Abstract
Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris, two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 105- to 106-fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed.
References
Abraham, G.N. and Podell, D.N. (1981). Pyroglutamic acid. Non-metabolic formation, function in proteins and peptides, and characteristics of the enzymes effecting its removal. Mol. Cell. Biochem.38, 181–190.10.1007/978-94-009-8027-3_11Search in Google Scholar
Awade, A.C., Cleuziat, P., Gonzales, T., and Robert-Baudouy, J. (1994). Pyrrolidone carboxyl peptidase (Pcp): an enzyme that removes pyroglutamic acid (pGlu) from pGlu-peptides and pGlu-proteins. Proteins20, 34–51.10.1002/prot.340200106Search in Google Scholar
Azarkan, M., Wintjens, R., Looze, Y., and Baeyens-Volant, D. (2004). Detection of three wound-induced proteins in papaya latex. Phytochemistry65, 525–534.10.1016/j.phytochem.2003.12.006Search in Google Scholar
Azarkan, M., Clantin, B., Bompard, C., Belrhali, H., Baeyens-Volant, D., Looze, Y., Villeret, V., and Wintjens, R. (2005). Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex. Acta Crystallogr. F Struct. Biol. Cryst. Commun.61, 59–61.10.1107/S1744309104025904Search in Google Scholar
Bateman, R.C., Temple, J.S., Misquitta, S.A., and Booth, R.E. (2001). Evidence for essential histidines in human pituitary glutaminyl cyclase. Biochemistry40, 11246–11250.10.1021/bi011177oSearch in Google Scholar
Caruso, C., Caporale, C., Chilosi, G., Vacca, F., Bertini, L., Magro, P., Poerio, E., and Buonocore, V. (1996). Structural and antifungal properties of a pathogenesis-related protein from wheat kernel. J. Protein Chem.15, 35–44.10.1007/BF01886809Search in Google Scholar
Cereghino, J.L. and Cregg, J.M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev.24, 45–66.10.1111/j.1574-6976.2000.tb00532.xSearch in Google Scholar
Choi, B.K., Bobrowicz, P., Davidson, R.C., Hamilton, S.R., Kung, D.H., Li, H., Miele, R.G., Nett, J.H., Wildt, S., and Gerngross, T.U. (2003). Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc. Natl. Acad. Sci. USA100, 5022–5027.10.1073/pnas.0931263100Search in Google Scholar
Cregg, J.M., Vedvick, T.S., and Raschke, W.C. (1993). Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology11, 905–910.10.1038/nbt0893-905Search in Google Scholar
Dahl, S.W., Slaughter, C., Lauritzen, C., Bateman, R.C. Jr., Connerton, I., and Pedersen, J. (2000). Carica papaya glutamine cyclotransferase belongs to a novel plant enzyme subfamily: cloning and characterization of the recombinant enzyme. Protein Expr. Purif.20, 27–36.10.1006/prep.2000.1273Search in Google Scholar
El Moussaoui, A., Nijs, M., Paul, C., Wintjens, R., Vincentelli, J., Azarkan, M., and Looze, Y. (2001). Revisiting the enzymes stored in the laticifers of Carica papaya in the context of their possible participation in the plant defence mechanism. Cell. Mol. Life Sci.58, 556–570.10.1007/PL00000881Search in Google Scholar
Fischer, W.H. and Spiess, J. (1987). Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides. Proc. Natl. Acad. Sci. USA84, 3628–3632.10.1073/pnas.84.11.3628Search in Google Scholar
Hamilton, S.R., Bobrowicz, P., Bobrowicz, B., Davidson, R.C., Li, H., Mitchell, T., Nett, J.H., Rausch, S., Stadheim, T.A., Wischnewski, H., et al. (2003). Production of complex human glycoproteins in yeast. Science301, 1244–1246.10.1126/science.1088166Search in Google Scholar
Hass, G.M. and Hermodson, M.A. (1981). Amino acid sequence of a carboxypeptidase inhibitor from tomato fruit. Biochemistry20, 2256–2260.10.1021/bi00511a029Search in Google Scholar
Huang, K.F., Liu, Y.L., Cheng, W.J., Ko, T.P., and Wang, A.H. (2005). Crystal structures of human glutaminyl cyclase, an enzyme responsible for protein N-terminal pyroglutamate formation. Proc. Natl. Acad. Sci. USA102, 13117–13122.10.1073/pnas.0504184102Search in Google Scholar
