RESUMEN
Chronic imbalance between production and degradation of the human amyloid-beta peptide (Abeta) is assumed to play an important role in pathogenesis of Alzheimer's disease (AD). Post-translational modifications of Abeta could influence its interactions with specifically cleaving proteases and, therefore, perturb the Abeta homeostasis. The angiotensin-converting enzyme (ACE) was previously shown to degrade non-modified Abeta in vitro and in cells. In the presented work, we investigated the effect of isomerization of Asp-7, a common non-enzymatic age-related modification found in AD-associated Abeta species, on hydrolysis of Abeta by ACE. Two synthetic peptides corresponding to the Abeta region 1-16 with either Asp or isoAsp residues in position 7 were examined as monomeric soluble substrates for the N- as well as for the C-domain of ACE. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with the (18)O-labeled internal standard approach has allowed us to show that (i) the N-domain of ACE (N-ACE), but not the C-domain, selectively cleaves the Arg-5-His-6 bond in both peptides, and that (ii) N-ACE hydrolyzes the isoAsp-7 analogue more efficiently than the non-modified one. Our results demonstrate a new endopeptidase activity of N-ACE as well as high preference of the domain to recognize and hydrolyze the isomerized Abeta species that were earlier suggested to promote AD pathogenesis. The results suggest the need for further analysis of biological effects of isomerized Abeta and its interaction with ACE in AD pathogenesis.
Asunto(s)
Péptidos beta-Amiloides/química , Arginina/química , Histidina/química , Fragmentos de Péptidos/química , Peptidil-Dipeptidasa A/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/etiología , Humanos , Hidrólisis , IsomerismoRESUMEN
Angiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) is a key enzyme in cardiovascular pathophysiology. A wide spectrum of monoclonal antibodies to different epitopes on the N and C domains of human ACE has been used to study different aspects of ACE biology. In this study we characterized the monoclonal antibody (mAb) 5F1, developed against the N domain of human ACE, which recognizes both the catalytically active and the denatured forms of ACE. The epitope for mAb 5F1 was defined using species cross-reactivity, synthetic peptide (PepScan technology) and phage display library screening, Western blotting, site-directed mutagenesis, and protein modeling. The epitope for mAb 5F1 shows no overlap with the epitopes of seven other mAbs to the N domain described previously and is localized on the other side of the N domain globule. The binding of mAb 5F1 to ACE is carbohydrate-dependent and increased significantly as a result of altered glycosylation after treatment with alpha-glucosidase-1 inhibitor, N-butyldeoxynojirimycin (NB-DNJ), or neuraminidase. Out of 17 species tested, mAb 5F1 showed strict primate ACE specificity. In addition, mAb 5F1 recognized human ACE in Western blots and on paraffin-embedded sections. The sequential part of the epitope for mAb 5F1 is created by the N-terminal part of the N domain, between residues 1 and 141. A conformational region of the epitope was also identified, including the residues around the glycan attached to Asn117, which explains the sensitivity to changes in glycosylation state, and another stretch localized around the motif 454TPPSRYN460. Site-directed mutagensis and inhibition assays revealed that mAb 5F1 inhibits ACE activity at high concentrations due to binding of residues on both sides of the active site cleft, thus supporting a hinge-bending mechanism for substrate binding of ACE.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Anticuerpos Monoclonales/farmacología , Mapeo Epitopo , Peptidil-Dipeptidasa A/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Inhibidores de la Enzima Convertidora de Angiotensina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Células CHO , Catálisis/efectos de los fármacos , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Epítopos/inmunología , Glicosilación , Humanos , Inmunoprecipitación , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Biblioteca de Péptidos , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/genética , Polisacáridos/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.
Asunto(s)
Mapeo Epitopo , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/inmunología , Secuencia de Aminoácidos , Animales , Epítopos/química , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Testículo/enzimologíaRESUMEN
The prediction of biological activity spectra for substances as an approach for searching compounds with complex mechanisms of action was studied. New compounds with dual mechanisms of antihypertensive action were found by this approach. Biological activity spectra for substances were predicted on the basis of their structural formulas by the computer program PASS. Thirty molecular mechanisms of action of compounds from the MDDR 99.2 database, which cause the antihypertensive effect and can be predicted by PASS, have been identified. The analysis of predictions for compounds with 15 dual antihypertensive mechanisms of action from the MDDR 99.2 database has confirmed high accuracy of prediction. This approach was applied to databases of commercially available compounds (AsInEx and ChemBridge) and allowed us to select four substances that are potential inhibitors of angiotensin converting enzyme (ACE) and of neutral endopeptidase (NEP). At a later time, all these compounds were found to be the inhibitors of both ACE and NEP. The most potent compounds had IC(50) of 10(-7)-10(-9) M for ACE and 10(-5) M for NEP. New combinations of dual mechanisms of action never before found for antihypertensive compounds were predicted.