Abstract
Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine β-naphthalamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.
References
Albiston, A.L., Mustafa, T., McDowall, S.G., Mendelsohn, F.A., Lee, J., and Chai, S.Y. (2003). AT(4) receptor is insulin-regulated membrane aminopeptidase: potential mechanisms of memory enhancement. Trends. Endocrinol. Metab.14, 72–77.10.1016/S1043-2760(02)00037-1Search in Google Scholar
Albiston, A.L., Pederson, E.S., Burns, P., Purcell, B., Wright, J.W., Harding, J.W., Mendelsohn, F.A., Weisinger, R.S., and Chai, S.Y. (2004a). Reversal of scopolamine-induced memory deficits by LVV-hemorphin 7 in rats in the passive avoidance and Morris water maze paradigms. Behav. Brain Res.154, 239–243.10.1016/j.bbr.2004.02.012Search in Google Scholar
Albiston, A.L., Ye, S., and Chai, S.Y. (2004b). Membrane bound members of the M1 family: more than aminopeptidases. Protein Pept. Lett.11, 491–500.10.2174/0929866043406643Search in Google Scholar
Braszko, J.J., Kupryszewski, G., Witczuk, B., and Wisniewski, K. (1988). Angiotensin II-(3-8)-hexapeptide affects motor activity, performance of passive avoidance and a conditioned avoidance response in rats. Neuroscience27, 777–783.10.1016/0306-4522(88)90182-0Search in Google Scholar
Cadel, S., Foulon, T., Viron, A., Balogh, A., Midol-Monnet, S., Noel, N., and Cohen, P. (1997). Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase. Proc. Natl. Acad. Sci. USA94, 2963–2968.10.1073/pnas.94.7.2963Search in Google Scholar
Chai, S.Y., Fernando, R., Peck, G., Ye, S.Y., Mendelsohn, F.A.O., Jenkins, T.A., and Albiston, A.L. (2004). The angiotensin IV/AT4 receptor. Cell. Mol. Life Sci.61, 2728–2737.10.1007/s00018-004-4246-1Search in Google Scholar
Fernando, R., Larm, J., Albiston, A.L., and Chai, S.Y. (2005). Distribution and cellular localization of the insulin regulated aminopeptidase (IRAP) in the rat central nervous system. J. Comp. Neurol.487, 372–390.10.1002/cne.20585Search in Google Scholar
Foulon, T., Cadel, S., and Cohen, P. (1999). Aminopeptidase B (EC 3.4.11.6). Int. J. Biochem. Cell. Biol.31, 747–750.10.1016/S1357-2725(99)00021-7Search in Google Scholar
Fukasawa, K.M., Fukasawa, K., Kanai, M., Fujii, S., and Harada, M. (1996). Molecular cloning and expression of rat liver aminopeptidase B. J. Biol. Chem.271, 30731–30735.10.1074/jbc.271.48.30731Search in Google Scholar PubMed
Funk, C.D., Radmark, O., Fu, J.Y., Matsumoto, T., Jornvall, H., Shimizu, T., and Samuelsson, B. (1987). Molecular cloning and amino acid sequence of leukotriene A4 hydrolase. Proc. Natl. Acad. Sci. USA84, 6677–6681.10.1073/pnas.84.19.6677Search in Google Scholar PubMed PubMed Central
Iturrioz, X., Rozenfeld, R., Michaud, A., Corvol, P., and Llorens-Cortes, C. (2001). Study of asparagine 353 in aminopeptidase A: characterization of a novel motif (GXMEN) implicated in exopeptidase specificity of monozinc aminopeptidases. Biochemistry40, 14440–14448.10.1021/bi011409jSearch 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
Laustsen, P.G., Vang, S., and Kristensen, T. (2001). Mutational analysis of the active site of human insulin-regulated aminopeptidase. Eur. J. Biochem.268, 98–104.10.1046/j.1432-1327.2001.01848.xSearch in Google Scholar
Lee, J.H., Mustafa, T., McDowall, S.G., Mendelsohn, F.A.O., Brennan, M.B., Lew, R., Albiston, A.L., and Chai, S.Y. (2003). Structure-activity study of LVV-hemorphin-7: angiotensin AT4 receptor ligand and inhibitor of insulin-regulated aminopeptidase (IRAP). J. Pharm. Exp. Ther.305, 205–211.10.1124/jpet.102.045492Search in Google Scholar
Lee, J., Albiston, A.L., Allen, A.M., Mendelsohn, F.A.O., Ping, S.E., Barrett, G.L., Murphy, M., Morris, M.J., McDowall, S.G., and Chai, S.Y. (2004). Effect of intracerebroventricular injection of AT4 receptor ligands, Nle1-angiotensin IV and LVV-hemorphin 7, on spatial learning in rats. Neuroscience124, 341–349.10.1016/j.neuroscience.2003.12.006Search in Google Scholar
Lew, R.A., Mustafa, T., Ye, S., McDowall, S.G., Chai, S.Y., and Albiston, A.L. (2003). Angiotensin AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP). J. Neurochem.86, 344–350.10.1046/j.1471-4159.2003.01852.xSearch in Google Scholar
Luciani, N., Marie-Claire, C., Ruffet, E., Beaumont, A., Roques, B.P., and Fournie-Zaluski, M.C. (1998). Characterization of Glu350 as a critical residue involved in the N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2): insights into its mechanism of action. Biochemistry37, 686–692.Search in Google Scholar
Nanus, D.M., Engelstein, D., Gastl, G.A., Gluck, L., Vidal, M.J., Morrison, M., Finstad, C.L., Bander, N.H., and Albino, A.P. (1993). Molecular cloning of the human kidney differentiation antigen gp160: human aminopeptidase A. Proc. Natl. Acad. Sci. USA90, 7069–7073.10.1073/pnas.90.15.7069Search in Google Scholar
Olsen, J., Cowell, G.M., Konigshofer, E., Danielsen, E.M., Moller, J., Laustsen, L., Hansen, O.C., Welinder, K.G., Engberg, J., Hunziker, W., et al. (1988). Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA. FEBS Lett.238, 307–314.10.1016/0014-5793(88)80502-7Search in Google Scholar
Rudberg, P.C., Tholander, F., Thunnissen, M.M., and Haeggstrom, J.Z. (2002). Leukotriene A4 hydrolase/aminopeptidase. Glutamate 271 is a catalytic residue with specific roles in two distinct enzyme mechanisms. J. Biol. Chem.277, 1398–1404.Search in Google Scholar
Song, L., Ye, M., Troyanovskaya, M., Wilk, E., Wilk, S., and Healy, D.P. (1994). Rat kidney glutamyl aminopeptidase (aminopeptidase A): molecular identity and cellular localization. Am. J. Physiol.267, F546–557.10.1152/ajprenal.1994.267.4.F546Search in Google Scholar PubMed
Thunnissen, M.M.G.M., Andersson, B., Samuelsson, B., Wong, C.H., and Haeggstrom, J.Z. (2002). Crystal structures of leukotriene A4 hydrolase in complex with captopril and two competitive tight-binding inhibitors. FASEB J.16, 1648–1650.10.1096/fj.01-1017fjeSearch in Google Scholar PubMed
Wright, J.W., Miller-Wing, A.V., Shaffer, M.J., Higginson, C., Wright, D.E., Hanesworth, J.M., and Harding, J.W. (1993). Angiotensin II(3-8) (ANG IV) hippocampal binding: potential role in the facilitation of memory. Brain Res. Bull.32, 497–502.10.1016/0361-9230(93)90297-OSearch in Google Scholar
©2007 by Walter de Gruyter Berlin New York