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.     

INVOLVEMENT OF PEPTIDOGLYCAN RECOGNITION PROTEIN L6 IN ACTIVATION OF IMMUNE DEFICIENCY PATHWAY IN THE IMMUNE RESPONSIVE SILKWORM CELLS

The immune deficiency (Imd) signaling pathway is activated by Gram‐negative bacteria for producing antimicrobial peptides (AMPs). In Drosophila melanogaster, the activation of this pathway is initiated by the recognition of Gram‐negative bacteria by peptidoglycan (PGN) recognition proteins (PGRPs), PGRP‐LC and PGRP‐LE. In this study, we found that the Imd pathway is involved in enhancing the promoter activity of AMP gene in response to Gram‐negative bacteria or diaminopimelic (DAP) type PGNs derived from Gram‐negative bacteria in an immune responsive silkworm cell line, Bm‐NIAS‐aff3. Using gene knockdown experiments, we further demonstrated that silkworm PGRP L6 (BmPGRP‐L6) is involved in the activation of E. coli or E. coli‐PGN mediated AMP promoter activation. Domain analysis revealed that BmPGRP‐L6 contained a conserved PGRP domain, transmembrane domain, and RIP homotypic interaction motif like motif but lacked signal peptide sequences. BmPGRP‐L6 overexpression enhances AMP promoter activity through the Imd pathway. BmPGRP‐L6 binds to DAP‐type PGNs, although it also binds to lysine‐type PGNs that activate another immune signal pathway, the Toll pathway in Drosophila. These results indicate that BmPGRP‐L6 is a key PGRP for activating the Imd pathway in immune responsive silkworm cells.     

Crystal structure of the C-terminal peptidoglycan-binding domain of human peptidoglycan recognition protein Ialpha

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyze, peptidoglycans (PGNs) on bacterial cell walls. These molecules, which are highly conserved from insects to mammals, participate in host defense against both Gram-positive and Gram-negative bacteria. We report the crystal structure of the C-terminal PGN-binding domain of human PGRP-Ialpha in two oligomeric states, monomer and dimer, to resolutions of 2.80 and 1.65 A, respectively. In contrast to PGRPs with PGN-lytic amidase activity, no zinc ion is present in the PGN-binding site of human PGRP-Ialpha. The structure reveals that PGRPs exhibit extensive topological variability in a large hydrophobic groove, located opposite the PGN-binding site, which may recognize host effector proteins or microbial ligands other than PGN. We also show that full-length PGRP-Ialpha comprises two tandem PGN-binding domains. These domains differ at most potential PGN-contacting positions, implying different fine specificities. Dimerization of PGRP-Ialpha, which occurs through three-dimensional domain swapping, is mediated by specific binding of sodium ions to a flexible hinge loop, stabilizing the conformation found in the dimer. We further demonstrate sodium-dependent dimerization of PGRP-Ialpha in solution, suggesting a possible mechanism for modulating PGRP activity through the formation of multivalent adducts.     

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.     

A peptidoglycan recognition protein from Sciaenops ocellatus is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn²⁺-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection.     

Structural insights into ligand binding of PGRP1 splice variants in Chinese giant salamander (<Emphasis Type="Italic">Andrias davidianus</Emphasis>) from molecular dynamics and free energy calculations

Peptidoglycan (PGN) recognition proteins (PGRPs) are important pattern recognition receptors of the innate immune system. A number of PGRP splicing variants produced by alternative splicing of PGRP genes have been reported. However, several important aspects of interactions between PGRP splice variants and their ligands are still unclear. In the present study, three dimensional models of salamander PGRP1 (adPGRP1) and its splice variant (adPGRP1a) were constructed, and their key amino acids involved in interacting with PGNs were analyzed. The results revealed that adPGRP1a has a typical PGRPs structure containing five β-sheets and four α-helices, while adPGRP1 contained five β-sheets and only one α-helix due to the lack of 51 amino acids at its C-terminus. Molecular docking revealed that van der Waals and Coulombic interactions contributed to interactions in the protein–ligand complex. Further binding energy of adPGRP-PGNs computed by the MM-PBSA method revealed that adPGRP1a and adPGRP1 might selectively bind to different PGNs; the former might selectively bind Dap-type PGNs and the latter both types of PGNs. In addition, the binding energy of each residue of adPGRP1a and adPGRP1 was also calculated, revealing that residues involved in the interaction of protein–ligand complexes were different in adPGRP1a and adPGRP1. These results provided a first insight into the potential basis for interaction between PGRPs generated by alternative splicing and PGN derivatives.     

