Functional Analysis Of Matrix Metalloproteinases And Tissue Inhibitor Of Metalloproteinases In Silkworm,Bombyx Mori

Insects possess a high degree of adaptability on the changeable environment during the long-term evolution,and which associate with the magical process of metamorphosis and efficient innate immune system.There are important practical significances to strengthen the study of insect metamorphosis and immune mechanisms.Meanwhile,it also provides important clues for the researchs of human stem cells differentiation,tissues and organs formation and immune mechanisms.Matrix metalloproteinase family(MMP)functions as one of the most important zinc-dependent endopeptidases,and plays important roles in the regulation of insect metamorphosis development and innate immunity response.MMPs can degrade allextracellular matrix(ECM)components and play important roles in multiple physiological and pathological processes,such as tissue remodeling,organ development,inflammation regulation,immune response,cell apoptosis,would repair,tumor invasion and metastasis,and so on.Tissue inhibitors of metalloproteinases(TIMPs)are the endogenous protein inhibitors of MMPs that inhibiting which degradation activity through direct combination with MMPs,and play an important role in maintaining the steady sate of ECM.However,there are few study on the function of MMPs and TIMPs on insects except Drosophila melanogaster.Number and function of MMPs and TIMPs in different insects also exist great differences.Thus,it is importance and necessity for further comprehensive study of MMPs and TIMPs functions in insects.Therefore,in current research,we studied the expression patterns of BmMMPs family and BmTIMP in silkworm model of lepidoptera insects.The roles of BmMMPs and BmTIMP in Bombyx mori nuclearpolyhedrovirus(BmNPV)resistance and silkworm metamorphosis were also researched in vitro and in vivo through using gene overexpression and CRISPR/Cas9 knockout models.Furthermore,the the action mechanisms of BmMMPs family were clarified by identifying the transcriptional regulation and protein interaction of BmMMPs.This research will provide an experimental basis and theoretical support for revealing the roles of BmMMP and BmTIMP in metamorphosis and innate immunity of silkworm.The main conclusions are as follows: 1.Characterization and expression analysis of Bm MMPs and Bm TIMPThree MMPs family genes have been characterized from silkworm genome,one of which contains two splice variants,and named them BmMMP1 a,BmMMP1b,BmMMP2,and BmMMP3.We also obtained a TIMP homologous gene,BmTIMP.Sequence analysis demonstrated that the silkworm MMPs family consiste the MMP typical domains,including pro-peptide domain,catalytic domain,hinge region,and hemopexin-like C-terminal domain.Phylogenetic tree analysis showed that BmMMPs clustered together as insect MMPs and BmTIMP clustered together as insect TIMPs with containing a typical NTR domain.Expression pattern analysis of BmMMPs and BmTIMP in silkworm various periods showed that BmMMPs and BmTIMP exhibited highest levels of expression on the first day of pupation.The pattern of BmMMPs and BmTIMP expressions were also analyzed in different tissues of third-day of fifth-instar larvae of Dazao.Results showed BmMMP1,BmMMP3,and BmTIMP were highest transcribed in midgut or fat body,BmMMP2 was highest transcribed in hemocyte.The subcellular localization of MMPs and TIMP were detected using immunofluorescence and protein deletion mutants.The results showed that the full length protein and transmembrane region truncated protein of BmMMP1a&b and BmMMP2 were detected in the cytoplasm;the full length protein MMP3 were in the cytoplasm,however,the nuclear localization signals(NLS)truncated protein was localized to the nucleus,N-terminal of BmMMP3 catalytic domain affect the NLS normal function.BmTIMP has a signal peptide region,while,it existed in the cytoplasm and extracellular.The expression analysis of BmMMPs and BmTIMP were also determined in different antigenic stimulation.During BmNPV infect the silkworm cell line,midgut,hemocyte and fat body,BmMMPs and BmTIMP exhibited overexpression in the early stages of viral infection,then showed down-regulated expression,and continuous up-regulated expression in the later stages of viral infection.BmMMPs and BmTIMP also exhibited overexpression during LPS and E.coli affect the silkworm cell line,midgut,hemocyte,and fat body.These results showed that BmMMPs and BmTIMP possessed a synergy on the expression pattern;furthermore,BmMMPs and BmTIMP may play important roles in metamorphosis and innate immunity of silkworm.2.Identification of interaction between Bm MMPs and BmTIMPWe confirmed the interactions involving BmMMP1 a,BmMMP2,BmMMP3 and BmTIMP by fluorescence co-localization experiments,bimolecular fluorescence complementation(BiFC),and co-immunoprecipitation(Co-IP).Fluorescence co-localization experiments and BiFC results showed that BmMMPs and BmTIMP were co-localized in the cytoplasm,respectively.Western blot analysis confirmed that BmMMPs and BmTIMP possessed interaction,respectively.These