THE ARRANGEMENT OF INTRAMOLECULAR AND INTERMOLECULAR DISULFIDE BONDS IN THE CARBOXYTERMINAL, NON-COLLAGENOUS AGGREGATION AND CROSS-LINKING DOMAIN OF BASEMENT-MEMBRANE TYPE-IV COLLAGEN
THE ARRANGEMENT OF INTRAMOLECULAR AND INTERMOLECULAR DISULFIDE BONDS IN THE CARBOXYTERMINAL, NON-COLLAGENOUS AGGREGATION AND CROSS-LINKING DOMAIN OF BASEMENT-MEMBRANE TYPE-IV COLLAGEN
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DOI：
10.1111/j.1432-1033.1988.tb14321.x
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发表时间：
1988-10-01
期刊：
影响因子：
--
通讯作者：
KUHN, K
中科院分区：
文献类型：
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作者：
SIEBOLD, B;DEUTZMANN, R;KUHN, K
用胶原酶消化人胎盘，分离出IV型胶原球状结构域的六聚体复合体，并对存在的不同单体和二聚体进行了层析分离。α1(IV)Nc1与α2(IV)Nc1的比例为2：1。约50%的Nc1结构域与二聚体相连。主要发现α1-α1二聚体。只有12%是α2-α2二聚体，没有α1-2α。可以检测到二聚体。大多数(88%)的分子间键是二硫键。其余的不能通过还原来切割。为了阐明二硫键的排列，未还原的α1(IV)NC1单体用溴化氰处理，分离出二硫键桥联的多肽，并以Edman降解为特征。单体中的两个同源亚区中的每一个都由一组相同的三个二硫键稳定。在亚结构域I中，位置20和53的半胱氨酸与C末端的半胱氨酸对108和111相连。这样形成的二硫化物结分别稳定了32个和54个残基的两个相互连接的环。由于半胱氨酸65和71之间的二硫键，出现了一个较小的五个残基的环。亚区II也有类似的二硫键排列，与亚区I相隔20个氨基酸残基。通过分离和表征α2(IV)NC1单体的二硫键桥联的胰酶片段，强烈建议其二硫键的排列方式与之相同。对二聚体α1(IV)NC1结构域的类似研究证实了分子间二硫键的排列。它们是由两个单体NC1结构域的相应二硫键结之间的完全二硫键交换形成的。
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.1-2.alpha. 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.