title: Molecular effects of homocysteine on cbEGF domain structure: insights into the pathogenesis of homocystinuria.
abstract: Homocystinuria is an inborn error of methionine metabolism that results in raised serum levels of the highly reactive thiol-containing amino acid homocysteine. Homocystinurics often exhibit phenotypic abnormalities that are similar to those found in Marfan syndrome (MFS), a heritable connective tissue disorder that is caused by reduced levels of, or defects in, the cysteine-rich extracellular matrix (ECM) protein fibrillin-1. The phenotypic similarities between homocystinuria and MFS suggest that elevated homocysteine levels may result in an altered function of fibrillin-1. We have used recombinant calcium binding epidermal growth factor-like (cbEGF) domain fragments from fibrillin-1, and an unrelated protein Notch1, to analyse the effects of homocysteine on the native disulphide (cystine) bonds of these domains. We show using analytical reverse phase, high performance liquid chromatography (HPLC), electrospray ionisation mass spectrometry (ESI-MS) and limited proteolysis that homocysteine attacks intramolecular disulphide bonds causing reduction of cystine and domain misfolding, and that the effects of homocysteine are dependent on its concentration. We also identify the importance of calcium binding to cbEGF domains for their stabilisation and protection against homocysteine attack. Collectively, these data suggest that reduction of intramolecular cbEGF domain disulphide bonds by homocysteine and the resulting disruption of this domain fold may contribute to the change in connective tissue function seen in homocystinuria. Furthermore, since we show that the effects of homocysteine are not unique to fibrillin-1, other cbEGF-containing proteins may be implicated in the pathogenic mechanisms underlying homocystinuria.
authors.affiliation: Division of Molecular and Cellular Biochemistry, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Hutchinson
authors.forename: Robin T
authors.initials: RT
authors.lastname: Aplin
authors.forename: Heather
authors.initials: H
authors.lastname: Webb
authors.forename: Susan
authors.initials: S
authors.lastname: Kettle
authors.forename: Janneke
authors.initials: J
authors.lastname: Timmermans
authors.forename: Godfried H J
authors.initials: GH
authors.lastname: Boers
authors.forename: Penny A
authors.initials: PA
authors.lastname: Handford
mesh_terms: D002118:Calcium / Q000378:metabolism; D003541:Cystathionine beta-Synthase / Q000187:drug effects / Q000235:genetics; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D006710:Homocysteine / Q000737:chemistry*; D006712:Homocystinuria / Q000209:etiology* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008382:Marfan Syndrome / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin; D054392:Adipokines
chemical_list: D002118:Calcium; D003541:Cystathionine beta-Synthase; D004815:Epidermal Growth Factor; D000071838:Fibrillin-1; D000071837:Fibrillins; D006710:Homocysteine; D008840:Microfilament Proteins; D010446:Peptide Fragments; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014157:Transcription Factors; D014357:Trypsin; C000605722:FBN1 protein, human; D054392:Adipokines; C497761:NOTCH1 protein, human