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

Abstract

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...

Silk based biomaterials to heal critical sized femur defects

“…Silk fibroin, the structural, hydrophobic, and approximately 370 kDa protein isolated from the cocoon of B. mori [43][44][45][46] that has emerged as a novel substrate for engineering biomaterials, has proven to be biocompatible [22,33], offers distinguishing mechanical properties when compared to other naturally derived or degradable polymeric biomaterials [47], and exhibits low inflammatory response [21,22].…”

Characterization of kinesin-like proteins in silkworm posterior silkgland cells

“…Fibroin, synthesized in PSG cells and secreted into the lumen, consists of a heavy chain, a light chain (L-chain) and a glycoprotein P25 [20]. To examine whether BmKinesin-1 interacts with fibroin-containing vesicles in PSG cells, we produced a rabbit polyclonal antibody of fibroin L-chain (FLch) (Supplementary information, Figure S8A and S8B) to perform the pulldown assay.…”

“…These highly specialized and polarized PSG cells secrete fibroin, one of the major silk components, into the PSG lumen [19,20]. Fibroin is composed of heavy chain (molecular weight 350 kDa), light chain (molecular weight 26 kDa), and P25 with a molar ratio of 6:6:1 [20].…”

Evaluating the impact of different exogenous factors on silk textiles deterioration with use of size exclusion chromatography

“…Regarding the first assumption, it is known that amorphous regions are easily degraded due to better accessibility of degradation agents such as VOCs, water vapour, oxygen [9][10][11]50], unlike tightly packed crystalline regions. The second assumption has its consequences in the calculation of DP.…”

“…Ordered crystalline regions making up about 60 % of the fibroin mass [6] are interspersed with amorphous regions located in the area of the light chain [7]. These regions are richer in the hydrophilic amino acids such as serine, threonine, arginine and lysine, which are more susceptible to degradation [8][9][10][11][12].…”