Identification Of Chitin-binding Proteins From The Larval Silk Gland And Function Analysis Of Cuticle Protein BmCPAP3 In The Silkworm(Bombyx Mori)

The silkworm, Bombyx mori, has been widely considered as one of the ideal model for the Lepidoptera investigation. Because it could spin cocoon, silkworm has a very important economic value due to that it could spin cocoon. The silk gland is the only spinning organ, and could be divided into three compartments according to the morphology and function, including anterior silk gland, middle silk gland, and posterior silk gland. Fibroins are produced by the posterior silk gland and the sericins are produced by the middle silk gland, the anterior silk gland cann’t secrete silk proteins. There is a membrane structure in the inner lumen of the silk gland when we observe from the cross-section of silk gland. The inner membrane is mainly composed of chitin and proteins. The inner membrane in anterior silk gland is more thicker than that in the middle silk gland and posterior silk gland. There are many channels in the inner membrane for secreting proteins by the silk gland cells. When the silk protein transport in anterior silk gland, the inner membrance can provide the shearing force when liquid fibroin transform from into gel phase and could protect the silk gland cells against injury.The inner membrane is mainly composed of chitin and proteins, while, the research about the proteins of the inner membrane is very rarely. The transcriptome and proteome studies about of anterior silk gland identified 53 and 33 cuticular proteins, but, the authors do not verified whether these proteins could bind chitin or not. Compare the anterior silk gland with the middle silk gland and the posterior silk gland, what is the difference between the protein compositions in the inner membrane? The epidermis and peritrophic membrane of silkworm are the typical membrane of chitin and proteins formed, however, what are differences of the epidermis and peritrophic membrane with the anterior silk gland? In order to clarify these issues, we observed an inner membrane of the silk gland by SEM and immunofluorescence, and we extracted the chitin binding proteins from different position of the silk gland by Chitin affinity chromatography and identified by LC-MS/MS. We compared chitin-binding proteins of the silk gland and the epidermal and peritrophic membrane, then revealing the special of the inner membrane of silk gland. Analysis of the Mass spectrometry results, we selected the cuticular protein BmCPAP3 of CPAP family, then we prokaryotic expression, purification, polyclonal antibody, the expression patterns analysis and the mode of chitin-binding of BmCPAP3. The main results are as follows:1. The observation of the inner membrane in silk glandWe observed an inner membrane in the anterior silk gland by SEM and found the membrane become thinner from anterior to posterior silk gland. It is difficult to observe the inner membrane in the middle and posterior silk gland. However, by immunofluorescence, we can observed a thin membrane in the the middle and the posterior silk gland and a thick membrane in the anterior silk gland.2. The chitin binding proteins extracted and identified from different compartments of silk glandThe chitin binding proteins were extracted from different position of the silk gland by chitin affinity chromatography and identified by LC-MS/MS. The results showed that these chitin binding proteins contain cuticular proteins, enzymes, and proteins of unknown function. The cuticular proteins were classified into several families, including CPR, CPAP, CPH, and CPG. By intensity based absolute quantification（iBAQ） method, we found that CPR proteins have the highest abundance and CPAP proteins come second. By comparing the abundance of chitin-binding proteins among different compartments of the silk gland, we found that they are more in the anterior silk gland than in the middle and posterior silk gland. By analyzing the cuticular protein families, we found that R&R-2 and CPAP proteins have higher abundance in the anterior silk gland than in the middle and posterior silk gland, on contrary, R&R-1 proteins are more in the middle silk gland.The chitin binding proteins in the silk gland are similar to that in the epidermis instead of that in the peritrophic matrix. Previous studies reported that R&R-2 domainwere involved in hard epidermis and R&R-1 domain were involved in the soft epidermis. The CPAPs have similar location with the R&R-2 proteins. So, we speculate that CPAPs and R&R-2 proteins bind with the chitin to form the inner membrane in the anterior silk gland. The protein-chitin complex could provide a shearing force for the conformational transition of silk proteins and also may protect the silk gland cells against injury.3. Prokaryotic expression and purification, expression pattern and chitin-binding mode analysis of Bm CPAP3.Bioinformatics analysis showed that the molecular weight of BmCPAP3 is 29 kDa and the isoelectric point is 4.82. There are three ChtBD2 domains in the BmCPAP3. We expressed the active recombinant protein BmCPAP3 and prepared polyclonal antibody. The expression patterns of BmCPAP3 in various tissues were analyzed by reverse transcription polymerase chain reaction（RT-PCR） and western blotting. At both the transcriptional and protein levels, BmCPAP3 was expressed in the head, epidermis and silk gland. In the silk gland, BmCPAP3 has a high expression in the 4th instar moltingand relatively lower expression at other stges. In the epidermis, the expression of BmCPAP3 decreased with the growth of the silkworm.BmCPAP3 consists of three tandem CPAP domains. In order to explore the binding mode of BmCPAP3 withthe chitin. We expressed the active recombinant protein domain₃, domain₁-2, domain₂-3. We found that all the domain₃, domain₁-2, domain₂-3 could bind to chitin in vitro but their binding ability were different. Individual domains could combined with chitin, but two domains have stronger chitin binding capacity than the single domain.