Peptidoglycan Recognition Protein 2 Research Articles

Novel role of peptidoglycan recognition protein 2 in activating NOD2-NFκB inflammatory axis in coronary artery disease

Backgrounds and aimsThe role of inflammation in driving atherosclerosis is well-established. It exerts systemic effects beyond the local site of plaque formation. In the context of coronary artery disease (CAD), the proteins that show altered levels in the plasma, are potentially important for understanding the key regulatory mechanism in the pathogenesis of atherosclerosis. A case-control study revealed that plasma soluble Peptidoglycan Recognition Protein 2 (PGLYRP2) primarily produced by the liver, is increased in subjects with CAD. Furthermore, the concentration of PGLYRP2 in the blood correlates with the severity of coronary artery disease. Thus, it raises interest in understanding the exact role of the protein in aortic inflammation and plaque progression. MethodsWe evaluated the plasma concentration of PGLYRP2 in three distinct groups: patients with CAD (N = 68), asymptomatic individuals (N = 34), and healthy volunteers (N = 20). Furthermore, we investigated the correlation between disease severity and PGLYRP2 levels in CAD patients. To identify potential binding partners of PGLYRP2, we employed computational analysis. We verified the PGLYRP2-NOD2 interaction in macrophage cells and elucidated the inflammatory pathways activated by PGLYRP2 within these cells. To assess the impact of PGLYRP2, we examined its effects in the atherosclerotic mice model (ApoE−/−). ResultsIn this study, we report for the first time that Nucleotide-binding Oligomerization domain 2 (NOD2) which is expressed on the surface of macrophages, is a receptor of PGLYRP2. The N-terminal domain of PGLYRP2 directly binds to NOD2 and activates the NOD2-RIP2-NFκB cascade that promotes the secretion of proinflammatory cytokines like TNFα, IL1β, and IL-8. In the atherosclerotic mice model (ApoE−/−) we demonstrate that elevated PGLYRP2 level is parallel with increased proinflammatory cytokines in the plasma when fed a High Cholesterol Diet (HCD). Immunohistochemical analysis reveals that PGLYRP2 is co-localized with NOD2 on the macrophages at the site of the lesion. ConclusionsTaken together, our data demonstrate that NOD2 acts as a receptor of PGLYRP2 on macrophages, which mediates the activation of the NOD2-RIP2-NFκB pathway and promotes inflammation, thus significantly contributing to the development and progression of atherosclerosis.

Afidopyropen suppresses silkworm growth and vitality by affecting carbohydrate metabolism and immune function

Afidopyropen has strong insecticidal toxicity to sucking pests by silencing the vanilloid-type transient receptor potential (TRPV) channels. However, the toxicity of afidopyropen to the Lepidoptera model insect silkworm remain unknown. In this study, the LC50 of afidopyropen to the silkworm at 72 h exposure was 256.82 mg/L. This indicates that afidopyropen is moderately toxic to the silkworm. Long-term exposure to concentrations of 100 mg/L, or less, of afidopyropen, significantly reduced silkworm growth, vitality, silk protein synthesis, and fecundity. A total of 220 differentially expressed genes (DEGs) were detected by transcriptome sequencing, among which 166 were downregulated and 54 were upregulated. Gene Ontology (GO) enrichment analysis showed that the DEGs were enriched in the immune system, immune response and carbohydrate metabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that DEGs were primarily concentrated in carbohydrate metabolism and biosynthesis of neomycin, kanamycin and gentamicin. Genes related to carbohydrate metabolism and immune system pathways in silkworm were detected by quantitative real-time PCR. The results showed that the genes related to carbohydrate metabolism, silk protein synthesis, and immune response were significantly downregulated. These genes included BCL-6 corepressor-like protein 1 (BCORL1), hexokinase type 2 (HEXO2), phosphoserine aminotransferase 1 (PSAT1), relish (Rel), peptidoglycan recognition protein 2 (PGRP2) and 27 kda glycoprotein precursor (P27K). The data demonstrated the toxic effects of afidopyropen against the silkworm and its regulation of genes responsible for immune function and abscissa carbohydrate metabolism.

Identification of PGRP2 and its three splice variants in grass carp Ctenopharyngodon idella

In this study, peptidoglycan recognition protein 2 (PGRP2) gene was cloned in grass carp Ctenopharyngodon idella, with the open reading frame (ORF) of PGRP2 being 1452 bp, encoding a protein of 483 amino acids. Three splice variants, PGRP2a, PGRP2b, and PGRP2c, were found also in grass carp with the absence of entire exon two and partial exon two of the PGRP2, and were predicted to have 124, 371 and 311 amino acids. But, they all have PGRP domain and signal peptide, except PGRP2a. The PGRP2 and its variants were expressed in all organs/tissues examined, and stimulated following PGN injection. It is further detected that the expression of gcPGRP2 and its variants was up-regulated after the single transfection of each of gcPGRP2 and its variant expression plasmids in CO cells. It is considered that the cloning of PGRP2 in grass carp provides a compositional completeness of PGRP members in this fish with the inclusion of previously reported PGRP5 and PGRP6.

