Properties and functions of the silk protein molecular complex

1.丝绸纤维素是由丝绸蠕虫、Bombyx Mori生产的，是一种含有三种蛋白质成分的分子复合体; 350 kDa的H链、25 kDa的L链和30 kDa的P25。使用抗肽抗体来对抗这些蛋白质成分，它展示了H和L链与干扰相连，但与P25相关的非关联、主要的羟基相互作用。H和L-链之间的差异位点是通过鉴别H-L复合体与淋巴结肽酶、免疫测定和隔离的差异化肽，以及在减少差异化连接后对差异化肽进行测序的。结果表明，Cys-172的L链形式与位于Cys第20个驻地的C终端的C-终端形成了混乱的债券。P25建议有三个Asn-连接的糖链，从其反应活性到ConA，通过N-糖基酶F在消化后减少分子尺寸，并从其cDNA序列中减少分子尺寸。在裸猪突变体中，H和L链不能形成一个中断连接，纤维素的秘密减少到低于正常水平的1%，L链是不可分割的，但H链和P25在纤维素保密的数量很小，建议P25对H链有更高的亲和力。在这些突变体含有糖链但在SDS-PAGE上迁移速度更快的P25中，关于形成H-L·P25分子复合体的条件下的建议，P25中的N-糖基化位点之一可能变得不可用。L链和P25的同源性在其他丝绸生产昆虫（Dendrolimus spectabilis和Papilio xuthus）中被识别，其cDNA序列被克隆。这些序列指示良好地使用半胱氨酸安置和N-糖基化位点（用于P25）。L-chain和P25没有找到，但H-H二聚体是在Anthaea物种中形成的。

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.