| Project/Area Number |
06556012
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Tohoku University |
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Principal Investigator |
MIZUNO Shigeki Tohoku University, Department of Applied Biological Chemistry, Professor, 農学部, 教授 (90112903)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAGI Takashi Tohoku University, Graduate School of Science, Assistant Professor, 理学研究科, 助教授 (00004474)
HARATA Masahiko Tohoku University, Department of Applied Biological Chemistry, Assistant, 農学部, 助手 (70218642)
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| Project Period (FY) |
1994 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1996: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1995: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Keywords | silkworm / silk-producing insect / naked-pupa mutant silkworm / fibroin molecular complex / anti-peptide antibody / disulfide bond / hydrophobic interaction / glycoprotein / 後部絹糸腺 / cDNAクローニング / フィブロイン低分子成分 / タンパク質糖鎖修飾 |
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| Research Abstract |
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.
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