A novel hexamerin with an unexpected contribution to the prophenoloxidase activation system of the Chinese oak silkworm,Antheraea pernyi

Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap-hexamerin), which shows high homology with reported insect methionine-rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap-hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap-hexamerin to immune responses. In further immune functional studies, Ap-hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen-associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap-hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein.     

Molecular cloning and mRNA expression of two peptidoglycan recognition protein (PGRP) genes from mollusk Solen grandis

Peptidoglycan recognition proteins (PGRPs) play crucial role in innate immunity for both invertebrates and vertebrates, owing to their prominent ability in detecting and eliminating invading bacteria. In the present study, two short PGRPs from mollusk Solen grandis (designated as SgPGRP-S1 and SgPGRP-S2) were identified, and their expression patterns, both in tissues and toward three PAMPs stimulation, were then characterized. The full-length cDNA of SgPGRP-S1 and SgPGRP-S2 was 1672 and 1285 bp, containing an open reading frame (ORF) of 813 and 426 bp, respectively, and deduced amino acid sequences showed high similarity to other members of PGRP superfamily. Both SgPGRP-S1 and SgPGRP-S2 encoded a PGRP domain. The motif of Zn²⁺ binding sites and amidase catalytic sites were well conserved in SgPGRP-S1, but partially conserved in SgPGRP-S2. The two PGRPs exhibited different tissue expression pattern. SgPGRP-S1 was highly expressed in muscle and hepatopancreas, while SgPGRP-S2 was highly in gill and mantle. The mRNA expression of SgPGRP-S1 could be induced acutely by stimulation of PGN, and also moderately by β-1,3-glucan, but not by LPS, while expression of SgPGRP-S2 was significantly up-regulated (P < 0.01) when S. grandis was stimulated by all the three PAMPs, though the expression levels were relatively lower than SgPGRP-S1. Our results suggested SgPGRP-S1 and SgPGRP-S2 could serve as pattern recognition receptors (PRRs) involved in the immune recognition of S. grandis, and they might perform different functions in the immune defense against invaders.     

Rice BiP3 regulates immunity mediated by the PRRs XA3 and XA21 but not immunity mediated by the NB-LRR protein,Pi5

Plant innate immunity is mediated by pattern recognition receptors (PRRs) and intracellular NB-LRR (nucleotide-binding domain and leucine-rich repeat) proteins. Overexpression of the endoplasmic reticulum (ER) chaperone, luminal-binding protein 3 (BiP3) compromises resistance to Xanthomonas oryzae pv. oryzae (Xoo) mediated by the rice PRR XA21 [12]. Here we show that BiP3 overexpression also compromises resistance mediated by rice XA3, a PRR that provides broad-spectrum resistance to Xoo. In contrast, BiP3 overexpression has no effect on resistance mediated by rice Pi5, an NB-LRR protein that confers resistance to the fungal pathogen Magnaporthe oryzae (M. oryzae). Our results suggest that rice BiP3 regulates membrane-resident PRR-mediated immunity.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

Characterization of arylalkylamine N-acetyltransferase from silkmoth (Antheraea pernyi) and pesticidal drug design based on the baculovirus-expressed enzyme

Arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) catalyzes the N-acetylation of arylalkylamines. A cDNA encoding AANAT (ApAANAT) was cloned from Antheraea pernyi by PCR. The cDNA of 1966 bp encodes a 261 amino acid protein. The amino acid sequence was found to have a high homology with Bombyx mori AANAT (BmNAT) but had very low homology with vertebrate AANATs. Amino acid sequence analysis revealed that four insect AANATs cloned from three species including ApAANAT formed a distinct cluster from the vertebrate group. A recombinant ApAANAT protein was expressed in Sf9 cells using a baculovirus expression system, having AANAT activity. The transformed cell extract acetylated tryptamine, serotonin, dopamine, tyramine, octopamine and norepinephrine. The AANAT activity was inhibited at over 0.03 mM tryptamine. Although insect AANATs have been considered as a target of insecticide, this type of insecticide has never been developed. Screening a chemical library of Otsuka Chemical Co., Ltd., we found a novel compound and its derivatives that inhibited the AANAT activity of ApAANAT. This may facilitate investigation of the monoamine metabolic pathway in insects and the development of new types of insecticides and inhibitors of AANATs.     

