title: Consequences of cysteine mutations in calcium-binding epidermal growth factor modules of fibrillin-1.
abstract: Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. The mRNA levels for the mutation constructs were similar to wild-type levels. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. The overall folds of the mutant polypeptides were indistinguishable from the wild-type as judged by the ultrastructural shape, CD analysis, and reactivity with a specific antibody sensitive for intact disulfide bonds. Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. These results expand and further strengthen the concept that proteolytic degradation of mutated fibrillin-1 might be an important potential mechanism in the pathogenesis of Marfan syndrome and other disorders caused by mutations in fibrillin-1.
authors.affiliation: Department of Medical Molecular Biology of the University of Lübeck, D-23538 Luebeck, Germany.
authors.forename: Tillman
authors.initials: T
authors.lastname: Vollbrandt
authors.forename: Kerstin
authors.initials: K
authors.lastname: Tiedemann
authors.forename: Ehab
authors.initials: E
authors.lastname: El-Hallous
authors.forename: Guoqing
authors.initials: G
authors.lastname: Lin
authors.forename: Jürgen
authors.initials: J
authors.lastname: Brinckmann
authors.forename: Harald
authors.initials: H
authors.lastname: John
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; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D004815:Epidermal Growth Factor / Q000378:metabolism*; D018604:Epitope Mapping; D000071838:Fibrillin-1; D000071837:Fibrillins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D014162:Transfection; D014466:Ultraviolet Rays; D054392:Adipokines
chemical_list: D002118:Calcium; D003545:Cysteine; D004815:Epidermal Growth Factor; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C000605722:FBN1 protein, human; D054392:Adipokines