Identification Of The Prolyl Oligopeptidase Family And In Vitro Expression, Activity Analysis Of BmAPH In Silkworm, Bombyx Mori

A group of serine peptidases, the prolyl oligopeptidase(POP) family, include prolyl oligopeptidase(POP), dipeptidyl peptidase(DDP), acylpeptide hydrolase(APH) and glutamyl endopeptidase(GEP). They regulate the activity of biologically active peptides or peptide hormones and are involved in many important physiological processes in organisms. They are implicated in diseases in mammals, such as amnesia, depression, diabetes, trypanosomiasis and so on. Prolyl oligopeptidases have a C-terminal ?/?-hydrolase catalytic domain that is similar to lipase and esterase. An N-terminal ?-propeller domain regulates access to the buried active site. There is much more research for POP family in mammals, but less research in insects. Silkworm, Bombyx mori(B. mori), was domesticated from its wild progenitor Bombyx mandarina(B. mandarina) about more than 5,000 years ago. Following the development of biology, as a model insect of Lepidoptera, silkworm is widely used for research in comparative genomics, genetics and other aspects of insects. In recent years, a growing number of gene families have been identified, the corresponding gene functions have been reported after the release of the silkworm genome sequence. The information and function of POP family genes in silkworm are still unclear.In this study, based on the silkworm genome sequence, protein and EST data, the POP family genes were identified. Evolutionary and expression patterns of the POP family genes had been investigated by comparative genomics and bioinformatics. The cloning and functional analyses of silkworm acylpeptide hydrolase gene(BmAPH) had been performed. The obtained main results were as follows:(1)Based on the predicted protein database of silkworm, nine candidate genes of the POP family in the silkworm genome were identified by hmmsearch. These identified genes could be divided into three subfamilies. There was only one member in POP or APH subfamily and seven members in DDP subfamily.(2)According to the nucleotide sequence of candidate genes, primers had been designed. The expression patterns of the POP family genes were investigated by RT-PCR. The results showed that there were eight genes with transcriptional evidence, Bm3 and Bm5 were highly expressed in larvae and late stages of pupae or adult, respectively. Bm3 showed a midgut-specific expression pattern in larvae. Bm5 and Bm7 were highly expressed in head, integument and gonad. The differences in expression pattern of the POP family genes provided a basis for function research in the future.(3)The full-length sequence of BmAPH gene(GenBank:KR094958) from the 3rd day of the 5th instar larvae was cloned by 5'-RACE, 3'-RACE and RT-PCR. The full-length c DNA of BmAPH gene was 2751 bp, containing an open reading frame of 2133 bp that encoded a protein with 710 amino acid residues. Furthermore, the 5'-RACE produced a 147 bp upstream-untranslated region and the 3'-RACE produced a 471 bp downstream-untranslated region. The calculated molecular mass(MW) and isoelectric point(pI) of the deduced protein were 78,481 Da and 6.31, respectively. This gene contained fourteen exons and fifteen introns, the splice boundary of intron had the conservative sequence “GT-AG”. Multiple sequence alignment revealed that the putative BmAPH sequence had the typical structural features of the POP family, such as the catalytic triad(Ser566-Asp654-His686) and the consensus sequence(G-X-S566-X-G-G), including the active site serine residue and three glycines. Moreover, the alignment also revealed that a conserved motif(HGGP) could form an oxyanion hole.(4) The prokaryotic expression vector BmAPH-pET28 a was constructed and heterologously induced expression in Escherichia coli. The best conditions for BmAPH protein expressed in a soluble form were obtained by exploring the influence on exogenous protein expression with different induction temperature, inducer concentration and induction time. The recombinant protein with His-tag was purified by affinity chromatography. It was confirmed that the purified protein was indeed exogenous expression product of BmAPH gene by Western blotting and Maldi-TOF-TOF MS analysis.(5)The expression patterns of BmAPH gene in nine tissues of the 3rd day of the 5th instar larvae and at various developmental stages were studied by RT-PCR. The results suggested that BmAPH gene was extensively expressed in all tested tissues and continuously expressed in all tested twenty-three development stages without preference. The polyclonal antibody was obtained by infecting mice with the purified protein. To extract the total protein of the 3rd day of the 5th instar larvae as antigen, the result showed that the prepared antibody was specificity by Western blotting analysis, it could be used in subsequent experiment. The tissue localization of BmAPH in the 3rd day of the 5th instar larvae head and midgut were studied by immunohistochemistry. The results showed that strong fluorescence signals for BmAPH were detected in the basal lamina in the both two tissues. Basal lamina not only plays a role in supporting cells and tissues, but also is the barriers of permeability. It can adjust the movement of cells and molecules and protect cells or tissues from damage in some extent.(6)The enzyme assays were performed using the purified protein. The results showed that BmAPH had acylpeptide hydrolase activity. It was found that BmAPH activity was susceptible to temperature or p H by measuring the change of enzyme activities under different reaction conditions. The optimum reaction temperature and pH were 50? and 7.7, respectively. Three organophosphate insecticides(chlorpyrifos, malathion and phoxim) were selected to investigate the influence on the activity of BmAPH in vitro or in vivo. The results showed that they all could inhibit the activity of BmAPH. The IC50 values of malathion and phoxim were close to each other, 7.62 mg/L and 7.90 mg/L, respectively. Relatively, chlorpyrifos was the most effective inhibitor for BmAPH, and its IC50 value was only 1.60 mg/L. Meanwhile APH acitvity in whole and midgut of silkworm treated with OP insecticides were also detected. APH activity significantly decreased in the whole body or midgut after exposure to different insecticides relative to the control.In conclusion, nine candidate genes of the POP family in silkworm were identified, and compared with the POP families of other species. The expression patterns of the POP family in various tissues and different development stages of silkworm were investigated in the molecular level, which provided some basis to understand the potential function and evolutionary relationship of them in silkworm and other insects. Meanwhile the molecular cloning, expression pattern, tissue localization and enzyme activity assays of the BmAPH gene were studied, which provided available information to study the function of APH in other insects. It was important that the organophosphate insecticides could inhibit the activity of BmAPH, which illustrated that BmAPH was a sensitive target for organophosphate insecticides.