Joosten, M.H., Bergmans, C.J., Meulenhoff, E.J., Cornelissen, B.J., and De Wit, P.J. (1990). Purification and serological characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato. Plant Physiol.94, 585–591.10.1104/pp.94.2.585Search in Google Scholar
Lee, J.E. and Raines, R.T. (2003). Contribution of active-site residues to the function of onconase, a ribonuclease with antitumoral activity. Biochemistry42, 11443–11450.10.1021/bi035147sSearch in Google Scholar
Messer, M. (1963). Enzymatic cyclization of L-glutamine and L-glutaminyl peptides. Nature197, 1299.10.1038/1971299a0Search in Google Scholar
Morty, R.E., Bulau, P., Pelle, R., Wilk, S., and Abe, K. (2006). Pyroglutamyl peptidase type I from Trypanosoma brucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts. Biochem. J.394, 635–645.10.1042/BJ20051593Search in Google Scholar
Oberg, K.A., Ruysschaert, J.M., Azarkan, M., Smolders, N., Zerhouni, S., Wintjens, R., Amrani, A., and Looze, Y. (1998). Papaya glutamine cyclase, a plant enzyme highly resistant to proteolysis, adopts an all-β conformation. Eur. J. Biochem.258, 214–222.10.1046/j.1432-1327.1998.2580214.xSearch in Google Scholar
Rep, M., Dekker, H.L., Vossen, J.H., de Boer, A.D., Houterman, P.M., Speijer, D., Back, J.W., de Koster, C.G., and Cornelissen, B.J. (2002). Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. Plant Physiol.130, 904–917.10.1104/pp.007427Search in Google Scholar
Schilling, S., Hoffmann, T., Rosche, F., Manhart, S., Wasternack, C., and Demuth, H.U. (2002a). Heterologous expression and characterization of human glutaminyl cyclase: evidence for a disulfide bond with importance for catalytic activity. Biochemistry41, 10849–10857.10.1021/bi0260381Search in Google Scholar
Schilling, S., Hoffmann, T., Wermann, M., Heiser, U., Wasternack, C., and Demuth, H.-U. (2002b). Continuous spectrometric assays for glutaminyl cyclase activity. Anal. Biochem.303, 49–56.10.1006/abio.2001.5560Search in Google Scholar
Schilling, S., Manhart, S., Hoffmann, T., Ludwig, H.-H., Wasternack, C., and Demuth, H.-U. (2003). Substrate specificity of glutaminyl cyclases from plants and animals. Biol. Chem.384, 1583–1592.10.1515/BC.2003.175Search in Google Scholar
Schilling, S., Hoffmann, T., Manhart, S., Hoffmann, M., and Demuth, H.U. (2004). Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions. FEBS Lett.563, 191–196.10.1016/S0014-5793(04)00300-XSearch in Google Scholar
Schilling, S., Cynis, H., von Bohlen, A., Hoffmann, T., Wermann, M., Heiser, U., Buchholz, M., Zunkel, K., and Demuth, H.U. (2005). Isolation, catalytic properties, and competitive inhibitors of the zinc-dependent murine glutaminyl cyclase. Biochemistry44, 13415–13424.10.1021/bi051142eSearch in Google Scholar
Song, I., Chuang, C.Z., and Bateman, R.C.J. (1994). Molecular cloning, sequence analysis and expression of human pituitary glutaminyl cyclase. J. Mol. Endocrinol.13, 77–86.10.1677/jme.0.0130077Search in Google Scholar
Svensson, B., Svendsen, I., Hojrup, P., Roepstorff, P., Ludvigsen, S., and Poulsen, F.M. (1992). Primary structure of barwin: a barley seed protein closely related to the C-terminal domain of proteins encoded by wound-induced plant genes. Biochemistry31, 8767–8770.10.1021/bi00152a012Search in Google Scholar
Wintjens, R., Belrhali, H., Clantin, B., Azarkan, M., Bompard, C., Baeyens-Volant, D., Looze, Y., and Villeret, V. (2006). Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases. J. Mol. Biol.357, 457–470.10.1016/j.jmb.2005.12.029Search in Google Scholar
Yang, J.T., Wu, C.S., and Martinez, H.M. (1986). Calculation of protein conformation from circular dichroism. Methods Enzymol.130, 208–269.10.1016/0076-6879(86)30013-2Search in Google Scholar
Zerhouni, S., Amrani, A., Nijs, M., Smolders, N., Azarkan, M., Vincentelli, J., and Looze, Y. (1998). Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation. Biochim. Biophys. Acta1387, 275–290.10.1016/S0167-4838(98)00140-XSearch in Google Scholar
©2007 by Walter de Gruyter Berlin New York