红螯螯虾酚氧化酶原及其特性研究

采用同源克隆策略和RACE技术, 从红螯螯虾Cherax quadricarinatus血细胞中克隆得到酚氧化酶原基因的全长cDNA序列, 共2951 bp, 开放读码框为1995 bp, 编码665个氨基酸. 预测的分子量和等电点分别为75.7 kD和6.23. 酚氧化酶原含有两个推测的tyrosinase copper-binding motifs (带有六个组氨酸残基)和一个thiol-ester-like motif, 这些特征和其他甲壳动物的酚氧化酶原特征相同. 红螯螯虾酚氧化酶原氨基酸序列与通讯螯虾Pacifastacus leniusculus、欧洲龙虾Homarus gammarus、美洲龙虾Homarus americanus 和克氏原螯虾Procambarus clarkii 酚氧化酶原的相似率分别为68%、63%、63%和59%. 酚氧化酶原基因双酶切后连接入pET-28a原核表达载体, 转化到大肠杆菌BL21后重组表达酚氧化酶原蛋白. 在重组蛋白纯化后, 免疫新西兰大耳兔制备得到的酚氧化酶原多克隆抗体, 其效价大于1:12800. 红螯螯虾血淋巴、肝和鳃组织中的酚氧化酶原mRNA表达和酚氧化酶活性较高, 而神经、心、肠和肌肉中较低. 中华绒螯蟹螺原体和嗜水气单胞菌免疫红螯螯虾后, 血淋巴细胞、肝和鳃组织中的酚氧化酶原和酚氧化酶活性在免疫后的不同时间均出现了显著性的增加, 此结果表明酚氧化酶原和酚氧化酶在红螯螯虾对抗细菌感染的过程中起到重要的免疫作用. 此结果为进一步深入研究酚氧化酶原基因和酚氧化酶的功能及其调控机理奠定基础.       

A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae

The prophenoloxidase (proPO) activation pathway, like the vertebrate complement system, consists of a protease cascade and functions as a non-self-recognition system in these animals. Determining the molecular mechanism by which pattern recognition molecules differentiate non-self from self and transduce signals that stimulate defense responses is a key for understanding the ways in which innate immune systems are regulated. However, the proPO system is poorly defined at the molecular level. The proPO-activating system of the insect Holotrichia diomphalia comprises several components, some of which have been cloned and characterized, such as the novel 27-kDa proPO-activating factor-III (PPAF-III) from the plasma of H. diomphalia larvae and two prophenoloxidases. The PPAF-III gene encodes an easter-type serine protease zymogen consisting of 351 amino acid residues with a mass of 40 kDa. The purified 27-kDa PPAF-III specifically cleaved a 55-kDa proPPAF-II to generate a 45-kDa PPAF-II with or without Ca2+ present. Furthermore, two Holotrichia prophenoloxidases (proPO-I and -II) have been characterized, and their structural changes during activation were examined by in vitro reconstitution experiments. When the proPOs were incubated with PPAF-I, the 79-kDa proPOs were converted to 76-kDa proPOs, which did not exhibit any phenoloxidase (PO) activity. However, when the proPOs were incubated simultaneously with PPAF-I, proPPAF-II, and PPAF-III in the presence of Ca2+, a 60-kDa protein (PO-1) with PO activity was detected in addition to the 76-kDa proPO-II protein. These results indicate that the conversion of Holotrichia proPOs to enzymatically active phenoloxidase is accomplished by PPAF-I, PAF-II, and PPAF-III through a two-step limited proteolysis in the presence of Ca2+.     

Prophenoloxidase activating enzyme-III from giant freshwater prawn Macrobrachium rosenbergii: characterization, expression and specific enzyme activity

The prophenoloxidase activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase, is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases. In this study, a cDNA encoding a prophenoloxidase activating enzyme-III from the giant freshwater prawn Macrobrachium rosenbergii, designated as MrProAE-III, was identified and characterized. The full-length cDNA contains an open reading frame of 1110 base pair (bp) encoding a predicted protein of 370 amino acids including an 22 amino acid signal peptide. The MrProAE-III protein exhibits a characteristic sequence structure of a long serine proteases-trypsin domain and an N- and C-terminal serine proteases-trypsin family histidine active sites, respectively, which together are the characteristics of the clip-serin proteases. Sequence analysis showed that MrProAE-III exhibited the highest amino acid sequence similarity (63%) to a ProAE-III from Atlantic blue crab, Callinectes sapidus. MrProAE-III mRNA and enzyme activity of MrProAE-III were detectable in all examined tissues, including hepatopancreas, hemocytes, pleopods, walking legs, eye stalk, gill, stomach, intestine, brain and muscle with the highest level of both in hepatopancreas. This is regulated after systemic infectious hypodermal and hematopoietic necrosis virus infection supporting that it is an immune-responsive gene. These results indicate that MrProAE-III functions in the proPO system and is an important component in the prawn immune system.     

