Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.     

The differentially spliced mouse tagL gene,homolog of tag7/PGRP gene family in mammals and Drosophila,can recognize Gram-positive and Gram-negative bacterial cell wall independently of T phage lysozyme homology domain

Tag7/PGRP, a recently characterized antimicrobial protein, is conserved from insects to mammals. Recently its involvement in Toll signalling in Drosophila was demonstrated. A number of genes representing a new family homologous to PGRP were identified in Drosophila and human. Here we describe a splicing pattern of the tagL gene, mouse member of tag7/PGRP family. Some of the identified splice variants lacked characteristics for the family T phage lysozyme homology domain (also known as PGRP domain). Accordingly to the predicted transmembrane domains, mouse TagL may be secreted as inducible proteins or retained on intracellular membranes. All detected splice variant isoforms of TagL bound Gram-positive, Gram-negative bacteria and peptidoglycan. This binding did not depend on the presence of T phage lysozyme homology domain but was associated with the C-terminal portion of the polypeptides. Thus, this variety of isoforms of a single gene may play a role in circulating bacteria recognition in mammals.     

A pattern recognition protein for peptidoglycan. Cloning the cDNA and the gene of the silkworm, Bombyx mori

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect. The PGRP is an essential component for peptidoglycan to trigger the prophenoloxidase cascade that is now recognized to be an important insect defense mechanism. We cloned cDNA encoding PGRP from the silkworm fat body cDNA library. Northern blot analysis showed that the PGRP gene is constitutively expressed in the fat body, epithelial cell, and hemocytes of naive silkworms. Furthermore, a bacterial challenge intensified the gene expression, with the maximal period being from 6 to 36 h after infection. The upstream sequence of the cloned PGRP gene was shown to contain putative cis-regulatory elements similar to the NF-kappaB-like element, interferon-response half-element, and GATA motif element, which have been found in the promoters of the acute phase protein genes of mammals and insects. A homology search revealed that the homologs of silkworm PGRP are present in mice, nematodes, and bacteriophages. This suggests that the recognition of peptidoglycan as foreign is effected in both vertebrates and invertebrates by PGRP homologs with an evolutionally common origin.     

Three peptidoglycan recognition protein (PGRP) genes encoding potential amidase from eri-silkworm, Samia cynthia ricini

Three cDNA clones encoding peptidoglycan recognition proteins (PGRP-B, -C and -D) were isolated from larval fat body of immunized Samia cynthia ricini. The deduced amino acid sequences show high homology to each other and also to Drosophila PGRP-LB, but rather lower homology to all of the known lepidopteran PGRPs including Samia PGRP-A, a receptor-type PGRP. The three PGRPs conserve the five amino acid residues which form the catalytic site of N-acetylmuramoyl L-alanine amidase as in Drosophila LB. The PGRP-C and -D genes were silent in naive larvae, but strongly induced in fat body by an injection of peptidoglycan. PGRP-B gene, in contrast, constitutively expressed at high levels in naive midgut, and the gene was weakly induced in fat body after injection of peptidoglycan.     

Bovine peptidoglycan recognition protein-S: antimicrobial activity, localization, secretion, and binding properties

Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern recognition molecules of innate immunity that are conserved from insects to humans. Various PGRPs are reported to have diverse functions: they bind bacterial molecules, digest PGN, and are essential to the Toll pathway in Drosophila. One family member, bovine PGN recognition protein-S (bPGRP-S), has been found to bind and kill microorganisms in a PGN-independent manner, raising questions about the identity of the bPGRP-S ligand. Addressing this, we have determined the binding and microbicidal properties of bPGRP-S in a range of solutions approximating physiologic conditions. In this study we show that bPGRP-S interacts with other bacterial components, including LPS and lipoteichoic acid, with higher affinities than for PCP, as determined by their abilities to inhibit bPGRP-S-mediated killing of bacteria. Where and how PGRPs act in vivo is not yet clear. Using Immunogold electron microscopy, PGRP-S was localized to the dense/large granules of naive neutrophils, which contain the oxygen-independent bactericidal proteins of these cells, and to the neutrophil phagolysosome. In addition, Immunogold staining and secretion studies demonstrate that neutrophils secrete PGRP-S when exposed to bacteria. Bovine PGRP-S can mediate direct lysis of heat-killed bacteria; however, PGRP-S-mediated killing of bacteria is independent of this activity. Evidence that bPGRP-S has multiple activities and affinity to several bacterial molecules challenges the assumption that the PGRP family of proteins recapitulates the evolution of TLRs. Mammalian PGRPs do not have a single antimicrobial activity against a narrow range of target organisms; rather, they are generalists in their affinity and activity.     