Crystal structure of the Drosophila peptidoglycan recognition protein (PGRP)-SA at 1.56 A resolution

Peptidoglycan recognition proteins (PGRPs) form a recently discovered protein family, which is conserved from insect to mammals and is implicated in the innate immune system by interacting with/or degrading microbial peptidoglycans (PGNs). Drosophila PGRP-SA is a member of this family of pattern recognition receptors and is involved in insect Toll activation. We report here the crystal structure of PGRP-SA at 1.56 A resolution, which represents the first example of a "recognition" PGRP. Comparison with the catalytic Drosophila PGRP-LB reveals an overall structure conservation with an L-shaped hydrophilic groove that is likely the PGN carbohydrate core binding site, but further suggests some possible functional homology between recognition and catalytic PGRPs. Consistent with sequence analysis, PGRP-SA does not contain the canonical zinc-binding residues found in catalytic PGRPs. However, substitution of the zinc-binding cysteine residue by serine, along with an altered coordinating histidine residue, assembles a constellation of residues that resembles a modified catalytic triad. The serine/histidine juxtaposition to a threonine residue and a carbonyl oxygen atom, along with conservation of the catalytic water molecule found in PGRP-LB, tantalizingly suggests some hydrolytic function for this member of receptor PGRPs.     

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.     

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.     

Elucidating the molecular interaction of Zebrafish (Danio rerio) peptidoglycan recognition protein 2 with diaminopimelic acid and lysine type peptidoglycans using in silico approaches

Abstract

Peptidoglycan recognition proteins (PGRPs) belong to the family of pattern recognition receptor, represent the major constituent of innate immunity. Although PGRPs are structurally conserved through evolution, their involvement in innate immunity is different in vertebrates and invertebrates. They are highly specific towards recognition of ligands and can hydrolyze bacterial peptidoglycans (PGNs). Zebrafish PGRPs (zPGRPs) have both peptidoglycans lytic amidase activity and broad-spectrum bactericidal activity, but far less is known about how these receptors recognize these microbial ligands. Such studies are hindered due to lack of structural and functional configuration of zPGRPs. Therefore, in this study, we predicted the three-dimensional structure of zPGRP2 through theoretical modeling, investigated the conformational and dynamic properties through molecular dynamics simulations. Molecular docking study revealed the microbial ligands, that is, muramyl pentapeptide–DAP , muramyl pentapeptide–LYS, muramyl tripeptide–DAP, muramyl tripeptide–Lys, muramyl tetrapeptide–DAP, muramyl tetrapeptide–LYS and tracheal cytotoxin interacts with the conserved amino acids of the ligand recognition site comprised of β1, α2, α4, β4 and loops connecting β1 ? α2, α2 ? β2, β3 ? β4 and α4 ? α5. Conserved His31, His32, Ala34, Ile35, Pro36, Lys38, Asp60, Trp61, Trp63, Ala89, His90, Asp106, His143 and Arg144 are predicted to essential for binding and provides stability to these zPGRP–PGN complexes. Our study provides basic molecular information for further research on the immune mechanisms of PGRP’s in Zebrafish. The plasticity of the zPGRP’s binding site revealed by these microbial ligands suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges in the future.

Communicated by Ramaswamy H. Sarma     

Involvement of Manduca sexta peptidoglycan recognition protein-1 in the recognition of bacteria and activation of prophenoloxidase system

Although the importance of peptidoglycan recognition proteins (PGRPs) in detecting bacteria and promoting immunity is well recognized in Drosophila melanogaster and other insect species, such a role has not yet been experimentally established for PGRPs in the tobacco hornworm, Manduca sexta. In this study, we purified M. sexta PGRP1 from the baculovirus-insect cell expression system, tested its association with peptidoglycans and intact bacteria, and explored its possible link with the prophenoloxidase activation system in larval hemolymph. Sequence comparison suggested that PGRP1 is not an amidase and lacks residues for interacting with the carboxyl group of meso-diaminopimelic acid-peptidoglycans (DAP-PGs). M. sexta PGRP1 gene was constitutively expressed at a low level in fat body, and the mRNA concentration became much higher after an injection of Escherichia coli. Consistently, the protein concentration in larval plasma increased in a time-dependent manner after the immune challenge. Purified recombinant PGRP1 specifically bound to soluble DAP-PG of E. coli but not to soluble Lys-type PG of Staphylococcus aureus. In addition, this recognition protein completely bound to insoluble PGs from Micrococcus luteus, Bacillus megaterium and Bacillus subtilis, whereas its association with the bacterial cells was low even though their peptidoglycans are exposed on the cell surface. After PGRP1 had been added to plasma of naïve larvae in the absence of microbial elicitor, there was a concentration-dependent increase in prophenoloxidase activation. Phenoloxidase activity, as usual, increased after the plasma was incubated with peptidoglyans or bacterial cells. These increases became more prominent when insoluble M. luteus or B. megaterium PG or soluble E. coli PG and PGRP1 were both present. Statistic analysis suggested a synergistic effect caused by interaction between PGRP1 and these PGs. Taken together, these results indicated that PGRP1 is a member of the M. sexta prophenoloxidase activation system, which recognizes peptidoglycans from certain bacteria and initiates the host defense response. The unexplained difference between the purified PGs and intact bacteria clearly reflects our general lack of understanding of PGRP1-mediated recognition and how it leads to proPO activation.     

