Primary structure and possible functions of a trypsin inhibitor of Bombyx mori

Abstract

A protein with a low molecular mass of 6027 was purified from cocoon shell of silkworm, Bombyx mori. Twodimensional polyacrylamide gel electrophoresis (2D/PAGE) resolved this protein into a single spot with pI 4.3 and M r 6000. Amino acid sequence analysis revealed that this protein consists of 55 amino acids, six of these being cysteine residues and is highly homologous to bovine pancreatic trypsin inhibitor-type inhibitors. The 6-kDa protein is heat stable and acid stable and inhibits bovine trypsin by forming a low-dissociation complex with trypsin in a 1 : 1 molar ratio (K i = 2.8 £ 10 ±10 ), but does not a-chymotrypsin. This cocoon shell-associated trypsin inhibitor (CSTI) was thus concluded to belong to the bovine pancreatic trypsin inhibitor class. CSTI was developmentally regulated in the silk gland at the final stage of larval growth, and its specific distribution in the middle silk gland, an organ in which silk proteins are stored during the final larval instar, occurred before the onset of spinning. This inhibitor protects the tryptic degradation of fibroin light (L) chain in vitro. These results suggest that this trypsin inhibitor may play an important part on regulating proteolytic activity in the silk gland or protecting silk proteins from degradation during histolysis.Keywords: amino acid sequence; Bombyx mori; cocoon; trypsin inhibitor.The silkworm Bombyx mori produces massive amounts of silk proteins during the final stage of larval development (fifth instar), and these proteins are stored in the middle silk gland until they are discharged through the anterior duct and the spinneret at the end of the fifth instar. As major components of cocoons, two kinds of silk proteins have been distinguished: fibroin, a fibrous protein composed of one heavy (H) chain of about 350 kDa and a L-chain of about 25 kDa linked by disulfide bond(s); and sericin, serving as an adhesive to unite fibroin for making cocoons. The crystalline structures, chemical compositions and physical properties of the silk proteins have been extensively studied, but it is believed that these proteins in the cocoon shell are biologically inactive molecules. We previously have reported that four low-molecular-mass proteins are present in a water-soluble extract from the cocoon shell as minor components. These proteins with relative molecular masses of 10 kDa, 6 kDa, 4 kDa and 3 kDa were found to be distinct from both types of silk proteins on the basis of amino acid composition analysis [1,2]. N-terminal amino acid sequence analysis revealed that the 4-kDa protein was highly homologous to Kazal-type trypsin inhibitors [3]. Biologically active proteins would thus appear to quite likely coexist with silk proteins in the cocoon shell. Although trypsin and a-chymotrypsin inhibitors in the hemolymph of insects such as B. mori [4,5], Antheraea pernyi [6], Drosophila melanogaster [7] and Manduca sexta [8] have been demonstrated, such protease inhibitor has yet to be shown present in the cocoon shell.We here report the purification, protein se...

Purification and characterization of cocoonase from the silkwormBombyx mori

“…They are designated cocoon shell-associated trypsin inhibitors (CSTIs). 36) Although the physiological role of CSTI is not fully known, it has been suggested that it plays a significant role in biological defense mechanisms. 23,37) CSE from the p50 strain of B. mori also inhibited the activity of bovine trypsin strongly, but it showed only very slight activity against bmCCN.…”

“…36) Although the physiological role of CSTI is not fully known, it has been suggested that it plays a significant role in biological defense mechanisms. 23,37) CSE from the p50 strain of B. mori also inhibited the activity of bovine trypsin strongly, but it showed only very slight activity against bmCCN. Hence it is likely that a CSTI protects the cocoon from accidental proteolysis by extrinsic factors such as a protease from pathogenic bacteria, 37) and that upon eclosion CSTI does not interfere with the CCNcatalyzed proteolysis of sericin.…”

“…The filtrate was concentrated to 10 mL by further boiling for 1-2 h, dialyzed overnight against water at 4°C, and used as a sericin solution. Cocoon shell extract (CSE) was prepared by the method of Kurioka et al 23) Each of the 11 cocoons (1.04 g) was cut into 8-10 pieces. These pieces were combined, suspended in 50 mL of 10 mM Tris-HCl buffer (pH 7.5), stirred at room temperature for 15 h, and filtered through a membrane filter (0.2 μm).…”

Silk-based biomaterials

“…Furthermore, protease cocktails [54] and chymotrypsin (known to be produced by macrophages) are capable of enzymatically degrading silk [55]. Of interest, the silkworm B. mori produces a protease inhibitor in the silk gland embedding it within the silk cocoon for protection against premature proteolytic degradation [56].…”

Purification and Identification of Flavonoids from the Yellow Green Cocoon Shell (Sasamayu) of the Silkworm,Bombyx mori

“…Recently bioactive compounds such as protease inhibitor polypeptides have been isolated from B. mori cocoons and their presence was shown to protect silk protein from proteolytic degradation. 4) In plants, ‰avonoids function as attractants of pollinators, 5) UV-protectants, 6,7) antimicrobial substances or phytoalexins, 8) and signaling molecules 9) in many species. Flavonoids in cocoon may thus be a protective agent and play an important role in protecting silk e.g.…”