Expression And Function Study Of Cathepsin B During Ontogenesis Of Helicoverpa Armigera

Proteases are the enzymes that hydrolyze proteins or peptides. There are many kinds of protease that distribute widely among animals, plants and microorganism. Usually, proteases play important roles in metabolism and biological regulation of various organisms. Proteases are categorized as endopeptidases (EC3.4 21-24, 99) and exopeptidases (EC3.4 11- 19) according to their cleavage sites on target peptides. Endopeptidases are further divided into serine endopeptidases, cysteine endopeptidases, aspartic endopeptidases and metalloendopeptidases based on the chemical characteristics or amino acids in the catalytic centers.Among these proteases, cathepsins (Cath.) are a large group of enzymes which are more important to life activities of organisms. Cathepsins can be activated under acid circumstance. Cathepsins normally exist in lysosomes, and can non-specifically degrade stock proteins into amino acids to supply the cells with amino acids for energy and glucose as needed. Cathepsins can also take part in many other physiological processes across species. Such as bone resorption and programmed cell death. If cathepsins expressed abnormally, it can invoke pathological reactions, for example, degradation of extracellular matrix (ECM) proteins by cathepsins is required for tumor cell invasion and metastasis.Usually, cathepsins are synthesized as inactive precursors (proenzyme), propeptide synthesized in proenzyme make cathepsins inactive. Proenzyme activation is a crucial step in the controlling of cathepsin activity. Proenzyme can be activated by proteolytic removal of the N-terminal propeptide. Removal of the propeptide can be facilitated either by activation by other proteases or by autocatalysis at acidic pH. Enzyme activity of cathepsins can be regulated by proteinase inhibitors also, all cathepsins have their corresponding inhibitors.Cathepsins have also been reported in numerous insects, most important of them are Cath.B, Cath.D and Cath.L. Insect cathepsins mainly distribute in fat bodies, mid gut oflarvae, early embryos and oocytes. They can play various roles in insect life processes, such as oogenesis, embryonic development, food digestion and histolysis and histogenesis during metamorphosis.The cotton boll worm, Helicoverpa armigera is belonged to Lepidoptera: Noctuidae, and is a kind of widely distributed and harmful pest. In previous work, Helicoverpa cathepsin B had been purified and its gene had been cloned from the cotton boll worm, named H. armigera cathepsin B (HCB;EC 3.4.22.1;GenBank AF222788). HCB is a cysteine proteinase. Pro-HCB is a 37 kDa protein and mature HCB is about 30 kDa, its optimum pH is 3.0—4.0, and activity can be inhibited by E-64, N-[N-(l,3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine. Many works had been done on the expression pattern, tissue distribution and substrate specificity of HCB. HCB has been demonstrated to be involved in the degradation of yolk proteins in embryonic developmental eggs.Based on the previous works, utilizing biochemical, molecular and cell biological method such as Western blotting, Northern blotting, immunoblotting, immunohistochemistry, in situ cell hybridization, in situ hydrolytic electrophoresis, light-microscope and electron-microscope etc, assosciating with monoclonal antibody technique, expression and function of HCB during H. armigera ontogenesis were studied in this work.Monoclonal antibody (McAb) against HCB was prepared. Cathepsin B, one was purified from cotton boll worm tissue homogenate (30kDa mature enzyme), another was purified from gene recombinant E.coli cells (37kDa proenzyme) were used as antigens to immunize male BALB/c mice, respectively. Through cell fusion of immunized mouse spleen cells and Sp2/0 myeloma cells, hybridoma screening and cloning, total 3 hybridoma cell lines were obtained, named 9D5, 100E2 and 100 H6 separately. McAbs produced by the three hybridomas were characterized by Western blotting method for their specificity. Results showed, 9D5 and 100E2 could recognize 30kDa mature HCB, and 100 H6 could recognize 37kDa pro-HCB. They were used for mature and zymogenic HCB recognition separately in the subsequent study.Expression and regulation of cathepsin B in enzyme activity during ontogenesis, including oogenesis, embryonic^ developmental stage and larval stages and metamorphosis were investigated. HCB expression was detected on both transcriptionaland translational level. Results showed, HCB activity was initially emerged in ovarian cells and oocytes, and maintained to early embryo stage. The