Studies on Chitin Recognition Mechanism and Biosynthesis of Cuticle Proteins in Silkworm, Bombyx mori

Insect cuticle is composed mainly of chitin, a polymer of N-acetylglucosamine (GIcNAc), and chitin-binding cuticle proteins. The physical and mechanical properties of cuticle arc highly diverse depending on the species, developmental stages, sex, body regions, and so on. These various properties are caused by cuticle protein composition. Therefore, to understand the mechanism by which cuticle maintains stable structure and obtains various mechanical properties, it is important to analyze chitin-recognition mechanism of cuticle proteins. On the other hand, the majority of cuticle protein genes is expressed in stage dependent manner. Moreover, it has been reported that genes for two of major cuticle proteins from silkworm, Bombyx moria, are transactivated by juvenile hormone (JH), whose mode of action has been unknown yet. These indicate that cuticle protein genes provide an excellent model system to study control of gene expression in post-embryonic development including JH signaling. I approached these two subjects, the chitin-recognition mechanism and the regulation mechanism of gene expression using cuticle proteins of B. mori. (1) Chitin recognition mechanism of cuticle proteins from Bombyx mori In larval cuticle of B. mori, four cuticle proteins, BMCP3O, 22, 18, and 17 have been identified. To analyze chitin-binding activity of cuticle proteins, I attempted purification of these proteins in native form. Soluble proteins were extracted with guanidine-HCI solution from fifth instar larval cuticle, followeded by dialysis against a guanidine-free buffer. Cuticle protein fraction was separated by ammonium sulfate precipitation (20% saturated ammonium sulfate), and each cuticle protein was purified by two cycles of ion-exchange chromatography. All purified cuticle proteins kept high affinity to chitin. The chitin-binding activity of purified BMCP30 was analyzed by chitin-affinity chromatography. It was revealed that pH optimum for binding to chitin of BMCP30 is pH 6.4, which corresponds to hemolymphal pH. Competition experiments using chitooligosaccharides showed that binding to chitin was not prevented by trimer of GIcNAc, but prevented by hexamer, indicating that BMCP3O recognizes 4-6 mer of GIcNAc as a unit for binding. BMCPs possess about 70 amino acid region conserved widely among cuticle proteins from different insect species. To determine the chitin binding domain of BMCP3O, I concerned my attention to the conserved region. The chitin-binding activities of BMCP3O and BMCP18 which have the conserved region in common were compared. They showed similar high chitin-binding activities, implying that the conserved region functions as a chitin binding domain. On the other hand, chitin-binding activity of BMCP30 showed resistance stronger than to urea solution than that of BMCP18. This result suggests that an additional chitin affinity region is present in another region of BMCP3O. To verify that the conserved region functions actually as chitin binding domain, a series of BMCP30 deletion mutants fused with glutathione S-transferase (GST) was synthesized in E. coli, and their chitin-binding activities were analyzed. The recombinant proteins bearing full length or only the conserved region of BMCP30 bound to chitin well. In contrast, the recombinant protein with only N-terminal region of BMCP30 and lacking the conserved region showed reduced chitin-binding activity. It indicated that the conserved region functions as the primary chitin binding domain. Some chitin-binding activity of the recombinant protein with only N-terminal of BMCP30 suggested an additional chitin-affinity region consistently with result from comparison of BMCP30 and BMCP18. The aromatic amino acid residues were highly conserved in the conserved region of cuticle proteins. The knowledge from chitin binding protein of other organisms suggested these aromatic residues in cuticle protein serve important role for binding to chitin. (2) Expression control of cuticle protein gene in Bombyx mori Although genomic clone for BMCP18 has been isolated previously, its structural analysis has been unsatisfactory. To study the control of cuticle protein gene expression, I analyzed the structure of BMCP18 genomic clone in detail. It was revealed that BMCP18 gene consists of three exons interspersed by two introns. The transcription initiation site was determined by primer extension analysis. A structural comparison of the BMCP18 gene and related cuticle protein genes of other lepidopteran species (MSCP14.6 and HCCP12) showed that the 5' upstream region of the BMCP18, MSCP14.6, and HCCP12 genes has a 12-bp identical sequence matching the recognition sequence for transcription factors COUP-TF and HNF-4. This implies that molecular mechanisms regulating expression of these cuticle protein genes are also conserved. mRNAs coding for Bmsvp, the B. mori homolog of Drosophila Seven-up, which is known as a homolog of vertebrate COUP TF, and BmHNF-4, a homolog of ver ...