Browsing by Author "Sturrock, E D"

Now showing 1 - 6 of 6
  • Thumbnail Image
    Item
    Open Access
    Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII
    (De Gruyter, 2006) Farias, S L; Sabatini, R A; Sampaio, T C; Hirata, I Y; Cezari, M S; Juliano, M A; Sturrock, E D; Carmona, A K; Juliano, L
    Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.
  • Thumbnail Image
    Item
    Restricted
    Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme
    (American Chemical Society, 2007) Danilov, S M; Watermeyer, J M; Balyasnikova, I V; Gordon, K; Kugaevskaya, E V; Elisseeva, Y E; Albrecht, R F; Sturrock, E D
    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 α-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.
  • Thumbnail Image
    Item
    Restricted
    Monoclonal antibodies 1B3 and 5C8 as probes for monitoring the integrity of the C-terminal end of soluble angiotensin-converting enzyme
    (Mary Ann Liebert, 2005) Balyasnikova, I V; Sun, Z L; Berestetskaya, Y V; Chubb, A J; Albrecht, R F; Sturrock, E D; Danilov, S M
    Angiotensin-converting enzyme (ACE) is a membrane-anchored ectoprotein that is proteolytically cleaved, yielding an enzymatically active soluble ACE. Two mouse monoclonal antibodies, MAbs 1B3 and 5C8, were generated to the C-terminal part of human soluble ACE. MAb 1B3 recognized the catalytically active ACE, as revealed by ELISA and precipitation assays, whereas Western blotting and immunohistochemisty on paraffin- embedded sections using MAb 5C8 detected denatured ACE. MAb 1B3 showed extensive cross-reactivity, recognizing 15 species out of the 16 tested. The binding of this MAb to ACE was greatly affected by conformational changes induced by adsorption on plastic, formalin fixation, and underglycosylation. Furthermore, MAb 1B3 binding to the mutated ACE (Pro1199Leu substitution in the juxtamembrane region, leading to a fivefold increase in serum ACE level) was markedly decreased. MAb 5C8 detected all the known expression sites of full-size ACE using formalin-fixed and paraffin-embedded human tissues. The sequential epitope for MAb 5C8 is formed by the last 11 amino acid residues of soluble ACE (Pro1193–Arg1203), whereas the conformational epitope for 1B3 is formed by a motif within these 11 amino acid residues and, in addition, by at least one stretch that includes Ala837–His839 located distal to the sequential epitope. Our findings demonstrated that MAbs 1B3 and 5C8 are very useful for the study of ACE shedding, for identification of mutations in stalk regions, and for studying alternatively spliced variants of ACE. In addition, binding of MAb 1B3 is a sensitive determinant of the integrity of soluble ACE.
  • Thumbnail Image
    Item
    Restricted
    Selective inclusion of proteins into urinary calcium oxalate crystals: comparison between stone-prone and stone-free population groups
    (Elsevier, 2003) Webber, D; Rodgers, A L; Sturrock, E D
    This study investigated whether incorporation of proteins into calcium oxalate urinary crystals is different in the black and white populations in South Africa and whether such differences could provide insight into the former group’s remarkably low stone incidence. CaOx monohydrate (COM) and dihydrate (COD) crystals were precipitated from each group’s urine after adjustment of the calcium concentrations to 0.5 and 12 mmol/l, respectively. Crystals were characterised by X-ray powder diffraction and scanning electron microscopy. Intracrystalline proteins were analysed by SDS-PAGE and immunodetected for urinary prothrombin fragment 1 (UPTF1) and osteopontin. Crystals precipitated from the black and white groups’ control urines comprised mainly COM and COD, respectively. In both race groups UPTF1 was the major protein included in pure COM crystals while in pure COD it was osteopontin, but in the black group osteopontin was also included in COM. The black group’s urine crystals incorporated significantly more intracrystalline protein. Selective inclusion of UPTF1 and osteopontin may be due to the unique crystal structure of COM and COD and the proteins’ conformation at the different calcium concentrations at which these hydrates precipitate. The greater amount of intracrystalline inhibitory protein in the black group may be a factor in their low stone incidence.
  • Thumbnail Image
    Item
    Restricted
    Structure of angiotensin I-converting enzyme
    (Springer, 2004) Sturrock, E D; Natesh, R; van Rooyen, J M; Acharya, K R
    Angiotensin-converting enzyme (ACE) is a zinc- and chloride-dependent metallopeptidase that plays a vital role in the metabolism of biologically active peptides. Until recently, much of the inhibitor design and mechanism of action of this ubiquitous enzyme was based on the structures of carboxypeptidase A and thermolysin. When compared to the recently solved structures of the testis isoform of ACE (tACE) and its Drosophila homologue (AnCE), carboxypeptidase A showed little structural homology outside of the active site, while thermolysin revealed significant but less marked overall similarity. The ellipsoid-shaped structure of tACE, which has a preponderance of α-helices, is characterised by a core channel that has a constriction approximately 10 Å from its opening where the zinc-binding active site is located. Comparison of the native protein with the inhibitor-bound form (lisinopril-tACE) does not reveal any striking differences in the conformation of the inhibitor binding site, disfavouring an open and closed configuration. However, the inhibitor complex does provide insights into the network of hydrogen-bonding and ionic interactions in the active site as well as the mechanism of ACE substrate hydrolysis. The three-dimensional structure of ACE now paves the way for the rational design of a new generation of domain-selective ACE inhibitors.
  • Thumbnail Image
    Item
    Open Access
    The structure of testis angiotensin-converting enzyme (tACE-g13) in complex with the inhibitor RXPA380
    (2006) Chitapi, Itai; Sewell, Bryan Trevor; Sturrock, E D
    Angiotensin-converting enzyme (ACE), a zinc metalloprotease, is a key regulator of the mammalian renin-angiotensin system (RAS) Primarily, ACF is a dipeptidl peptidase which cleaves angiotensin I to produce angiotensin II, a potent vasoconstrictor. By the same enzymatic mechanism, ACE also inactivates the vasodilator bradykinin. The main overall effect of these actions is an increase in blood pressure. Several ACF inhibitors have been developed as drugs for the treatment of myocardial infarction, hypertension, kidney failure and heart failure.