The arrangement of intra- and intermolecular disulfide bonds in the carboxyterminal, non-collagenous aggregation and cross-linking domain of basement-membrane type IV collagen.
The hexameric complex of globular domains of type IV collagen was isolated after collagenase digestion of human placenta and the different monomers and dimers present were chromatographically separated. The ratio of alpha 1(IV)NC1 to alpha 2(IV)NC1 was 2:1. About 50% of the NC1 domains were connected to dimers. Predominantly alpha 1-alpha 1 dimers were found. Only 12% were alpha 2-alpha 2 dimers and no alpha 1-alpha 2 dimers could be detected. The majority (88%) of the intermolecular bonds was found to be disulfide bridges. The remainder could not be cleaved by reduction. To elucidate the arrangement of the disulfide bonds, the unreduced alpha 1(IV)NC1 monomers were treated with cyanogen bromide, the disulfide-bridged peptides isolated and characterized by Edman degradation. Each of the two homologous subdomains within a monomer is stabilized by an identical set of three disulfide bonds. In subdomain I, cysteines at positions 20 and 53 are connected with the C-terminal cysteine pair 108 and 111. Thus formed, the disulfide knot stabilizes two interconnected loops of 32 and 54 residues, respectively. A smaller loop of five residues occurs due to a disulfide bond between the cysteines 65 and 71. A similar disulfide arrangement is indicated for subdomain II which is separated from subdomain I by a segment of 20 amino acid residues. The same arrangement of disulfide bonds has been strongly suggested for the alpha 2(IV)NC1 monomer by the isolation and characterization of its disulfide-bridged tryptic fragments. Similar investigations on the dimeric alpha 1(IV)NC1 domain established the arrangement of the intermolecular disulfide bonds. They are formed by a complete disulfide exchange between corresponding disulfide knots of two monomeric NC1 domains.
Siebold
B
B
Max Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Deutzmann
R
R
Kühn
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001485:Basement Membrane / Q000032:analysis*; D002845:Chromatography; D003094:Collagen / Q000032:analysis*; D004220:Disulfides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D046911:Macromolecular Substances; D003012:Microbial Collagenase; D010920:Placenta / Q000032:analysis; D011247:Pregnancy
D000596:Amino Acids; D004220:Disulfides; D046911:Macromolecular Substances; D003094:Collagen; D003012:Microbial Collagenase