findings indicated that BmTIMP may interact with BmMMPs and inhibit the MMPs activities.3.Effects of BmMMPs and BmTIMP on BmNPV proliferationThe gene overexpression and CRISPR/Cas9 knockout models were created in silkworm BmN-SWU1 cell line.The effects of BmMMPs and BmTIMP on BmNPV proliferation were further confirmed by using these cell models.The results showed that BmMMPs overexpression and BmTIMP knockout could induce reproduction of BmNPV;BmMMPs knockout and BmTIMP overexpression could inhibit reproduction of BmNPV.The Collagen-IV degradation,broad spectrum inhibitors of MMPs,and protein truncated experiments of MMPs activity areas were used to determine the effects of MMPs activy on BmNPV proliferation.The results showed that overexpression activity areas of MMPs could enhance degradation of Collagen-IV of silkworm cells lysate.The broad spectrum inhibitors of MMPs,BB-94 and GM6001,could inhibit reproduction of BmNPV.The truncated activity areas of MMPs showed higher antiviral effects than the full length proteins.These findings indicated that MMPs may regulate reproduction of BmNPV in the activity form.4.Effects of transgenic overexpression of Bm MMP3 and BmTIMP on BmNPV proliferatio and silkworm metamorphosisBmMMP3-OE and Bm TIMP-OE transgenic silkworm strains were created by microinjection technology.The effects of BmMMP3 and BmTIMP on BmNPV proliferation and silkworm metamorphosis were further studied in individual level.We analyzed the phenotype statistics,fatality rates after the infection of BmNPV,and transcription of BmNPV key gene of transgenic silkworm strains,and the results showed that BmMMP3-OE exhibited size increases and weight gain than normal Dazao;BmMMP3-OE also induce reproduction of BmNPV;and BmTIMP-OE possessed developmental retardation,larva lethal,pupate deletion and so on.These results indicated that BmMMP3-OE and BmTIMP-OE may regulate the growth and development of silkworm;overexpression of BmMMP3 in vivo could induce reproduction of BmNPV.5.Effects of transgenic knockout of BmMMPs on BmNPV proliferatio and silkworm metamorphosisCRISPR / Cas9 gene editing technology and transgenic technology were used to generate transgenic silkworm strain,Cas9-OE,which only overexpressing Cas9 protein,and other three transgenic silkworm lines,which solely overexpressing target sequence of sgRNA,and named them BmMMP1-sgRNA,BmMMP2-sgRNA,and BmMMP3-sgRNA.The hybrid strains,BmMMP1-KO,BmMMP2-KO,and BmMM3-KO,were generated with BmMMPs genes knocking out by crossbreeding Cas9-OE and BmMMP1-sgRNA,respectively.The phenotype statistics of BmMMPs-KO developmental process showed that BmMMP1-KO possessed developmental retardation,larva lethal and especially affect extensions and branches of the tracheas.BmMMP2-KO exhibited developmental retardation,low viability of adults and not mating.The female moths of BmMMP2-KO showed protruding of jaw and tail,easy to fall off of coat,abdominal water,abnormal eggs,marble tube abnormalities,and so on.The male moths of BmMMP2-KO showed same phenotypes with female moths in addition to protruding of jaw and tail.BmMMP3-KO showed developmental retardation,larva lethal,low viability of adults,not spawning,and so on.Meanwhile,we found that BmMMPs-KO could inhibit transcription of VP39 during oral infection and injection infection.These findings further showed BmMMPs play important roles in metamorphosis and innate immunity of silkworm.6.Study on the mechanism of BmMMPs and BmTIMP and the identification of interaction proteinsTo investigate the roles of the key genes of BmNPV proliferation related on the transcription of BmMMPs and BmTIMP,the genes,IE1,FGF,GP64,and VP39,were overexpressed in BmN-SWU1 cells.The results showed that IE1,FGF,and GP64 could induce BmMMPs and BmTIMP up-regulation transcription,in addition to VP39.In order to explore the regulation of relations between BmMMPs and BmTIMP,the BmMMPs and BmTIMP were overexpressed in BmN-SWU1 cells,respectively.The data show that they can be adjusted between each other.We also found BmMMPs overexpression could induce the activity of Caspase3,7,and 9.The above results further suggest that BmMMPs and BmTIMP are involved in the regulation of BmNPV infection in silkworm,and there may be a relationship between BmMMPs and BmTIMP.Six proteins interacting with BmMMP1 a and six proteins interacting with BmMMP3 were screened and identified from BmN-SWU1 cells through the Co-IP and mass spectrometry.It has been found that ATP synthase interacts with BmMMP1 a and PP6 interacts with BmMMP3 after preliminary verification.These findings suggested that BmMMPs may be associated with energy metabolism,and BmMMP3 may interact with PP6 to participate in the regulation of cell physiological process.In summary,BmMMPs and BmTIMP play important roles in the innate immunity of silkworm fight against virus infection and the metamorphosis of silkworms.This study not only provides a solid basis for a comprehensive analysis of the functions of BmMMPs and BmTIMP in silkworm,but also provides a new clue for the research of silkworm innate immunity and metamorphosis.