RESUMEN
Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn(2+)-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX(18)E (Zn(2+)-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G(298)XM(300)E(301)N(302) motif and one mutant of the HEIS(328)HX(18)E motif were expressed in Escherichia coli. All mutations except G(298)P, G(298)S, and S(328)A abolished the aminopeptidase activity. The S(328)A mutant mimics the sequence of bovine Ap-B Zn(2+)-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G(298)S and G(298)P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl(-) anions. Moreover, the G(298)P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G(298) plays in the catalytic mechanism of Ap-B. Our results show that G(298) is essential to Ap-B activity and participates to the substrate specificity of the enzyme.
Asunto(s)
Aminopeptidasas/química , Aminopeptidasas/genética , Mutación/genética , Secuencias de Aminoácidos , Aminopeptidasas/antagonistas & inhibidores , Animales , Sitios de Unión , Dominio Catalítico , Bovinos , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estabilidad Proteica , Ratas , Especificidad por Sustrato , Zinc/químicaRESUMEN
BACKGROUND: Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. RESULTS: The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions. CONCLUSION: Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing.
Asunto(s)
Aminopeptidasas/metabolismo , Glucagón/metabolismo , Modelos Moleculares , Mutagénesis/fisiología , Zinc/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Aminopeptidasas/genética , Animales , Sitios de Unión/fisiología , Cristalografía por Rayos X , Glucagón/genética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína/fisiología , RatasRESUMEN
Using a monoclonal antibody against the entire C-terminal end of human APP(695) (643-695 sequence) and a monoclonal antibody directed against human beta[1-40] amyloid peptide (betaA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of betaA. According to the nature of N- and C-terminal amino acids we identified endogenous beta-, gamma-, epsilon-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the beta-amyloid sequence at same sites as those observed in human betaA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study betaA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human betaA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.
Asunto(s)
Precursor de Proteína beta-Amiloide/aislamiento & purificación , Precursor de Proteína beta-Amiloide/metabolismo , Exudados y Transudados/química , Piel/química , Proteínas de Xenopus/aislamiento & purificación , Xenopus laevis , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/inmunología , Animales , Cromatografía en Gel/métodos , Cromatografía Líquida de Alta Presión/métodos , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Proproteína Convertasas/metabolismo , Análisis de Secuencia de Proteína , Proteínas de Xenopus/inmunología , Proteínas de Xenopus/metabolismoRESUMEN
Dactylysin (EC 3.5.24.60) is a metalloendopeptidase first isolated from the skin granular gland secretions of Xenopus laevis. This peptidase hydrolyzes bonds on the amino-terminus of singlets and between doublets of hydrophobic amino acids and was considered to play a role in the in vivo inactivation of biologically active regulatory peptides. Here, we show that dactylysin has also the ability to cleave human beta[1-40]-amyloid peptide and related peptides. Cleavage of the wild type beta[1-40]-amyloid peptide form, and to a lesser extent Flemish and Dutch mutants, occurred predominantly at the His14-Glu15 bond. We demonstrate that frog skin exudate contains a full-length amyloid protein precursor detected by immunochemical cross-reactivity with monoclonal antibody against C-terminal human amyloid protein precursor. The possibility that dactylysin, might be involved in normal catabolism of beta amyloid peptide of Xenopus laevis is discussed.