Proteomics Of Ectoperitrophic Space And Biochemical Characterization Of Chitin Deacetylases From Bombyx Mori

Insect peritrophic membrane(PM)is a chitin-protein layer secreted by midgut epithelial cells,playing two important physiological roles.First,the PM functions as a barrier to separate epithelial cells from food particles,thus defending the invasion of exogenous harmful substances.Interference or destruction of the PM structure will promote invasion of pathogenic microorganisms or pesticides,affecting the normal development of insects or even leading to death.Therefore,the PM and the proteins which involved in PM formation and maintaining,are considered promising targets for pest control.Second,the PM provides structural basis for the functional differentiation of different compartments within midgut.The PM creates three distinct compartments within the midgut.including the epithelial cell,the ectoperitrophic space(EcPS)and the endoperitrophic space.The EcPS.,which is between the insect midgut epithelial cells and the PM.is an unexplored resource for concentrated secreted proteins of the midgut.These EcPS proteins may be involved in the formation and dynamic changes of PM structure.Therefore,the study of EcPS protein is of great significance in the agrochemical discovery targeting peritrophic membrane.For two reasons,Bombyx mori is an appropriate model to study the functions of peritrophic membrane.On the one hand,the genome of B.mori has been fully sequenced and annotated.On the other hand.the midgut of B.mori is well developed.In this dissertation,we performed proteomic analyses of the secreted proteins in the EcPS from B.mori.Meanwhile,we focused on the midgut chitin deacetylase(CDA,EC 3.5.1.41)for in-depth molecular,biochemical and structural study.The results will provide important information for the EcPS function and new strategies for pest control.I.Proteomic analyses of the EcPSThe tandem mass tag(TMT)-labelled comparative proteomic analyses combining with bioinformatics methods(gene classification and enrichment analysis)were performed to compare the protein components of the EcPS of the fifth instar Day-5(5L5)larvae and the wandering larvae.The main results obtained include:(1)A total of 70 secreted proteins were identified.According to the GO annotation,the proteins are mainly divided into five groups including digestion,nutrition storage,defense,chitin metabolism and other functions.Among the chitin metabolism related proteins,a midgut CDA(BmCDA8)was identified.(2)The temporal and spatial expression patterns of the specific proteins were then analyzed and demonstrated that all the digestive proteins were up-regulated at 5L5 stage and exclusively expressed in the posterior of the midgut.Beyond that,proteins with nutrient reservoir activity and defense activity were found to be up-regulated at wandering stage and expressed in the anterior of the midgut.It is very clear that the EcPS undergoes dynamic changes in its composition of proteins in response to the changing needs of the insect at different developmental stages.(3)The transcript level of BmCDA8 was upregulated at the wandering stage,indicating that the BmCDA8 might be involved in the dissociation of PM structure at this stage.?.The study of B.mori CD AsTo get insight into the function of BmCDA8.the gene expression pattern analysis,recombinant protein expression,biochemical characterization and crystal structure analysis were performed.Compared with the other two midgut CDAs(BmCDA6 and BmCDA7)found in Silkworm Gene Database,the catalytic mechanism and structure-function relationship of midgut CDAs were revealed.We also elaborated the catalytic mechanism and structure-function relationship of epidermal CDA(BmCDA1)and compared them with midgut CDAs.The main results obtained include;(1)Gene expression analyses and enzymatic activityGene expression pattern indicated that BmCDA6 was expressed almost exclusively at the mid-molt stage,the stage when the PM was thick and with multiple chitin layers.Unlike BmCDA6,high expression levels of BmCDA7 and BmCDA8 were observed only at the feeding stage,the stage when the PM is thin and with fewer chitin layers.The different expression pattern of the three CDA s suggested their functional specialization.To further verify the functional specialization,after