Signal transduction of the prophenoloxidase activating system of prawn haemocytes triggered by CpG oligodeoxynucleotides

Intracellular phenoloxidase (PO) activity in haemocyte lysate supernatant (HLS) of giant freshwater prawn (Macrobrachium rosenbergii) was shown to be enhanced by CpG oligodeoxynucleotide (ODN) 2006, but not by so-ODN13. When haemocytes were treated in vitro with 50 microg/ml of ODN2006 for 30 min, the increases in both intra- and extracellular stimulated PO activity (POS) and extracellular total PO activity (POT) and the reduction of POT suggest that the PO activity of haemocytes is enhanced by ODN2006 stimulation, but new prophenoloxidase (proPO) is not synthesised. In an attempt to determine which signal transduction pathway is involved in the activation of the proPO system, haemocytes were separately treated with activators or inhibitors of specific signalling components. The results show that there was an increase in both intra- and extracellular POT of haemocytes treated with sodium fluoride (a G-protein activator); the addition of phosphokinase A (PKA)-activating 8-bromo-cAMP to haemocytes only increased intracellular POT, and the addition of either phorbol-12-myristate-13-acetate (PMA; a phosphokinase C (PKC) activator) or caffeine (a phosphodiesterase inhibitor) only increased extracellular POT. When PMA-stimulated haemocytes were treated with chelerythrine (a PKC inhibitor), the induced extracellular POT was significantly reduced. Furthermore, the study of ODN2006-stimulated haemocytes treated with chelerythrine or palmitoyl-DL-carnitine (a PKC inhibitor) showed that the enhancement effects of ODN2006 on the intra- and extracellular POS and extracellular POT were significantly decreased. ODN-stimulated haemocytes treated with genistein (an inhibitor of protein tyrosine kinase) showed a further increase in extracellular POT, but the other PO activities remained the same as those of the ODN-stimulated group. These results suggest that the activation of the proPO system of prawn haemocytes, including degranulation and PO activity, is induced by ODN2006 via a PKC-activating signalling pathway, but negatively regulated via the tyrosine kinase pathway.     

Purification and characterization of a small cationic protein from the tobacco hornworm Manduca sexta

The prophenoloxidase (proPO) activation system is an important defense mechanism in arthropods, and activation of proPO to active phenoloxidase (PO) involves a serine proteinase cascade. Here, we report the purification and characterization of a small cationic protein CP8 from the tobacco hornworm, Manduca sexta, which can stimulate proPO activation. BLAST search showed that Manduca CP8 is similar to a fungal proteinase inhibitor-1 (AmFPI-1), an inducible serine proteinase inhibitor-1 (ISPI-1), and other small cationic proteins with unknown functions. However, we showed that Manduca CP8 did not inhibit proteinase activity, but stimulated proPO activation in plasma. When small amount (0.1 μg) of purified native CP8 or BSA was added to cell-free plasma samples and incubated for 20 min, low PO activity was observed in both groups. But significantly higher PO activity was observed in the CP8-group than in the BSA-group when more proteins (0.5 μg) were added and incubated for 20 min. However, when the plasma samples were incubated with proteins for 30 min, high PO activity was observed in both the CP8 and BSA groups regardless of the amount of proteins added. Moreover, when PO in the plasma was pre-activated with Micrococcus luteus, addition of CP8 did not have an effect on PO activity, and CP8/bacteria mixture did not stimulate PO activity to a higher level than did BSA/bacteria. These results suggest that CP8 helps activate proPO more rapidly at the initial stage. CP8 mRNA was specifically expressed in fat body and its mRNA level decreased when larvae were injected with saline or bacteria. However, CP8 protein concentration in hemolymph did not change significantly in larvae injected with saline or microorganisms.     

The effects of age and social interactions on innate immunity in a leaf-cutting ant

Both developmental and environmental factors shape investment in costly immune defences. Social insect workers have different selection pressures on their innate immune system compared to non-social insects because workers do not reproduce and their longevity affects the fitness of relatives. Furthermore, hygienic behavioural defences found in social insects can result in increased survival after fungal infection, although it is not known if there is modulation in physiological immune defence associated with group living vs. solitary living.Here we investigated whether physiological immune defence is affected by both age and the short-term presence or absence of nestmates in the leaf-cutting ant Acromyrmex octospinosus. We predicted that older ants would show immune senescence and that group living may result in prophylactic differences in immune defence compared to solitarily kept ants. We kept old and young workers alone or in nestmate groups for 48 h and assayed a key innate immune system enzyme, expressing phenoloxidase (PO) and its stored precursor (proPO), a defence that acts immediately, i.e. it is constitutive. Short-term solitary living did not affect PO or proPO levels relative to group living controls and we found no evidence for immunosenescence in proPO. However, we found a significant increase in active PO in older workers, which is consistent with two non-mutually exclusive explanations: it could be an adaptive response or indicative of immunosenescence. Our results suggest that future studies of immunosenescence should consider both active immune effectors in the body, such as PO, and the stored potential to express immune defences, such as proPO.