Identification And Functional Analysis Of Cathepsin L In Silkworm(Bombyx Mori)

Proteases are major participants which play an important role in the proteins degradation in organisms.Cathepsin is a type of proteases that is widely present in various plants and animals cells or tissues(especially in lysosomal)and it plays a very important role in all living organisms.Initial views to cathepsins just as a protease,but recently cathepsins have been considered as the most important group within the papain superfamily for their functional roles in intracellular protein degradation.Cathepsin L(Cat L)is an important proteolytic enzyme which belongs to the lysosomal papain family of cysteine proteases.Cathepsin L belongs to the lysosomal papain family cysteine protease and it as the main member of the papain family(C1)in the protease Clan CA which is present in the lysosomes in the form of zymogens.Cathepsin L is involved in many physiological and pathological processes,such as tumor invasion and regulation,immune response,even the regulation of certain cancer stem cells radiation sensitivity.Cathepsins is a large family of proteases with many family members whose functions are also different.At present,the research on insect cathepsins has lagged behind mammals,and the study on cathepsin in silkworms is still in infancy.Bombyx mori as a typical representative biological model of lepidoptera insects which has a great guiding role in the study of insects.Therefore,in this study,we selected the gene of BmCathepsin L in silkworm,which is essential to further reveal the function and mechanism of cathepsin L gene.In this study,the Cat L primers were designed based on the relatve sequences'analysis of Silkworm Genome Database,NCBI database and EST database,and we successfully obtained the full-length cDNA of BmCat L through RACE technology.Two isoforms of cathepsin L were identified by bioinformatics analysis.The expression of the gene in different ages and tissues of the silkworm was analyzed.Finally,the function of BmCat L was further explored to reveal its potential role in the immune regulation in silkworm.The main research results are as follows:1.The full-length cloning and bioinformatics analysis of BmCat LThe basic information of BmCat L was compared and analyzed using online tools.The primers were designed through Primer 5.0 software,then the full-length cDNA of cathepsin L was successfully cloned using RACE technology.By comparison analysis,two different lengths Cathepsin L were identified in silkworm,and were named BmCat L-L and BmCat L-S respectively.Both the two splices contained a complete open reading frame(ORF)with 1664 bp and 687 bp,encoding 556 and 228 amino acids,respectively.The predicted molecular weights were 62.471 KDa and 26.132 KDa,and the theoretical isoelectric points were 4.86 and 4.57.The predicted protein structures of the two splices were very similar,and they all have a signal peptide that is identical in composition,and a Pept_C1 domain,the BmCat L-L has a specific Pept_C1 domain.The NJ phylogenetic tree was constructed by comparing with other species,the results showed that the gene was high conservative during long-term evolution.The three-dimensional structure of BmCat L were predicted and calculated by Robetta software,and then compared through the protein database.The results showed that BmCat L protein is very similar to the Cathepsin L3 preenzyme structure of Tenebrio molitor midgut.It was predicted that the proteinase has the function for protein digestion.2.Expression analysis of BmCat LThe qRT-PCR was used to detect the expression of BmCat L in different tissues of the 3rd day of 5th instar larvae and its expression levels in haemocyte from the 2nd day of 4th instar larvae to the 2nd day of prepupa.The results of different tissues showed that both the BmCat L-L and BmCat L-S were highly and specifically expressed in haemocyte,and the two spliceosomes were low expression in the head,the BmCat L-L was slightly expressed in testis,and both BmCat L-L and BmCat L-S were almost not detected in other tissues.In order to further study why this gene was specifically and highly expressed in haemocyte,the qRT-PCR was used to detect the expression of BmCat L in haemocyte of differernt ages,and the results showed that BmCat L maintained a relatively high expression from the larval L4D2 to PP2.With the advancement of metamorphosis,the expression changes of the two kinds of spliced bodies showed a differential change.The expression of BmCat L-S reached a peak in larval L4M,while that of BmCat L-L also reached a peak in L4D4,then the expression level of BmCat L-S gradually decreased followed the age changes,and finally approaches non-expression.Relative to BmCat L-S,the expression level of BmCat L-L also decreased with ages,but its expression level is always maintained at a stable level.Through the detection and analysis of the expression profiles of BmCat L at different ages in haemocyte,which predicted that the two spliceosomes of BmCat L seem to play a complementary role in the haemocyte in silkworm,and it may have an impact on the metamorphosis process in Bombyx mori.3.Prokaryotic expression,protein purification and antibody preparation of BmCat LThe specific sequence of BmCat L was selected to construct a prokaryotic expression vector.The optimum expression conditions were explored under different temperature and IPTG concentration,then the recombinant protein was large-scale induction under the conditions.After that it was purified by Ni~+affinity chromatography.In order to further verify the purified recombinant protein,the His-antibody was used to western blots,and the corresponding size of the target protein was successfully detected.Finally,the purified recombinant protein was used to immunize rats for several times to obtain the anti-serum protein of BmCat L,which laid a foundation for the later study of BmCat L's functions and mechanisms.4.The activity,stability and influencing factors detection of BmCat LThe cathepsin was extracted from the haemocyte of L5D3 larvaes,then its endogenous cathepsin activity and stability characteristics were detected and analyzed.The enzyme activity of different cathepsins in the haemocyte of silkworm larvaes were measured through different cathepsins specific detection substrates.The results showed that the activity of the cathepsins(B,L,H)were affected by the factors such as temperature and pH;The enzyme stability test showed that almost all enzymes were inactive when the temperature was higher than 75°C.When the enzyme was placed at room temperature,its activity gradually weakened with the time changes.The results of the comprehensive detection showed that cathepsin B is an acid-specific enzyme,the optimum pH is approximately 5.5,the optimum temperature is 37°C;The optimum pH of cathepsin L is about 4.0-5.0,the optimum temperature is 37°C;However the cathepsin H has an optimum pH about 8.0 and an optimum temperature is 37°C.The purified recombinant cathepsin L protein was detected by the same method,and results showed that only the specific fluorescent substrate Z-phe-Arg-Nmec can be catalyzed by the recombinant protease,further demonstrated that the purified protease is active.These results of the enzyme activity,the stability of BmCat L showed that recombinatnt cathepsin L also has the similar enzymatic characteristics to endogenous cathepsin L.Our research will provide a theoretical reference for the preventing and controlling silkworm diseases,especially the haemocyte diseases.Besides,our research has also enriched the contents of the cathepsins enzymes'research at the same time.5.Preliminary research on the function of BmCat LCathepsin L is a protease that plays an important role in proteins digestion and degradation.Besides,according to many studies reported that it has an indispensable role in the immune system.However,it has been shown in previous studies that the cathepsin L is highly and specifically expressed in the haemocyte,as we know,the haemocyte is the most important immune organ of insects,many studies had reported that its components play an very important role in the immune regulation in insects.The silkworm,likes other insects,the innate immune system is also the most critical defense system to against pathogens intrusion,So does high expression of cathepsin L in haemocyte also be related to immune functions?In fact,it is unclear on purpose.Therefore,the gram-negative pathogens and gram-positive pathogenic factors were used to infect silkworm,then the BmCat L relative mRNAs were detected by RT-PCR.The results showed that the expression level of BmCat L will reach a peak with the time of attack,and then its expression level will return to a normal expression level.By comparison and analysis,the two spliceosomes'mRNA expression level of BmCat L were basically the same trend,presumably it may be involved in the function of immune regulation in silkworm.