Purification and characterization of a 1,3-β-D-glucan recognition protein from Antheraea pernyi larve that is regulated after a specific immune challenge

Pattern recognition receptors are known to participate in the activation of Prophenoloxidase system. In this study, a 1,3-β-D-glucan recognition protein was detected for the first time in Antheraea pernyi larvae (Ap-βGRP). Ap-βGRP was purified to 99.9% homogeneity from the hemolymph using traditional chromatographic methods. Ap-βGRP specifically bind 1,3-β-D-glucan and yeast, but not E. coli or M. luteus. The 1,3-β-D-glucan dependent phenoloxidase (PO) activity of the hemolymph inhibited by anti-Ap-βGRP antibody could be recovered by addition of purified Ap-βGRP. These results demonstrate that Ap-βGRP acts as a biosensor of 1,3-β-Dglucan to trigger the Prophenoloxidase system. A trace mount of 1,3-β-D-glucan or Ap-βGRP alone was unable to trigger the proPO system, but they both did. Ap-βGRP was specifically degraded following the activation of proPO with 1,3-β-Dglucan. These results indicate the variation in the amount of Ap-βGRP after specific immune challenge in A. pernyi hemolymph is an important regulation mechanism to immune response. [BMB Reports 2013; 46(5): 264-269]     

Enzymatic characteristics of an ApaH-like phosphatase,PrpA, and a diadenosine tetraphosphate hydrolase,ApaH, from Myxococcus xanthus

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap₄A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards Ap_nA and ATP. In the presence of Mn²⁺, PrpA hydrolyzed Ap₄A into AMP and ATP, whereas in the presence of Co²⁺ PrpA hydrolyzed Ap₄A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards Ap_nA and ATP. Mn²⁺ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co²⁺ was required for Ap_nA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an Ap_nA hydrolase, respectively.     

Two Complementary Enzymes for Threonylation of tRNA in Crenarchaeota: Crystal Structure of Aeropyrum pernix Threonyl-tRNA Synthetase Lacking a cis-Editing Domain

In protein synthesis, threonyl-tRNA synthetase (ThrRS) must recognize threonine (Thr) from the 20 kinds of amino acids and the cognate tRNA^Thr from different tRNAs in order to generate Thr-tRNA^Thr. In general, an organism possesses one kind of gene corresponding to ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in the threonylation of tRNA^Thr, one for catalysis and the other for trans-editing of misacylated Ser-tRNA^Thr. In order to clarify their three-dimensional structures, we performed X-ray analyses of two putatively assigned ThrRSs from Aeropyrum pernix (ApThrRS-1 and ApThrRS-2). These proteins were overexpressed in Escherichia coli, purified, and crystallized. The crystal structure of ApThrRS-1 has been successfully determined at 2.3 Å resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for trans-editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus.     

Identification and characterization of complement factor H in Branchiostoma belcheri

Complement factor H (CFH) is an essential regulator of the complement system and plays very important roles in animal innate immunity. Although the complement system of amphioxus has been extensively studied, the expression in amphioxus and evolution of CFH gene remain unknown. In this study, we identified and characterized an amphioxus (Branchiostoma belcheri) CFH gene (designated as AmphiCFH). Our results showed that the full-length cDNA of AmphiCFH gene consists of 1295 bp nucleotides containing an 855 bp open reading frame (ORF) that was predicted to encode a 284 amino acid protein. The putative AmphiCFH protein possessed the characteristic of the CFH protein family, including typical CCP (complement control protein) domain. Real-time PCR analysis showed that the AmphiCFH was ubiquitously and differentially expressed in five investigated tissues (intestine, gills, notochord, muscles, and hepatic cecum). The expression level of the AmphiCFH gene was induced upon lipopolysaccharide stimulation, indicating that the AmphiCFH gene might be involved in innate immunity. In addition, phylogenetic analysis showed that the AmphiCFH gene was located between that of invertebrates and vertebrates, suggesting that the AmphiCFH gene is a member of the CFH gene family. In conclusion, our findings provided an insight into animal innate immunity and evolution of the CFH gene family.     

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus)

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys⁶¹-Cys¹⁵⁸, Cys¹³⁴-Cys¹⁵⁰) and a Ca²⁺/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca²⁺-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca²⁺-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.     