activity declined as the embryos developed and was absent when the embryos hatched into 1st instar larvae. Cathepsin B activity was not observed in larval fat bodies during whole larval stage. In the pupal stage, fat bodies did not show cathepsin B activity until preemergence. Cathepsin B enzyme activity remained after emergence and enzyme activity remarkably increased along with the aging of adult. The variation of enzyme activity was correlated with cotton boll worm ontogenesis, and activity was focused on two stages, from oocytes to early embyos and from pupa to adult. The HCB transcript was detected at all stages from larva to adult fat bodies. Pro-HCB was also detected in fat bodies at these stages. However, mature HCB and its activity were only detected in fat bodies of pre-adults and adults. This evidence suggested that HCB is regulated post-translationally by activation of the pro-enzyme during the pupa-adult metamorphosis.HCB expression and distribution in hemocytes were observed also. Cotton boll worm larval hemocytes was classified by acridine orange and propidium staining, Giemsa staining primarily. Based on their morphology and stain characteristics, total five types of hemocytes were identified: prohaemocytes, plasmatocytes, granular haemocytes, spherulocyte and oenocytoids. Size and concentration of different hemocytes types are calculated. HCB expressed in hemocytes was found to distribute in plasmatocytes and granular haemocytes detected on both transcriptional level and translational level. Expression of HCB in hemocytes exhibited developmental specificity, and increased before pupation. It suggested the relationship between the HCB expressed in hemocyte and larva-pupa metamorphosis.Study of enzyme expression profile during cotton boll worm ontogenesis suggested that HCB was not only functional during embryogenesis, but also correlated to pupa-adult metamorphosis. Previous work has revealed that HCB played a key role in the degradation of yolk proteins during embryogenesis. This study investigated the function and regulatory activation of HCB in adult fat bodies during aging and oogenesis. It is well known that ecdysone is extensively involved in larva molting cascade and larva-pupa metamorphosis. However, the mechanism underlying ecdysonregulation of pupa-adult metamorphosis, and related events, is not well understood. So, the probable role of ecdysone in pupa-adult metamorphosis and the relationship between ecdysone and HCB activation was studied: In fat bodies of different developmental stage, the activation of HCB during pupa-adult metamorphosis was coupled with the expression of H. armigera hormone receptor 3 (HHR3), a useful indicator of ecdysone during molting. In vitro experiment showed, HCB activity was up-regulated by the ecdysteroid agonist, RH-2485, suggesting that HCB activation is related to the ecdysone regulatory system. Other experiment revealed that HCB activation in adult resulted in the decomposition of the adult fat bodies during aging and oogenesis, and the decomposition of the adult fat bodies was found to occur via programmed cell death. It was found that compared with early adult, the fat body structure of late stage adult became loose and vacuolated, the nucleus exhibited distinct chromosome condensation, a typical phenomena of programmed cell death, observed by electron microscopy. Further evidence to support the occurrence of programmed cell death in fat body decomposition was obtained by examining DNA fragmentation. All these observation suggested that programmed cell death occurred in the adult fat body during its decomposition. Correspondingly, HCB activity dramatically increased along with the adult aging. To demonstrate the relationship between the DNA fragmentation and HCB activity, fat bodies from early adults were cultured in vitro with or without E-64. After 24 h in culture, fat bodies cultured with E-64 exhibited decreased HCB activity and did not display DNA fragmentation. In contrast, when E-64 was absent from the culture media, HCB activity was maintained and fat bodies displayed DNA fragmentation. This indicated that HCB is involved in DNA fragmentation, which results in the decomposition of adult fat bodies during aging and oogenesis.Summarily, HCB expression and actity variation were explored in this study. Regulation of HCB activation by ecdysone agonist was deteced. The correlation between HCB expression and pupa-adult metamorphosis was found. Decomposition of adult fat body via programmed cell death and active HCB involved in adult fat body apoptosis was also verified. Expression of HCB in hemocyte were also detected.