title: Modification of the structure and function of fibrillin-1 by homocysteine suggests a potential pathogenetic mechanism in homocystinuria.
abstract: Homocystinuria, a disorder originating in defects in the methionine metabolism, is characterized by an elevated plasma concentration of homocysteine. Most patients have a defect in the cystathionine-beta-synthase, the key enzyme in the conversion of homocysteine to cysteine. Many abnormalities in the connective tissue of patients with homocystinuria resemble those seen in Marfan syndrome, caused by mutations in fibrillin-1. These observations led to the hypothesis that the structure and function of fibrillin-1 is compromised in patients with homocystinuria. To test this hypothesis we produced recombinant human fibrillin-1 fragments spanning the central portion of the molecule (8-Cys/transforming growth factor-beta binding domain 3 to calcium binding EGF domain 22) and extensively analyzed the potential of homocysteine to modify structural and functional properties of these proteins. Circular dichroism spectroscopy revealed moderate changes of their secondary structures after incubation with homocysteine. Equilibrium dialysis demonstrated a number of high affinity calcium binding sites in the tandemly repeated calcium binding epidermal growth factor-like domains 11-22. Calcium binding of homocysteine-modified fragments was completely abolished. Incubation of the recombinant proteins with homocysteine rendered the analyzed calcium binding EGF domains as well as the 8-Cys/transforming growth factor-beta binding domain 3 significantly more susceptible to proteolytic degradation. Furthermore, data were obtained demonstrating that homocysteine can covalently modify fibrillin-1 via disulfide bonds. These data strongly suggest that structural and functional modifications as well as degradation processes of fibrillin-1 in the connective tissues of patients with homocystinuria play a major role in the pathogenesis of this disorder.
authors.affiliation: Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A2B2, Canada.
authors.forename: Dirk
authors.initials: D
authors.lastname: Hubmacher
authors.forename: Kerstin
authors.initials: K
authors.lastname: Tiedemann
authors.forename: Rainer
authors.initials: R
authors.lastname: Bartels
authors.forename: Jürgen
authors.initials: J
authors.lastname: Brinckmann
authors.forename: Tillman
authors.initials: T
authors.lastname: Vollbrandt
authors.forename: Boris
authors.initials: B
authors.lastname: Bätge
authors.forename: Holger
authors.initials: H
authors.lastname: Notbohm
authors.forename: Dieter P
authors.initials: DP
authors.lastname: Reinhardt
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000494:pharmacology; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006710:Homocysteine / Q000737:chemistry*; D006712:Homocystinuria / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D008382:Marfan Syndrome / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D054392:Adipokines
chemical_list: D002118:Calcium; D002918:Chymotrypsin; D004815:Epidermal Growth Factor; D000071838:Fibrillin-1; D000071837:Fibrillins; D006710:Homocysteine; D008840:Microfilament Proteins; D010455:Peptides; D011994:Recombinant Proteins; D014357:Trypsin; C000605722:FBN1 protein, human; D054392:Adipokines