Downregulation of the Drosophila immune response by peptidoglycan-recognition proteins SC1 and SC2

Peptidoglycan-recognition proteins (PGRPs) are evolutionarily conserved molecules that are structurally related to bacterial amidases. Several Drosophila PGRPs have lost this enzymatic activity and serve as microbe sensors through peptidoglycan recognition. Other PGRP family members, such as Drosophila PGRP-SC1 or mammalian PGRP-L, have conserved the amidase function and are able to cleave peptidoglycan in vitro. However, the contribution of these amidase PGRPs to host defense in vivo has remained elusive so far. Using an RNA-interference approach, we addressed the function of two PGRPs with amidase activity in the Drosophila immune response. We observed that PGRP-SC1/2-depleted flies present a specific over-activation of the IMD (immune deficiency) signaling pathway after bacterial challenge. Our data suggest that these proteins act in the larval gut to prevent activation of this pathway following bacterial ingestion. We further show that a strict control of IMD-pathway activation is essential to prevent bacteria-induced developmental defects and larval death.     

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.     

昆虫肽聚糖识别蛋白研究进展

在脊椎动物和非脊椎动物中,识别非己是天生免疫反应中的第一步。肽聚糖是细菌细胞壁的必需成分,属于进化上保守的微生物表面病原相关分子模式(pathogen-associated molecular pattern, PAMP),可以被模式识别蛋白(pattern recognition proteins, PRRs)如肽聚糖识别蛋白(peptidoglycan recognition proteins, PGRPs)识别。 在昆虫的天生免疫系统中,有些PGRPs能够利用细菌独有的肽聚糖识别入侵细菌,并将细菌入侵信号传递给下游的抗菌肽(antimicrobial peptide, AMP)合成途径,启动抗菌肽基因的转录及合成;PGRPs对肽聚糖的识别也会启动酚氧化酶原途径的激活,引起黑化反应。有些具有酰胺酶活性的PGRPs可以促进吞噬作用;有些可以抑制抗菌肽合成以减弱过度免疫反应带来的损伤。还有一些PGRPs作为效应因子直接作用于细菌将细菌杀死。本文主要从昆虫PGRPs作为识别受体(recognition receptor)、调节子(regulator)和效应因子(effector) 3个方面进行了综述,并分析了目前PGRPs研究中仍不清楚的问题和未来研究的方向。     

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.     

大鲵短型肽聚糖识别蛋白PGRP-S的克隆及其功能研究

实验构建了大鲵短型肽聚糖识别蛋白PGRP-S的真核表达质粒, 并对其功能进行了初步的研究。序列分析显示, 所克隆的大鲵PGRP-S的N端不含有信号肽; 其编码的氨基酸序列具有2个相距较近的半胱氨酸残基以及2个Zn2+结合位点。Western-blotting检测结果显示大鲵PGRP-S既可分泌到胞外, 也可滞留在胞内。体外抗菌实验的结果表明, 过表达大鲵PGRP-S能显著抑制迟缓爱德华氏菌(Edwardsiella tarda)在HEK293T细胞内和胞外培养基中的增殖。此外, 过表达大鲵PGRP-S能诱导NF-κB启动子的活性; 能结合Lys-type和Dap-type的肽聚糖但不能降解它们。研究结果表明大鲵PGRP-S在功能上类似于哺乳动物而有别于硬骨鱼类短型的肽聚糖识别蛋白。     

Cloning and analysis of peptidoglycan recognition protein‐LC and immune deficiency from the diamondback moth,Plutella xylostella

Peptidoglycan (PGN) exists in both Gram‐negative and Gram‐positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP‐LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP‐LC and IMD (PxPGRP‐LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP‐LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP‐LC and IMD homologs. PxPGRP‐LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP‐LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full‐length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP‐LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.     