Expression analysis of proteins encoded by genes of the tag7/tagL (PGRP-S,L) family in human peripheral blood cells

Various types of human blood cells were tested for expression of the Tag7/PGRP-SA and TagL/PGRP-L proteins, which belong to the family of proteins possessing the lysozyme-like peptidoglycan recognition protein (PGRP) domain. Expression regulation by several factors was demonstrated.     

Peptidoglycan recognition protein (PGRP) from eri-silkworm, Samia cynthia ricini; protein purification and induction of the gene expression

Peptidoglycan recognition protein (PGRP) was isolated from immunized hemolymph of the wild silkworm, Samia cynthia ricini, detecting the biding activity with (125)I-labeled peptidoglycan (PGN). The binding specificity of PGRP was tested by competitive inhibition of the binding to (125)I-labeled-PGN by a large excess amount of non-labeled-PGN or other glucans. The binding to labeled uncross-linked Lys-type PGN from Micrococcus luteus was strongly inhibited by non-labeled-PGN of the same structure and meso-diaminopimelic acid (DAP)-type cross-linked PGN from Bacillus cell wall, but only a little by cross-linked PGN from M. luteus cell wall. The PGRP cDNA encodes a 193 amino acid open reading frame. The deduced amino acid sequence had 62 to 91% identities to known lepidopteran PGRPs, but less than 40% to Drosophila PGRPs. The PGRP gene constitutively expressed at a low level in naive fat body, and strongly induced by an injection of DAP-type cross-linked and Lys-type uncross-linked PGNs, but only weakly by Lys-type cross-linked PGN from M. luteus. The silkworm possibly distinguish between PGNs based on the structure of cross-linking peptide, but has less if any preference for the diamino acid residue of the stem peptide.     

Analysis of sequence variability and protein domain architectures for bovine peptidoglycan recognition protein 1 and Toll-like receptors 2 and 6

The mammalian Toll-like receptors (TLRs) recognize invading pathogens, thereafter provoking innate immune responses, whereas peptidoglycan recognition protein 1 (PGLYRP1) is directly microbicidal. The primary objective of this study was to characterize single-nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (indels) within bovine TLR2, TLR6, and PGLYRP1, thereby facilitating future TLR signaling, association, and PGLYRP1 microbicidal assays relevant to bovine innate immunity. Comparative sequence analysis for 10 bovine breeds revealed 83 polymorphisms (82 SNPs, 1 indel), with 15 nonsynonymous SNPs located within predicted functional domains. Of the 83 polymorphisms detected, 72 (87%) are reported here for the first time. Several predicted amino acid replacements encoded by bovine TLR2 and TLR6, but not PGLYRP1, resulted in the confident prediction of protein domain alterations. Prediction and comparison of protein domain architectures for TLR2 and TLR6 revealed six regions of leucine-rich-repeat patterning that was conserved among multiple species. Collectively, differences in the patterns and frequencies of polymorphism were noted between bovine TLRs that predominantly recognize viral ligands (TLRs 3, 7, 8, 9) and those that recognize microbial and/or unknown ligands (TLRs 1, 2, 5, 6, 10).     

Expression Analysis of Proteins Encoded by Genes of the tag7/tagL (PGRP-S, L) Family in Human Peripheral Blood Cells

Various types of human blood cells were tested for expression of the Tag7/PGRP-SA and TagL/PGRP-L proteins, which belong to the family of proteins possessing the lysozyme-like peptidoglycan recognition protein (PGRP) domain. Expression regulation by several factors was demonstrated.     

