Chemical composition and biosynthesis of silk proteins

Shimura, Kensuke

doi:10.1007/bf01965160

Cited by 37 publications

(21 citation statements)

References 22 publications

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“…4). In this situation the metamorphosing insect synthesizes a methioninepoor protein that by electrophoretic properties resembles a silk [Shimura, 1983]. It presumptively uses a The gland is negative for single-strand breaks until day 3, when intense labeling is seen.…”

Section: Discussionmentioning

confidence: 99%

“…The labial glands of Manduca sexta are homologues of the Bombyx mori silk glands. Silk fibroin, the major product of the silk glands, contains a high proportion of glycine and alanine but very little methionine [Shimura, 1983]. Therefore, we tested whether total protein synthesis in Manduca labial glands measured by incorporation of radioactive glycine would differ from that revealed by incorporation of [ 35 S]-methionine.…”

Section: Drop In Total Protein Synthesismentioning

confidence: 99%

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Protein synthesis, DNA degradation, and morphological changes during programmed cell death in labial glands ofManduca sexta

et al. 1997

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Labial glands of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingiidae), homologues of Drosophila salivary glands, undergo programmed cell death (PCD) in a 4‐day period during larva‐to‐pupa metamorphosis. The programmed death of the labial gland was examined by electron microscopy and measurement of protein synthesis as well as measurement of DNA synthesis, end‐labeling of single strand breaks, and pulsed‐field gel electrophoresis. One of the earliest changes observed is a sharp drop in synthesis of most proteins, coupled with synthesis of a glycine‐rich protein, reminiscent of silk‐like proteins. From a morphological standpoint, during the earliest phases the most prominent changes are the formation of small autophagic vacuoles containing ribosomes and an apparent focal dissolution of the membranes of the endoplasmic reticulum, whereas later changes include differing destruction at the lumenal and basal surfaces of the cell and erosion of the basement membrane. By the fourth day of metamorphosis, individual cells become rapidly vacuolated in a cell‐independent manner. In the vacuolated cells on day 3, chromatin begins to coalesce. It is at this period that unequivocal nucleosomal ladders are seen and end‐labeling in situ or electrophoretic techniques document single or double‐strand breaks, respectively. DNA synthesis ceases shortly after the molt to the fifth instar, as detected by incorporation of tritiated thymidine and weak TUNEL labeling. Large size fragments of DNA are seen shortly after DNA synthesis ceases and thence throughout the instar, raising the possibility of potential limitations built into the cells before their final collapse. Dev. Genet. 21:249–257, 1997. © 1997 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

Section: Drop In Total Protein Synthesismentioning

confidence: 99%

Protein synthesis, DNA degradation, and morphological changes during programmed cell death in labial glands ofManduca sexta

et al. 1997

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“…D, materials in peak fractions in A, and purified Hchain, L-chain, and P25 as controls, were subjected to ELISA with the anti-P25 antibody. tification were 350 kDa for H-chain (12,19), 26 kDa for L-chain (1), and 30 kDa (in the breeds producing the normal level fibroin) or 27 kDa (in the naked pupa mutants) for P25.…”

Section: Fig 3 Separation Of P25 Peptidesmentioning

confidence: 99%

Silk Fibroin of Bombyx mori Is Secreted, Assembling a High Molecular Mass Elementary Unit Consisting of H-chain, L-chain, and P25, with a 6:6:1 Molar Ratio

Inoue¹,

Tanaka²,

Arisaka³

et al. 2000

Journal of Biological Chemistry

657

501

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Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.A vast amount of silk fibroin is synthesized within the cells of a pair of PSGs 1 of the silkworm, Bombyx mori, during the larval fifth instar, secreted into the lumen of PSG, and transported through the middle silk gland, where heterogeneous molecules of sericin are added, and further toward the anterior part of the silk gland, where the silk fiber is formed and spun. Both the efficient secretion of the vast amount of fibroin from PSG cells into the lumen and the maintenance of solubility of fibroin during the luminal transport have been speculated to be facilitated by the formation of a molecular complex consisting of fibroin heavy chain (H-chain) of 350 kDa and two lower molecular mass protein components: fibroin light chain (Lchain) of 26 kDa (1) and P25, which is a glycoprotein of about 30 kDa (2).H-chain and L-chain are linked by a single disulfide bond between Cys-172 of L-chain and Cys-c20 (twentieth residue from the C terminus) of H-chain (3). The H-L linkage is essential for the secretion of a vast amount of fibroin, because the silkworm carrying the Nd-s or Nd-s D mutation of the L-chain gene (fibL) could not form the disulfide linkage with H-chain, and less than 1% of the normal level fibroin is secreted (4).This tremendous reduction of fibroin secretion has been suggested to be caused by the appearance of a free sulfhydryl group of Cys-c20 on H-chain, which prevents the transport of H-chain from ER to Golgi (4).On the other hand, P25 has been shown to associate with the H-L complex by noncovalent interactions, i.e. mainly by hydrophobic interactions with the H-chain moiety (2, 5). However, the role of P25 in the formation of the fibroin molecular complex has not been elucidated, mainly because a silkworm carrying a mutation of the P25 gene has not been available.In the present study, a m...

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“…Then, the fibroin is secreted into the cocoon via the anterior silk gland (ASG). The silk fibroin contains three polypeptides: a 350-kDa heavy chain (H-chain, Shimura, 1983), a 26-kDa light chain (L-chain, Yamaguchi et al, 1989), and fibrohexamerin (fhx), originally named P25 (Inoue et al, 2000). In the PSG, these proteins form a large protein complex called an elementary unit of fibroin, which consists of a 6:6:1 molar ratio of the H-chain:Lchain:fibrohexamerin (Inoue et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

A fibroin secretion-deficient silkworm mutant, , provides an efficient system for producing recombinant proteins

Inoue

Kanda

Imamura

et al. 2005

Insect Biochemistry and Molecular Biology

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Chemical composition and biosynthesis of silk proteins

Cited by 37 publications

References 22 publications

Protein synthesis, DNA degradation, and morphological changes during programmed cell death in labial glands ofManduca sexta

Protein synthesis, DNA degradation, and morphological changes during programmed cell death in labial glands ofManduca sexta

Silk Fibroin of Bombyx mori Is Secreted, Assembling a High Molecular Mass Elementary Unit Consisting of H-chain, L-chain, and P25, with a 6:6:1 Molar Ratio

A fibroin secretion-deficient silkworm mutant, , provides an efficient system for producing recombinant proteins

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