Properties and functions of the silk protein molecular complex

1. Silk fibroin produced by the silkworm, Bombyx mori是一个molecular complex containing three protein componentsH-chain of 350 kDa，L-chain of 25 kDa and P25 of approximately 30 kDa. Using anti-peptide antibodies against theseprotein components,这应该是H and L-chains were disulfide linked but P25 associated with them by non-covalent，2. primarily hydrophobic interactions. Sites of disulfide linkage between H and L-chains weredetermined by digesting the H-L complex with lysylendopeptidase免疫detecting and isolating the disulfide-linked peptidesand sequencing peptides after reducing the disulfide linkage. the results indicate that Cys-172 ofl chain forms a disulfide bond with the Cys located at the 20th residue from the C-terminus of3. P25 was suggested to have three Asn-linked sugar chains from its reactivity toConA,reduction of molecular size after digestion with N-glycosidaseF and from its cDNA sequence. Innaked pupa mutants，in which H and L-chains do not form a disulfide linkage and the secretion of fibroin is reduced toless than 1% of the normal levelL-chain was undetectable but H-chain and P25 were present in the small amount of fibroin secreted，P25在这些mutants contains sugar chains but P25有高的affnitiy to H-chainmigrated faster on sd - page,which suggests that under the conditions to form H-L P25 molecularcomplex)》,one of the N-glycosylation sites in P25 may become unavailable.4. Homologues of L-chain and P25 wereidentified in其他silk producing insects (Dendrolimus spectabilis and Papilio xuthus) and theircDNA sequences是cloned. Those sequences indicate well conserved cysteine residues andN-glycosylation sites (for P25). L-chain and P25 were not found but H-H dimer was formed instead inantheraea species。

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.