Abstract
Lysosomal dipeptidase catalyzes the hydrolysis of dipeptides with unsubstituted terminals. It is a homodimer and binds zinc. Dimerization is an important issue in understanding the enzyme's function. In this study, we investigated the influence of the propeptide on the folding and dimerization of recombinant lysosomal dipeptidase. For this purpose, we separately cloned and overexpressed the mature protein and the proenzyme. The overexpressed proteins were localized exclusively to insoluble inclusion bodies. Refolding of the urea-solubilized inclusion bodies showed that only dipeptidase lacking the propeptide was dimeric. The soluble renatured proenzyme was a monomer, although circular dichroism and fluorescence spectra of the proenzyme indicated the formation of secondary and tertiary structure. The propeptide thus controls dimerization, as well as activation, of lysosomal dipeptidase.
References
Baker, D., Shiau, A.K., and Agard, D.A. (1993). The role of pro regions in protein folding. Curr. Opin. Cell Biol.5, 966–970.10.1016/0955-0674(93)90078-5Search in Google Scholar
Bendetowicz, A.V., Morris, J.A., Wise, R.J., Gilbert, G.E., and Kaufman, R.J. (1998). Binding of factor VIII to von Willebrand factor is enabled by cleavage of the von Willebrand factor propeptide. Blood92, 529–538.10.1182/blood.V92.2.529Search in Google Scholar
Burton, S.J., Quirk, A.V., and Wood, P.C. (1989). Refolding human serum albumin at relatively high protein concentration. Eur. J. Biochem.17, 379–387.10.1111/j.1432-1033.1989.tb14564.xSearch in Google Scholar
Chatterjee, A., Mridula, P., Mishra, R.K., Mittal, R., and Hosur, R.V. (2005). Folding regulates autoprocessing of HIV-1 protease precursor. J. Biol. Chem.280, 11369–11378.10.1074/jbc.M412603200Search in Google Scholar
Clark, E.D.B. (1998). Refolding of recombinant proteins. Curr. Opin. Biotechnol.9, 157–163.10.1016/S0958-1669(98)80109-2Search in Google Scholar
Davis, M.I., Bennett, M.J., Thomas, L.M., and Bjorkman, P.J. (2005). Crystal structure of prostate-specific membrane antigen, a tumor marker and peptidase. Proc. Natl. Acad. Sci. USA102, 5981–5986.10.1073/pnas.0502101102Search in Google Scholar PubMed PubMed Central
Della Fazia, M.A., Piobbico, D., Bartoli, D., Castelli, M., Brancorsini, S., Magni, M.V., and Servillo, G. (2002). lal-1: a differentially expressed novel gene during proliferation in liver regeneration and in hepatoma cells. Genes Cells7, 1183–1190.10.1046/j.1365-2443.2002.00593.xSearch in Google Scholar PubMed
Dolenc, I. and Mihelič, M. (2003). Purification and primary structure determination of human lysosomal dipeptidase. Biol. Chem.384, 317–320.10.1515/BC.2003.036Search in Google Scholar PubMed
Dunn, A.D., Myers, H.E., and Dunn, J.T. (1996). The combined action of two thyroidal proteases releases T4 from the dominant hormone-forming site of thyroglobulin. Endocrinology137, 3279–3285.10.1210/endo.137.8.8754751Search in Google Scholar PubMed
Fortenberry, S.C. and Chirgwin, J.M. (1995). The propeptide is nonessential for the expression of human cathepsin D. J. Biol. Chem.270, 9778–9782.10.1074/jbc.270.17.9778Search in Google Scholar PubMed
Gingras, R., Richard, C., El-Alfy, M., Morales, C.R., Potier, M., and Pshezhetsky, A.V. (1999). Purification, cDNA cloning, and expression of a new human blood plasma glutamate carboxypeptidase homologous to N-acetyl-aspartyl-α-glutamate carboxypeptidase/prostate-specific membrane antigen. J. Biol. Chem.274, 11742–11750.10.1074/jbc.274.17.11742Search in Google Scholar
Hed, J. and Stendahl, O. (1982) Differences in the ingestion mechanisms of IgG and C3b particles in phagocytosis by neutrophils. Immunology45, 727–736.Search in Google Scholar
Imamoto, Y., Tamura, C., Kamikubo, H., and Kataoka, M. (2003). Concentration-dependent tetramerization of bovine visual arrestin. Biophys. J.85, 1186–1195.10.1016/S0006-3495(03)74554-8Search in Google Scholar
