Initial steps in assembly of microfibrils. Formation of disulfide-cross-linked multimers containing fibrillin-1.
Fibrillins are the major constituents of extracellular microfibrils. How fibrillin molecules assemble into microfibrils is not known. Sequential extractions and pulse-chase labeling of organ cultures of embryonic chick aortae revealed rapid formation of disulfide-cross-linked aggregates containing fibrillin-1. These results demonstrated that intermolecular disulfide bond formation is an initial step in the assembly process. To identify free cysteine residues available for intermolecular cross-linking, small recombinant peptides of fibrillin-1 harboring candidate cysteine residues were analyzed. Results revealed that the first four cysteine residues in the unique N terminus form intramolecular disulfide bonds. One cysteine residue (Cys(204)) in the first hybrid domain of fibrillin-1 was found to occur as a free thiol and is therefore a good candidate for intermolecular disulfide bonding in initial steps of the assembly process. Furthermore, evidence indicated that the comparable cysteine residue in fibrillin-2 (Cys(233)) also occurs as a free thiol. These free cysteine residues in fibrillins are readily available for intermolecular disulfide bond formation, as determined by reaction with Ellman's reagent. In addition to these major results, the cleavage site of the fibrillin-1 signal peptide and the N-terminal sequence of monomeric authentic fibrillin-1 from conditioned fibroblast medium were determined.
Reinhardt
D P
DP
Shriners Hospital for Children, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Gambee
J E
JE
Ono
R N
RN
Bächinger
H P
HP
Sakai
L Y
LY
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000196:embryology; D002642:Chick Embryo; D003432:Cross-Linking Reagents / Q000378:metabolism*; D016895:Culture Media, Serum-Free / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000737:chemistry; D000071838:Fibrillin-1; D000071840:Fibrillin-2; D000071837:Fibrillins; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D020894:Microfibrils / Q000737:chemistry / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009924:Organ Culture Techniques; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D054392:Adipokines
D003432:Cross-Linking Reagents; D016895:Culture Media, Serum-Free; D003545:Cysteine; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071840:Fibrillin-2; D000071837:Fibrillins; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C000605722:FBN1 protein, human; D054392:Adipokines; C000605723:FBN2 protein, human