Determination of the site of disulfide linkage between heavy and light chains of silk fibroin produced by Bombyx mori.

The analysis of fibroin secretion-deficient 'naked-pupa' mutant silkworms has suggested that the disulfide linkage between heavy (H) and light (L) chains of fibroin, produced by the silkworm, Bombyx mori, is essential in its efficient large-scale secretion from the posterior silk gland cells. However, the site of disulfide-linkage between H- and L-chains has not been determined. In this study, cysteine residues involved in the single disulfide linkage between H- and L-chains were identified as the twentieth residue from the carboxyl terminus of H-chain (Cys-c20) and Cys-172 of L-chain by sequencing of genomic clones and peptide analysis. Furthermore, Cys-c4 (fourth residue from the carboxyl terminus) and Cys-c1 at the carboxyl terminus of H-chain were shown to form an intramolecular disulfide bond.

[1]  P. Couble,et al.  Structural organization of the P25 gene of Bombyx mori and comparative analysis of its 5' flanking DNA with that of the fibroin gene. , 1985, Nucleic acids research.

[2]  R. F. Manning,et al.  Internal structure of the silk fibroin gene of Bombyx mori. I The fibroin gene consists of a homogeneous alternating array of repetitious crystalline and amorphous coding sequences. , 1980, The Journal of biological chemistry.

[3]  K. Kimura,et al.  Reduced level of secretion and absence of subunit combination for the fibroin synthesized by a mutant silkworm, Nd(2) , 1984, The Journal of cell biology.

[4]  Y. Kikuchi,et al.  Further evidence for importance of the subunit combination of silk fibroin in its efficient secretion from the posterior silk gland cells , 1987, The Journal of cell biology.

[5]  C. Alberini,et al.  Developmental regulation of IgM secretion: The role of the carboxy-terminal cysteine , 1990, Cell.

[6]  Toshio Yamamoto,et al.  Genetic analysis of the Nd-s mutation in the silkworm, Bombyx mori , 1984 .

[7]  K Tanaka,et al.  Hydrophobic interaction of P25, containing Asn-linked oligosaccharide chains, with the H-L complex of silk fibroin produced by Bombyx mori. , 1999, Insect biochemistry and molecular biology.

[8]  D. Russell,et al.  Transport-deficient mutations in the low density lipoprotein receptor. Alterations in the cysteine-rich and cysteine-poor regions of the protein block intracellular transport. , 1988, The Journal of biological chemistry.

[9]  Y. Tsujimoto,et al.  The dna sequence of bombyx mori fibroin gene including the 5′ flanking, mRNA coding, entire intervening and fibroin protein coding regions , 1979, Cell.

[10]  J. Miyazaki,et al.  Intracellular transport blockade caused by disruption of the disulfide bridge in the third external domain of major histocompatibility complex class I antigen. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[11]  K. Mita,et al.  Specific codon usage pattern and its implications on the secondary structure of silk fibroin mRNA. , 1988, Journal of molecular biology.

[12]  S. Waga,et al.  Different behavior of chromatin domains encompassing fibroin heavy-chain gene in active, temporarily inactive, and permanently inactive transcriptional states in silk gland nuclei. , 1993, The Journal of biological chemistry.

[13]  T. Toyo’oka,et al.  Application of a fluorogenic reagent, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate for detection of cystine-containing peptides. , 1985, Journal of Biochemistry (Tokyo).

[14]  T. Tobe,et al.  Complete determination of disulfide bonds localized within the short consensus repeat units of decay accelerating factor (CD55 antigen). , 1992, Biochimica et biophysica acta.

[15]  A. Kikuchi,et al.  The Occurrence of Small Component Proteins in the Cocoon Fibroin of Bombyx mori , 1982 .

[16]  M. Alting-Mees,et al.  pBluescriptII: multifunctional cloning and mapping vectors. , 1992, Methods in enzymology.

[17]  T. Tamura,et al.  Preferential codon usage and two types of repetitive motifs in the fibroin gene of the Chinese oak silkworm, Antheraea pernyi , 1997, Insect molecular biology.

[18]  Shigeru Sato,et al.  Ultrastructural study of the posterior silkgland in the Nd, Nd-s and Nd-sD mutants with a defect of fibroin synthesis , 1985 .

[19]  T. Takagi,et al.  Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis. , 1989, Journal of molecular biology.

[20]  G. Wahl,et al.  Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[21]  P. Couble,et al.  Complete nucleotide sequence of the gene encoding the Bombyx mori silk protein P25 and predicted amino acid sequence of the protein. , 1986, Nucleic acids research.

[22]  U. Rüegg,et al.  [10] Reductive cleavage of cystine disulfides with tributylphosphine , 1977 .

[23]  C. Earland,et al.  A study of the cystine residues in Bombyx mori and other silks. , 2009, International journal of peptide and protein research.

[24]  K. Tanaka,et al.  Immunological identification of the major disulfide-linked light component of silk fibroin. , 1993, Journal of biochemistry.

[25]  G. Deléage,et al.  Aminoacid sequence and putative conformational characteristics of the 25 KD silk protein of Bombyx mori , 1986 .

[26]  K. Tanaka,et al.  Production of a chimeric fibroin light-chain polypeptide in a fibroin secretion-deficient naked pupa mutant of the silkworm Bombyx mori. , 1995, Journal of molecular biology.