Med.317, 909–916.10.1056/NEJM198710083171501Search in Google Scholar
Takayama, T.K., McMullen, B.A., Nelson, P.S., Matsumura, M., and Fujikawa, K. (2001). Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase. Biochemistry40, 15341–15348.10.1021/bi015775eSearch in Google Scholar
Umezawa, H. (1976). Structures and activities of protease inhibitors of microbial origin. Methods Enzymol.45, 678–695.10.1016/S0076-6879(76)45058-9Search in Google Scholar
Underwood, L.J., Tanimoto, H., Wang, Y., Shigemasa, K., Parmley, T.H., and O'Brien, T.J. (1999). Cloning of tumor-associated differentially expressed gene-14, a novel serine protease overexpressed by ovarian carcinoma. Cancer Res.59, 4435–4439.Search in Google Scholar
Valaitis, A.P. (1995). Gypsy moth midgut proteinases: purification and characterization of luminal trypsin, elastase and the brush border membrane leucine aminopeptidase. Insect Biochem. Mol. Biol.25, 139–149.10.1016/0965-1748(94)00033-ESearch in Google Scholar
Villoutreix, B.O., Getzoff, E.D., and Griffin, J.H. (1994). A structural model for the prostate disease marker, human prostate-specific antigen. Protein Sci.3, 2033–2044.10.1002/pro.5560031116Search in Google Scholar
Walt, H., Hornung, R., Fink, D., Dobler-Girdziunaite, D., Stallmach, T., Spycher, M.A., Maly, F., Haller, U., and Burki, N. (2001). Hypercalcemic-type of small cell carcinoma of the ovary: characterization of a new tumor line. Anticancer Res.21, 3253–3259.Search in Google Scholar
Watt, K.W., Lee, P.J., M'Timkulu, T., Chan, W.P., and Loor, R. (1986). Human prostate-specific antigen: structural and functional similarity with serine proteases. Proc. Natl. Acad. Sci. USA83, 3166–3170.10.1073/pnas.83.10.3166Search in Google Scholar
Yoshida, S., Taniguchi, M., Hirata, A., and Shiosaka, S. (1998). Sequence analysis and expression of human neuropsin cDNA and gene. Gene213, 9–16.10.1016/S0378-1119(98)00232-7Search in Google Scholar
Yousef, G.M. and Diamandis, E.P. (2001). The new human tissue kallikrein gene family: structure, function, and association to disease. Endocr. Rev.22, 184–204.Search in Google Scholar
Yousef, G.M. and Diamandis, E.P. (2002). Human tissue kallikreins: a new enzymatic cascade pathway?Biol. Chem.383, 1045–1057Search in Google Scholar
Yousef, G.M., Polymeris, M.E., Grass, L., Soosaipillai, A., Chan, P.C., Scorilas, A., Borgono, C., Harbeck, N., Schmalfeldt, B., Dorn, J., et al. (2003). Human kallikrein 5: a potential novel serum biomarker for breast and ovarian cancer. Cancer Res.63, 3958–3965.Search in Google Scholar
Yousef, G.M., Borgono, C.A., Popalis, C., Yacoub, G.M., Polymeris, M.E., Soosaipillai, A., and Diamandis, E.P. (2004a). In silico analysis of kallikrein gene expression in pancreatic and colon cancers. Anticancer Res.24, 43–51.Search in Google Scholar
Yousef, G.M., Yacoub, G.M., Polymeris, M.E., Popalis, C., Soosaipillai, A., and Diamandis, E.P. (2004b). Kallikrein gene downregulation in breast cancer. Br. J. Cancer90, 167–172.10.1038/sj.bjc.6601451Search in Google Scholar PubMed PubMed Central
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