无脊椎动物先天免疫模式识别受体研究进展

免疫系统的基本功能是“自己”与“非己”识别.对入侵物的识别是免疫防御的起始,最终引发效应物反应系统,包括吞噬作用、包被作用、激活蛋白酶级联反应和黑化作用以及诱导抗菌肽的合成等,从而清除或消灭入侵物.研究证明,这种“非己”识别是因为存在某些特异性的、可溶的或与细胞膜结合的模式识别受体,可以识别或结合微生物表面保守的、而在宿主中又不存在的病原相关分子模式.模式识别受体通过对病原相关分子的识别启动先天免疫防御.近年来这方面的研究进展很快,已经在无脊椎动物中确定了多种模式识别受体,包括肽聚糖识别蛋白、含硫酯键蛋白、革兰氏阴性菌结合蛋白、清除受体、C型凝集素、硫依赖型凝集素、Toll样受体和血素等,并对其性质和功能进行了研究.     

Innate immunity: structure and function of TLRs

The innate immune system provides the first line of defence against infection. Through a limited number of germline-encoded receptors called pattern recognition receptors (PRRs), innate cells recognize and are activated by highly conserved structures expressed by large group of microorganisms called pathogen-associated molecular patterns (PAMPs). PRRs are involved either in recognition (scavenger receptors, C-type lectins) or in cell activation (Toll-like receptors or TLR, helicases and NOD molecules). TLRs play a pivotal role in cell activation in response to PAMPs. TLR are type I transmembrane proteins characterized by an intracellular Toll/IL 1 receptor homology domain that are expressed by innate immune cells (dendritic cells, macrophages, NK cells), cells of the adaptive immunity (T and B lymphocytes) and non immune cells (epithelial and endothelial cells, fibroblasts). In all the cell types analyzed, TLR agonists, alone or in combination with costimulatory molecules, induce cell activation. The crucial role played by TLR in immune cell activation has been detailed in dendritic cells. A TLR-dependent activation of dendritic cells is required to induce their maturation and migration to regional lymph nodes and to activate na?ve T cells. The ability of different cell types to respond to TLR agonists is related to the pattern of expression of the TLRs and its regulation as well as their intracellular localization. Recent studies suggest that the nature of the endocytic and signaling receptors engaged by PAMPs may determine the nature of the immune response generated against the microbial molecules, highlighting the role of TLRs as molecular interfaces between innate and adaptive immunity. In this review are summarized the main biological properties of the TLR molecules.     

A Mechanism for the Inhibition of DNA-PK-Mediated DNA Sensing by a Virus

The innate immune system is critical in the response to infection by pathogens and it is activated by pattern recognition receptors (PRRs) binding to pathogen associated molecular patterns (PAMPs). During viral infection, the direct recognition of the viral nucleic acids, such as the genomes of DNA viruses, is very important for activation of innate immunity. Recently, DNA-dependent protein kinase (DNA-PK), a heterotrimeric complex consisting of the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs was identified as a cytoplasmic PRR for DNA that is important for the innate immune response to intracellular DNA and DNA virus infection. Here we show that vaccinia virus (VACV) has evolved to inhibit this function of DNA-PK by expression of a highly conserved protein called C16, which was known to contribute to virulence but by an unknown mechanism. Data presented show that C16 binds directly to the Ku heterodimer and thereby inhibits the innate immune response to DNA in fibroblasts, characterised by the decreased production of cytokines and chemokines. Mechanistically, C16 acts by blocking DNA-PK binding to DNA, which correlates with reduced DNA-PK-dependent DNA sensing. The C-terminal region of C16 is sufficient for binding Ku and this activity is conserved in the variola virus (VARV) orthologue of C16. In contrast, deletion of 5 amino acids in this domain is enough to knockout this function from the attenuated vaccine strain modified vaccinia virus Ankara (MVA). In vivo a VACV mutant lacking C16 induced higher levels of cytokines and chemokines early after infection compared to control viruses, confirming the role of this virulence factor in attenuating the innate immune response. Overall this study describes the inhibition of DNA-PK-dependent DNA sensing by a poxvirus protein, adding to the evidence that DNA-PK is a critical component of innate immunity to DNA viruses.