非洲爪蟾中一种长型肽聚糖识别蛋白的克隆与表达分析(英文)

肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是固有免疫系统中一类重要的模式识别受体。该文首次从两栖类模式生物-非洲爪蟾(Xenopus tropicalis)中克隆得到了一个长型PGRP(XtPGRP-L)基因。XtPGRP-L具有5个外显子和4个内含子的基因组结构,该结构在进化的过程中比较保守。序列比对与系统进化分析显示XtPGRP-L具有保守的酰胺酶活性位点。蛋白质建模显示XtPGRP-L拥有保守的3-D结构。实时定量PCR检测显示,XtPGRP-L在非洲爪蟾胚胎早期不表达,到72h蝌蚪期开始表达。在成体的肝脏、肺、肠和胃高表达。同时,在LPS刺激后,XtPGRP-L在肝脏、肠和胃中呈明显上调表达。结果表明,XtPGRP-L在非洲爪蟾固有免疫系统中可能具有重要的作用。     

A scavenger function for a Drosophila peptidoglycan recognition protein

Recent studies of peptidoglycan recognition protein (PGRP) have shown that 2 of the 13 Drosophila PGRP genes encode proteins that function as receptors mediating immune responses to bacteria. We show here that another member, PGRP-SC1B, has a totally different function because it has enzymatic activity and thereby can degrade peptidoglycan. A mass spectrometric analysis of the cleavage products demonstrates that the enzyme hydrolyzes the lactylamide bond between the glycan strand and the cross-linking peptides. This result assigns the protein as an N-acetylmuramoyl-l-alanine amidase (EC ), and the corresponding gene is thus the first of this class to be described from a eukaryotic organism. Mutant forms of PGRP-SC1B lacking a potential zinc ligand are enzymatically inactive but retain their peptidoglycan affinity. The immunostimulatory properties of PGRP-SC1B-degraded peptidoglycan are much reduced. This is in striking contrast to lysozyme-digested peptidoglycan, which retains most of its elicitor activity. This points toward a scavenger function for PGRP-SC1B. Furthermore, a sequence homology comparison with phage T7 lysozyme, also an N-acetylmuramoyl-l-alanine amidase, shows that as many as six of the Drosophila PGRPs could belong to this class of proteins.     

Mammalian peptidoglycan recognition protein binds peptidoglycan with high affinity, is expressed in neutrophils, and inhibits bacterial growth

Peptidoglycan recognition protein (PGRP) is conserved from insects to mammals. In insects, PGRP recognizes bacterial cell wall peptidoglycan (PGN) and activates prophenoloxidase cascade, a part of the insect antimicrobial defense system. Because mammals do not have the prophenoloxidase cascade, its function in mammals is unknown. However, it was suggested that an identical protein (Tag7) was a tumor necrosis factor-like cytokine. Therefore, the aim of this study was to identify the function of PGRP in mammals. Mouse PGRP bound to PGN with fast kinetics and nanomolar affinity (K(d) = 13 nm). The binding was specific for polymeric PGN or Gram-positive bacteria with unmodified PGN, and PGRP did not bind to other cell wall components or Gram-negative bacteria. PGRP mRNA and protein were expressed in neutrophils and bone marrow cells, but not in spleen cells, mononuclear cells, T or B lymphocytes, NK cells, thymocytes, monocytes, and macrophages. PGRP was not a PGN-lytic or a bacteriolytic enzyme, but it inhibited the growth of Gram-positive but not Gram-negative bacteria. PGRP inhibited phagocytosis of Gram-positive bacteria by macrophages, induction of oxidative burst by Gram-positive bacteria in neutrophils, and induction of cytokine production by PGN in macrophages. PGRP had no tumor necrosis factor-like cytotoxicity for mammalian cells, and it was not chemotactic on its own or in combination with PGN. Therefore, mammalian PGRP binds to PGN and Gram-positive bacteria with nanomolar affinity, is expressed in neutrophils, and inhibits growth of bacteria.     