The peptidoglycan recognition proteins (PGRPs)

Peptidoglycan recognition proteins (PGRPs) are innate immunity molecules present in insects, mollusks, echinoderms, and vertebrates, but not in nematodes or plants. PGRPs have at least one carboxy-terminal PGRP domain (approximately 165 amino acids long), which is homologous to bacteriophage and bacterial type 2 amidases. Insects have up to 19 PGRPs, classified into short (S) and long (L) forms. The short forms are present in the hemolymph, cuticle, and fat-body cells, and sometimes in epidermal cells in the gut and hemocytes, whereas the long forms are mainly expressed in hemocytes. The expression of insect PGRPs is often upregulated by exposure to bacteria. Insect PGRPs activate the Toll or immune deficiency (Imd) signal transduction pathways or induce proteolytic cascades that generate antimicrobial products, induce phagocytosis, hydrolyze peptidoglycan, and protect insects against infections. Mammals have four PGRPs, which are secreted; it is not clear whether any are directly orthologous to the insect PGRPs. One mammalian PGRP, PGLYRP-2, is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and reduces its proinflammatory activity; PGLYRP-2 is secreted from the liver into the blood and is also induced by bacteria in epithelial cells. The three remaining mammalian PGRPs are bactericidal proteins that are secreted as disulfide-linked homo- and hetero-dimers. PGLYRP-1 is expressed primarily in polymorphonuclear leukocyte granules and PGLYRP-3 and PGLYRP-4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus, stomach, and intestine. These three proteins kill bacteria by interacting with cell wall peptidoglycan, rather than permeabilizing bacterial membranes as other antibacterial peptides do. Direct bactericidal activity of these PGRPs either evolved in the vertebrate (or mammalian) lineage or is yet to be discovered in insects.     

Mammalian PGRPs: novel antibacterial proteins

Peptidoglycan recognition proteins (PGRPs) are innate immunity molecules conserved from insects to mammals. Insects have up to 19 PGRPs, which activate Toll or Imd signal transduction pathways or induce proteolytic cascades that generate antimicrobial products, induce phagocytosis, hydrolyse peptidoglycan, and protect insects against infections. Mammals have four PGRPs, which were hypothesized to function as signal-transducing pattern recognition receptors. However, all mammalian PGRPs are secreted, usually as disulphide-linked homo- and heterodimers. One mammalian PGRP, PGLYRP-2, is an N-acetylmuramoyl-L-alanine amidase that hydrolyses bacterial peptidoglycan and reduces its proinflammatory activity. PGLYRP-2 is secreted from liver into blood, and is also induced by bacteria in epithelial cells. The three remaining mammalian PGRPs are bactericidal or bacteriostatic proteins. PGLYRP-1 is expressed primarily in the granules of polymorphonuclear leucocytes (PMNs) , and PGLYRP-3 and PGLYRP-4 are expressed in the skin, eyes, salivary glands, throat, tongue, esophagus, stomach and intestine, and protect the host against infections. They kill bacteria by interacting with their cell wall peptidoglycan, rather than permeabilizing their membranes. These PGRPs therefore are a new class of bactericidal and bacteriostatic proteins that have different structure, mechanism of action, and expression pattern from currently known vertebrate antimicrobial peptides. Direct bactericidal activity of these PGRPs either evolved in vertebrates or mammals, or it is yet to be discovered in insects.     

Distribution of multiple peptidoglycan recognition proteins in the tissues of Pacific oyster, Crassostrea gigas

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors that specifically bind to peptidoglycans, a major component of bacterial cell wall. Generally, PGRPs are responsible for recognition of bacterial invasion in invertebrates. Full length cDNAs of PGRP, designated as CgPGRP-S1S, -S1L, -S2 and -S3, were identified from the Pacific oyster, Crassostrea gigas. Homology and domain searches classified these CgPGRPs as short-type PGRPs for extracellular PGN recognition. Amidase activity was predicted in all CgPGRPs, and defensin-like domains were found in CgPGRP-S1S and -S1L, suggesting that they may also function as antimicrobial proteins. Although phylogenetic analysis indicated that CgPGRPs are closely related to each other, they showed different tissue expression patterns; CgPGRP-S1S in the mantle and the gill, -S1L in the mantle, -S2 in the hemocytes and -S3 in the digestive diverticula. The CgPGRPs seem to survey bacterial invasion in their corresponding expression tissues. This is the first report of the possibility that bivalve mollusks have PGN recognition systems as suggested by the identification of multiple PGRPs distributed in various tissues.