Expression And Activity Of Carboxypeptidase A(Bm-CPA) In Silkworm Molting Fluid

Molting is an important physiological phenomenon in the life cycle of many metamorphosis insects.Molting fluid is secreted between the old and new epidermis during this process,which plays an important role in the formation of new epidermis and the ecdysis of old epidermis.Relevant studies have shown that molting fluid contains a large number of proteases related to chitin metabolism,which are involved in the synthesis and degradation of epidermal proteins.Proteome studies have helped people to further understand the composition of molting fluid in recent years.Researchers have identified several proteases from molting fluid of different developmental stages,including serine proteases,cysteine proteases,aminopeptidases and carboxypeptidases,which are suspected to be involved in the molting process of silkworm.Carboxypeptidase A is a typical exopeptidase and the most widely studied metalloproteinase.It can degrade amino acids one by one from the carboxyl-terminal of protein,which is widely involved in the growth and development process of plants or animals.Many studies have shown that carboxypeptidase A is involved in the treatment of tumors,cancer and other diseases at present.In this study,the carboxypeptidase A（Bm-CPA）BGIBMGA008910 in silkworm molting fluid was used as the research object.The basic characteristics of Bm-CPA in the molting fluid were analyzed by preparation of antibody,Real-time PCR and Western Blotting,which aimed to reveal the role of Bm-CPA in molting development.Subsequently,the eukaryotic expression system was used to express the Bm-CPA in vitro and the effect of glycosylation modification on Bm-CPA was investigated.The activation experiment demonstrated that the Bm-CPA can be induced by trypsin.The activation mechanism of Bm-CPA in vivo was investigated by using recombinant molting fluid p37K trypsin and predicted the causes of Bm-CPA activity and activation sites.Also,a carboxypeptidase inhibitor（Bm-CPAi）was successfully identified in silkworm molting fluid.These findings can provide evidence for further study of metamorphosis and development of Bombyx mori,and give some reference for the research of protease functions involved in the degradation of old epidermis and formation of new epidermis.1.Analyze the basic characteristics of Bm-CPAWe analyzed sequence of Bm-CPA using online tools including Ex PASy,SMART and PROSECT.It shows that the total length of Bm-CPA was 1440 bp,encodes 479amino acids,and has a predicted molecular weight of about 53 k Da.The predicted amino acid sequence contains an extracellularly secreted signal peptide,a carboxypeptidase activation peptide and M14 zinc carboxypeptidase domain.The results of site prediction showed that Bm-CPA had one potential amidation site,three potential N-type glycosylation sites and five potential phosphorylation sites.Protein multi-sequence alignment showed that Bm-CPA had 39%similarity to mammalian carboxypeptidase A and 59%similarity to Helicoverpa armigera carboxypeptidase A,especially the M14 zinc carboxypeptidase domain and glycosylation site are conserved.RT-q PCR and Western Blotting were used to detect the expression level of Bm-CPA in each tissue,and the results showed that Bm-CPA expression was higher in the epidermis and wing disc on the first day of wandering stage.At the same time,Spatio-temporal expression profiles of Bm-CPA in the epidermis showed that Bm-CPA was highly expressed in the wandering stage and the dormancy stage.Meanwhile,Bm-CPA can be induced up-regulated by 20E.The results of immunofluorescence staining of epidermal tissue sections showed that Bm-CPA was localized in the epidermal layer and enriched during the pre-ecdysis phase.A commercial carboxypeptidase A inhibitor was used to inject the Silkworm at wandering stage.The results showed that as the concentration gradient of the inhibitor increased,pupation was delayed and the number of unsuccessful pupation increased.2.Study on Eukaryotic expression and Glycosylation modification of Bm-CPATo better understand the function of Bm-CPA,the Pichia pastoris eukaryotic expression system X-33 was used to express Bm-CPA.The results of Western Blotting and Mass spectra showed that the expression was successful but the main molecular weight bands were 60 k Da,62 k Da,66 k Da and 70 k Da,respectively.The glycoprotein staining experiment of Bm-CPA showed that Bm-CPA could be successfully stained by the Periodate acid-Schiff method,suggesting the presence of glycation.When using glycosidase PNGase F to digest Bm-CPA,the molecular weight was significantly reduced,which further proved the existence of glycosylation.To identify the glycosylation sites,LC-MS/MS identification of the glycosylation modification sites was performed on Bm-CPA.The results showed that there were significant glycosylation in four sites,including an additional glycosylation site except for the predicted three glycosylation sites.The identified glycosylation sites were performed to single-point mutations and then using Pichia pastoris to detect small-scale expression,it was found that there was no significant change after the 122^th and 137^th amino acids were respectively mutated,only the expression level was decreased,and mutation respectively the 284^th,360^th amino acids expressed only 70 k Da band,These results indicated that there was a heterogeneous glycosylation phenomenon in Bm-CPA and the glycosylation sites may affect the protein status.3.Preliminary study on the activation and activity of Bm-CPATo explore the activation mechanism of Bm-CPA,a protease fluorescence detection kit with fluorescein isothiocyanate（FITC）labeled casein as a substrate was used for determination.The results showed that Bm-CPA in the initial state of Pichia pastoris expression was almost inactive.But when Bm-CPA was activated with trypsin,the protease activity of Bm-CPA increased significantly,and a 35 k Da band appeared after activation.In order to better explore the activity of Bm-CPA protein,recombinant baculovirus was used to transfect insect cell SF9 for expression.The results showed that the activity of Bm-CPA expressed in insect cells was significantly increased after activated with trypsin than that of Pichia pastoris expression.Meanwhile,the activity of Bm-CPA was also significantly improved after incubated with recombinant molting fluid p37K trypsin,suggesting that the molting fluid p37K trypsin is the protease that may activate Bm-CPA in vivo.By homology modeling analysis,it is predicted that the M14 zinc carboxypeptidase domain is the active region,and its predicted molecular weight was 35 k Da,which was consistent with the size of the band generated after in vitro activation treatment,implying that the arginine site between the carboxypeptidase activation peptide and the M14 zinc carboxypeptidase domain was the potential activation site.4.Identification and analysis of carboxypeptidase inhibitor in molting fluidTo explore the regulation mechanism of Bm-CPA activity in molting fluid,through the analysis of the relevant data of silkworm molting fluid proteomics,and found a protein defined as carboxypeptidase inhibitor,Which consists of 342 bp nucleotides,encodes 113 amino acids,and predicted molecular weight was 12.5 k Da.It is numbered KWMTBOMO09342 in the Silkbase database.Sequence analysis showed that it contained an extracellular signal peptide and a carboxypeptidase inhibitor domain and named it was Bm-CPAi.The homologous comparison found that the protein contained12 conservative cysteines,and the carboxypeptidase inhibitor domain was conservative.Spatio-temporal expression profiles analysis in the epidermal showed that Bm-CPAi was low in the ecdysis stage,and the expression pattern of Bm-CPAi was opposite to that of Bm-CPA.By constructing a prokaryotic expression vector,the Bm-CPAi protein was successfully obtained,and the expression was successfully identified by Mass spectrometry.When incubating it with the activated Bm-CPA protein,the activity of Bm-CPA can be successfully inhibited.It implied that the silkworm Bm-CPAi could regulate the activity of Bm-CPA.These results provide important information for further elucidating the active mechanism of Bm-CPA.