Peptidoglycan recognition protein LF: a negative regulator of Drosophila immunity

Peptidoglycan recognition proteins (PGRPs) play important roles in the innate immune defence. Each PGRP detects a distinct subset of peptidoglycans and initiate immune signalling or enzymatic degradation of peptidoglycans. Here we characterize one of the 13 Drosophila PGRPs, PGRP-LF. PGRP-LF is membrane bound and has its two PGRP domains, z and w, localized outside the cell. Our data demonstrate that the z-and w-domain differ in their affinities to peptidoglycan. The z-domain has affinity to several groups of peptidoglycans while the w-domain only recognizes peptidoglycan from Escherichia coli. In addition, we observed that overexpression of PGRP-LF in Drosophila melanogaster Schneider 2 cells (S2 cells) promotes aggregation of cells. Furthermore, following immune stimulation of S2 cells overexpressing PGRP-LF, we noticed a reduced up-regulation of expression of antimicrobial peptide genes, in consonance with an immune suppressive role for PGRP-LF.     

Molecular cloning and characterization of peptidoglycan recognition proteins from the rockfish,Sebastes schlegeli

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are structurally conserved through evolution in both invertebrate and vertebrate animals. Here we report the identification and characterization of two long forms of PGRP (SsPGRP-L1 and SsPGRP-L2) from the rockfish, Sebastes schlegeli. The deduced amino acid sequences of SsPGRP-L1 and SsPGRP-L2, 466 and 482 residues respectively, contain the conserved PGRP domain and the four Zn²⁺-binding amino acid residues required for amidase activity. In addition to peptidoglycan-lytic amidase activity, recombinant SsPGRPs have broad-spectrum antimicrobial activity like zebrafish PGRPs. RT-PCR analysis of total RNA shows that the expression patterns of SsPGRP-L1 and SsPGRP-L2 genes are different, though they are widely expressed in the tissues that come in contact with bacteria. Overall, these data suggest that rockfish PGRPs are involved in the innate host defense of S. schlegeli against bacterial infections.     

Human peptidoglycan recognition protein-L is an N-acetylmuramoyl-L-alanine amidase

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.     

PGRP-SB1: an N-acetylmuramoyl L-alanine amidase with antibacterial activity

The peptidoglycan recognition protein (PGRP) family is conserved from insects to mammals and is involved in immune regulation and bacterial clearance. They form at least three functional classes; receptors required for immune gene expression; amidases that degrade peptidoglycan and scavenge the tissues from immune-stimulating peptidoglycan; and as proteins with antibacterial activity. We here report that PGRP-SB1 is an N-acetylmuramoyl l-alanine amidase, which (in contrast to the previously described PGRP-amidases) shows antibacterial activity. PGRP-SB1 is highly active against peptidoglycans that have a diaminopimelic acid (DAP) residue in the cross-linking peptide, but lack activity to most lysine-containing peptidoglycans. The antibacterial activity is pronounced against Bacillus megaterium with an LD(50) of 1.5microg ml(-1). The bactericidal effect of PGRP-SB1 is dependent on its enzymatic activity, as the zinc co-factor is essential. The bactericidal mode of action is thus different from non-enzymatic vertebrate PGRPs that have been reported to be antibacterial.     