Katoh, S., Murata, K., Kubota, Y., Kumeta, H., Ogura, K., Inagaki, F., Asayama, M., and Katoh, E. (2005). Refolding and purification of recombinant OsNifU1A domain II that was expressed by Escherichia coli. Protein Expr. Purif.43, 149–156.10.1016/j.pep.2005.04.019Search in Google Scholar
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227, 680–685.10.1038/227680a0Search in Google Scholar
Lawrence, C.M. (1999). Crystal structure of the ectodomain of human transferrin receptor. Science286, 779–782.10.1126/science.286.5440.779Search in Google Scholar
Louis, J.M., Clore, G.M., and Gronenborn, A.M. (1999). Autoprocessing of HIV-1 protease is tightly coupled to protein folding. Nat. Struct. Biol.6, 868–875.Search in Google Scholar
Mansfeld, J., Petermann, E., Dürrschmidt, P., and Ulbrich-Hofmann, R. (2005). The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus. Protein Expr. Purif.39, 219–228.10.1016/j.pep.2004.10.008Search in Google Scholar
McDonald, J.K. and Schwabe, C. (1977). Intracellular exopeptidases. In: Proteinases in Mammalian Cells and Tissue, A.J. Barrett, ed. (Amsterdam, The Netherlands: North-Holland Publishing), pp. 311–391 (see pp. 346–348).Search in Google Scholar
McDonald, J.K., Zeitman, B.B., Reilly, T.J., and Ellis, S. (1969). New observations on the substrate specificity of cathepsin C (dipeptidyl aminopeptidase I). Including the degradation of β-corticotropin and other peptide hormones. J. Biol. Chem.244, 2693–2709.10.1016/S0021-9258(18)83453-6Search in Google Scholar
McDonald, JK., Zeitman, B.B., and Ellis, S. (1972). Detection of a lysosomal carboxypeptidase and a lysosomal dipeptidase in highly-purified dipeptidyl aminopeptidase I (cathepsin C) and the elimination of their activities from preparations used to sequence peptides. Biochem. Biophys. Res. Commun.46, 62–70.10.1016/0006-291X(72)90630-4Search in Google Scholar
Price, N.C. and Stevens, E. (1983). The refolding of denatured rabbit muscle pyruvate kinase. Biochem. J.209, 763–770.10.1042/bj2090763Search in Google Scholar PubMed PubMed Central
Raffyet, S. Sassoon, N. Hofnung, M., and Betton, J.M. (1998). Tertiary structure-dependence of misfolding substitutions in loops of the maltose-binding protein. Protein Sci.7, 2136–2142.10.1002/pro.5560071010Search in Google Scholar PubMed PubMed Central
Rawlings, N.D. and Barrett, A.J. (1999). MEROPS: the peptidase database. Nucleic Acids Res.27, 325–331.10.1093/nar/27.1.325Search in Google Scholar
Soares, M.B., Bonaldo, M.D.F., Jelene, P., Su, L., Lawton, L., and Efstratiadis, A. (1994). Construction and characterization of a normalized cDNA library. Proc. Natl. Acad. Sci. USA91, 9228–9232.10.1073/pnas.91.20.9228Search in Google Scholar
Song, L. and Fricker, L.D. (1997). The pro region is not required for the expression or intracellular routing of carboxypeptidase E. Biochem. J.323, 265–271.10.1042/bj3230265Search in Google Scholar
Vincentelli, R., Canaan, S., Campanacci, V., Valencia, C., Maurin, D., Frassinetti, F., Scappucini-Calvo, L., Bourne, Y., Cambillau, C., and Bignon, C. (2004). High-throughput automated refolding screening of inclusion bodies. Protein Sci.13, 2782–2792.10.1110/ps.04806004Search in Google Scholar
Wicher, K.B., Abou-Hachem, M., Halldorsdottir, S., Thorbjarnadottir, S.H., Eggertsson, G., Hreggvidsson, G.O., Karlsson, E.N., and Holst, O. (2001). Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus. Appl. Microbiol. Biotechnol.55, 578–584.10.1007/s002530000559Search in Google Scholar
Yasuda, M., Murakami, Y., Sowa, A., Ogino, H., and Ishikawa, H. (1998). Effect of additives on refolding of a denatured protein. Biotechnol. Prog.14, 601–606.10.1021/bp9800438Search in Google Scholar
Zwickl, P. Lottspeich, F., and Baumeister, W. (1992). Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli. FEBS Lett.312, 157–160.10.1016/0014-5793(92)80925-7Search in Google Scholar
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