Properties and functions of the silk protein molecular complex

丝蛋白分子复合物的性质和功能

基本信息

批准号：
06556012
负责人：
MIZUNO Shigeki
金额：
$ 3.97万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

1. Silk fibroin produced by the silkworm, Bombyx mori, is a molecular complex containing three protein components ; H-chain of 350 kDa, L-chain of 25 kDa and P25 of approximately 30 kDa. Using anti-peptide antibodies against these protein components, it was shown that H and L-chains were disulfide linked but P25 associated with them by non-covalent, primarily hydrophobic interactions.2. Sites of disulfide linkage between H and L-chains were determined by digesting the H-L complex with lysylendopeptidase, immunodetecting and isolating the disulfide-linked peptides, and sequencing peptides after reducing the disulfide linkage. The results indicate that Cys-172 of L-chain forms a disulfide bond with the Cys located at the 20th residue from the C-terminus of H-chain.3. P25 was suggested to have three Asn-linked sugar chains from its reactivity to ConA,reduction of molecular size after digestion with N-glycosidaseF and from its cDNA sequence. In naked pupa mutants, in which H and L-chains do not form a disulfide linkage and the secretion of fibroin is reduced to less than 1% of the normal level, L-chain was undetectable but H-chain and P25 were present in the small amount of fibroin secreted, suggesting that P25 has higher affnitiy to H-chain. P25 in these mutants contains sugar chains but migrated faster on SDS-PAGE,which suggests that under the conditions to form H-L・P25 molecular complex, one of the N-glycosylation sites in P25 may become unavailable.4. Homologues of L-chain and P25 were identified in other silk producing insects (Dendrolimus spectabilis and Papilio xuthus) and their cDNA sequences were cloned. Those sequences indicate well conserved cysteine residues and N-glycosylation sites (for P25). L-chain and P25 were not found but H-H dimer was formed instead in Antheraea species.

1。蚕bombyx mori产生的丝纤维蛋白是一种分子络合物，包含三个蛋白质成分。 H链为350 kDa，L链为25 kDa，p25的H链约为30 kDa。使用针对这些蛋白质成分的抗肽抗体，结果表明H和L链是二硫键的，但P25与非共价，一级疏水相互作用相关。2。 H和L链之间的二硫键部位是通过用赖氨酸肽酶消化H-L复合物，免疫检测和分离二硫键连接的Petides的，并在降低二硫键链接后进行测序的Petides来确定的。结果表明，L链的Cys-172与位于H链的C端位于第20居住地的Cys形成二硫键。3。建议p25具有三个ASN连接的糖链，从其对CONA的反应性，用N-糖苷酶消化后的分子大小降低，并从其cDNA序列。在裸露的pupa突变体中，其中H和L链不会形成二硫键，并且肌动蛋白的分泌降低至正常水平的小于1％，L链是无法检测到的，但H-Chain和P25是少量的fibroin分泌的，这表明P25具有更高的Affnitiy和H-Chainy。这些突变体中的p25含有糖链，但在SDS-PAGE上迁移得更快，这表明在形成H-L p25分子复合物的条件下，P25中的N-糖基化位点之一可能变得不可用。4。在其他丝产生的绝缘（树枝状镜和帕皮里奥Xuthus）中鉴定出L链和p25的同源物，并克隆其cDNA序列。这些序列表明保守的半胱氨酸保留和N-糖基化位点（对于P25）。找不到L链和p25，但是在花药物种中形成了H-H二聚体。