桔小实蝇肽聚糖识别蛋白基因BdPGRP-SB1的克隆及功能鉴定

【目的】为探究肽聚糖识别蛋白(PGRP)基因BdPGRP-SB1在桔小实蝇Bactrocera dorsalis免疫中的作用。【方法】本研究利用PCR克隆桔小实蝇BdPGRP-SB1全长cDNA序列;利用生物信息学软件对该基因核苷酸序列及其编码的氨基酸序列特征进行分析。采用RT-qPCR分析BdPGRP-SB1在桔小实蝇不同发育阶段(卵、幼虫、蛹、成虫)及5日龄成虫不同组织(中肠、马氏管、后肠、脂肪体、卵巢和精巢)中的表达模式;对桔小实蝇5日龄雌成虫分别注射大肠杆菌Escherichia coli 0111:B4肽聚糖(PGN-EB)和金黄色葡萄球菌Staphylococcus aureus肽聚糖(PGN-SA)后检测BdPGRP-SB1表达水平变化。利用RNAi沉默BdPGRP-SB1的表达,测定大肠杆菌和金黄色葡萄球菌诱导后桔小实蝇雌成虫的死亡率及大肠杆菌诱导后抗菌肽(AMP)基因attacin-A, defensin和diptercin表达变化情况。【结果】克隆获得桔小实蝇BdPGRP-SB1的全长cDNA序列(GenBank登录号: MN892482),开放阅读框长558 bp,编码185个氨基酸,其编码蛋白预测分子量为21.45 kD,等电点为8.57。序列分析表明,BdPGRP-SB1无跨膜结构域,具有PGRP保守结构域,前端具有信号肽,为分泌型蛋白;具有Zn²⁺依赖性酰胺酶活性和DAP型肽聚糖识别位点。系统进化分析发现,BdPGRP-SB1与辣椒实蝇B. latifrons的PGRP-SB1亲缘关系最近,氨基酸序列一致性达96%。发育表达模式表明,BdPGRP-SB1在桔小实蝇3日龄幼虫和成虫期高表达;组织表达谱结果显示BdPGRP-SB1在5日龄成虫各组织中均有表达,在脂肪体内表达量最高。PGN-EB和PGN-SA均能诱导桔小实蝇雌成虫体内BdPGRP-SB1表达水平变化。通过RNAi抑制BdPGRP-SB1表达后,注射大肠杆菌导致桔小实蝇雌成虫死亡率显著升高,以及attacin-A, defensin和diptercin表达量显著上调。【结论】结果说明桔小实蝇BdPGRP-SB1参与识别革兰氏阴性细菌,并可能参与桔小实蝇Imd途径调控其免疫反应。     

The Drosophila peptidoglycan-recognition protein LF interacts with peptidoglycan-recognition protein LC to downregulate the Imd pathway

The peptidoglycan (PGN)‐recognition protein LF (PGRP‐LF) is a specific negative regulator of the immune deficiency (Imd) pathway in Drosophila. We determine the crystal structure of the two PGRP domains constituting the ectodomain of PGRP‐LF at 1.72 and 1.94 Å resolution. The structures show that the LFz and LFw domains do not have a PGN‐docking groove that is found in other PGRP domains, and they cannot directly interact with PGN, as confirmed by biochemical‐binding assays. By using surface plasmon resonance analysis, we show that the PGRP‐LF ectodomain interacts with the PGRP‐LCx ectodomain in the absence and presence of tracheal cytotoxin. Our results suggest a mechanism for downregulation of the Imd pathway on the basis of the competition between PRGP‐LCa and PGRP‐LF to bind to PGRP‐LCx.     

小菜蛾PGRP-S2基因的克隆表达及功能分析

肽聚糖识别蛋白(peptidoglycan recognition protein,PGRP)对于昆虫来说是一种高度保守的病原识别蛋白。为阐明PGRP-S2在小菜蛾Plutella xylostella抵抗病原微生物过程中的作用,本研究结合RT-PCR和RACE技术克隆得到小菜蛾PGRP-S2基因的cDNA全长序列,命名为PGRP-S2(GenBank登录号:MG570190)。生物信息学分析结果表明,PGRP-S2的开放阅读框为588 bp,编码195个氨基酸;蛋白质预测分子量为21.46 kDa,理论等电点为8.46;编码蛋白具有PGRP超家族保守结构域和酰胺酶结构域,是典型的肽聚糖识别蛋白,包含一条信号肽,不存在跨膜结构;同源序列比对和系统进化树分析表明PGRP-S2与家蚕Bombyx mori的BmPGRP-S 1进化距离最近。利用大肠杆菌Escherichia coli BL21(DE3)高效表达重组蛋白PxPGRP-S2,利用倒置显微镜及平板涂布观察重组蛋白对大肠杆菌E.coli和金黄色葡萄球菌Staphylococcus aureus的作用,结果表明PxPGRP-S2蛋白能够与两种细菌发生结合并凝集细菌,但不具备直接杀菌功能。本研究为进一步研究基于PGRP-S2介导的小菜蛾免疫防御反应提供基础。