gene cloning,recombinantly expression and purification of the three CDAs,their enzymatic activity and chitin-binding ability were characterized and compared.They exhibited comparable chitin-binding abilities,while they showed different enzymatic activities.Of the three enzymes.BmCDA7 exhibited the highest activity.BmCDA8 showed weaker activity.However,BmCDA6 was inactive when using peritrophic membrane chitin as the substrate.Based on the spatial and temporal gene expression patterns together with the enzyme activities,we hypothesize that BmCDA6 functions as a chitin-binding protein for PM chitin protection at the mid-molt stage,while BmCDA7 and BmCDA8 involve in PM chitin modification at feeding stages.(2)Crystal structureThe crystal structure of BmCDA8(PDB entry 5Z34)were obtained using the hanging-drop technique and selenmethionine incorporation.Diffraction data were collected to 2.4 A.Compared with the well-studied microbial CDAs,BmCDA8 contains two unique structural elements:a loop insertion of the(?/?)7 barrel and a C-terminal loop region.The two structural elements help forming two ends of the substrate-binding cleft,conferring the different characteristics of the substrate-binding cleft of insect CDAs compared with those of microbal CDAs.The structures of BmCDA6 and BmCDA7 were simulated using the structure of BmCDA8 as the model.The comparison of the three structures reveled that the different amino acids in the loop region may contribute to the functional differences of the three CDAs.(3)Deacetylation pattern and its structural basisBmCDA8 was inactive toward GlcNAc and(GlcNAc)2.However,BmCDA8 exhibit activities toward(GlcNAc)3-6 that were positively related to the length of the oligomeric substrates,which indicated that BmCDA8 contains at least three substrate-binding subsites.The structure of the products of(GlcNAc)3.4 showed that the reducing-end two GlcNAc residues always remain intact.The above results indicated that the activity of BmCDA8 requires the substrate occupying the 0.+1 and +2 subsites,in which 0 refers to the catalytic site and+refers to the reducing-end subsites.Based on the deacetylation pattern,the binding mode of(GlcNAc)3 to BmCDA8 and the asymmetric substrate-binding sites were verified by structural molecular dynamics simulation and mutagenesis analysis.The simulation results indicate that(GlcNAc)3 binds to subsites 0.+1,+2 of BmCDA8 via multiple interactions.In subsite +1.the acetyl moiety of the mid GlcNAc residue provides a crucial contribution to a hydrogen bond with Gln125.In subsite +2,two interactions occur between the enzyme and the reducing GlcNAc residue.The C3 hydroxyl group hydrogen bonds with the side-chain oxygen atom of Ser241,and the C5 hydroxyl group hydrogen bonds with Gln125.The further activity determination results showed the mutation of Ser241 caused much more serious impairment than the mutation of Gln125.This difference suggests that the Ser241 in +2 subsite might be more crucial for(GlcNAc)3 binding.(4)The catalytic mechanism and crystal structure of epidermal CDA(BmCDAl)In vitro enzymatic assay showed that the deacetylation activity of BmCDAl was undetectable towards various chitinous substrates even with prolonged incubation time or with higher enzyme concentrations.Surprisingly,the activity of BwCDA1 was boosted when molting fluid(MF)was added into the reaction mixture.Further.we found that a cuticular chitin-binding protein CPAP-3A1 significantly increased the deacetylation activity of BmCDA1.The in vitro pull-down assay illustrated that CPAP3-A1 can pull down BmCDA1.Taken together,these data indicated that BmCDAl requires specific accessary proteins to achieve activity.The crystal structure of BmCDA1 catalytic domain(PDB entry 5ZNS)were obtained.Diffraction data were refined to 2.4 A.Comparing with BmCDA8.two significant structural differences were found.First,the replacement of Ser by Ala at +2 site of BmCDA1 abolished the interaction between enzyme and substrates.Second.BmCDA1 possess a more open substrate-binding cleft with few hydrophobic residues.These two differences might confer BmCDA1 lower deacetvlation activity than that of BmCDA8.In summary,this work provides important information for the EcPS function and structural basis for the development of green pesticides for pest control.