PGLYRP2 as a novel biomarker for the activity and lipid metabolism of systemic lupus erythematosus

BackgroundTo assess the value of peptidoglycan recognition protein 2 (PGLYRP2) in assessing the disease activity and lipid metabolism in patients with systemic lupus erythematosus (SLE).MethodsSLE patients with stable disease (n = 15), active lupus nephritis (LN) (n = 15) and neuropsychiatric systemic lupus erythematosus (NP-SLE) (n = 15) admitted to Northern Jiangsu People’s Hospital (Jiangsu, China) in 2019–2020 were recruited. In addition, volunteers with matched age and sex (n = 15) were enrolled as controls. The level of PGLYRP2 in the serum and its expression in peripheral blood mononuclear cells (PBMCs) were measured. The link between PGLYRP2 level and clinical parameters (including lipid profile) was described.ResultsSerum PGLYRP2 level in SLE cases exceeded that in healthy volunteers (3938.56 ± 576.07 pg/mL), and significantly higher in active LN (5152.93 ± 446.13 pg/mL) and NP-SLE patients (5141.52 ± 579.61 pg/mL). As shown by quantitative real-time PCR results, the expression of PGLYRP2 in PBMCs of SLE patients with active LN and NP-SLE surpassed that in healthy volunteers (P < 0.01). Receiver operating characteristic (ROC) curves demonstrated that PGLYRP2 was capable of distinguishing stable SLE from active LN (AUC = 0.841, 95%CI = 0.722–0.960, P = 0.000). PGLYRP2 level positively correlated with SLEDAI of SLE patients (r = 0.5783, P < 0.01). Moreover, its level varied with serological and renal function parameters (complement 3, complement 4, estimated glomerular filtration rate and 24-h urine protein) and immunoglobulin A (IgA) of SLE. A potential correlation between PGLYRP2 level and lipid profile (HLD-c, Apo-A1 and Apo B/A1) was determined in SLE patients. The linear regression analysis indicated SLEDAI as an independent factor of PGLYRP2 level, with a positive correlation in between (P < 0.05).ConclusionsSerum PGLYRP2 level significantly increases in SLE patients, and is positively correlated to SLEDAI. Moreover, serum PGLYRP2 level is correlated with renal damage parameters and the abnormal lipid profile of SLE. PGLYRP2 could be used to predict SLE activity, dyslipidemia and cardiovascular disease risks in SLE patients.

Contribution of plasma protein PGLYRP2 in the inflammatory pathway in atherosclerosis

Background and Aims: Peptidoglycan Recognition Protein 2 (PGLYRP2) belongs to highly conserved group of Peptidoglycan Recognition Protein (PGLYRPs) whose major function identified till date is recognition and cleaving of PGN by its amidase activity, by means of which it initiates an inflammatory response. Recently, it has been identified as one of the circulating proteins that showed increased expression in ST segment Elevated Myocardial Infarction patients (STEMI). In this study we aimed to find out its role in atherosclerosis in the context of inflammation.

Antibody to peptidoglycan recognition protein (PGLYRP)-2 as a novel biomarker in rheumatoid arthritis.

To identify novel autoantigens from circulating immune complexes (CICs) in rheumatoid arthritis (RA) patients and further explore their clinical significance. From serum samples of 10 early RA (ERA) patients and 10 healthy donors, CICs were isolated and subjected to orbitrap mass spectrometry for autoantigen identification. Antibodies against the peptidoglycan recognition protein-2 (PGLYRP-2) derived from CICs were further detected by indirect enzyme-linked immunosorbent assay (ELISA) in 178 patients with RA, compared with 59 osteoarthritis (OA), 59 systemic lupus erythematosus (SLE), 55 ankylosing spondylitis (AS), 95 primary Sjögren's syndrome (pSS) and 50 healthy controls (HC). Thirty-three potential antigens out of 323 proteins were identified from CICs of RA patients. The autoantibodies to PGLYRP-2 were significantly increased in RA patients with 42.70% sensitivity and 85.20% specificity in comparison to other rheumatic diseases and healthy controls. The prevalence of anti-PGLYRP-2 was also elevated in subgroups of RA, with 34.72% in ERA, 35.29% in RF negative and 42.86% in anti-CCP negative patients. Further analysis suggested that anti-PGLYRP-2 was potentially accompanied with production of other autoantibodies in RA. In addition, we found by homology analysis that an epitope of PGLYRP-2442-447 mimics amino acid residues 431-436 of N-acetylmuramoyl-L-alanine amidase (NAMLAA) in actinomyces naeslundii. Autoantibody against PGLYRP-2 was identified as a promising biomarker in RA, especially in early and seronegative patients.

Tumor-Derived Peptidoglycan Recognition Protein 2 Predicts Survival and Antitumor Immune Responses in Hepatocellular Carcinoma.

Background and AimsHepatocellular carcinoma (HCC) is linked to immunosuppression. Relieving immunosuppression has been an attractive strategy to improve the efficacy of cancer immunotherapy. Peptidoglycan recognition protein 2 (PGLYRP2) is a pattern recognition receptor which is specifically expressed in liver and implicated in the regulation of innate immunity and immunosurveillance. However, the role of hepatic PGLYRP2 in modulating immune responses against HCC remains to be investigated.Approach and ResultsIn this study, we investigated whether PGLYRP2 is able to influence HCC progression through regulating host antitumor immune responses. We demonstrated that PGLYRP2 was down‐regulated in HCC, which was linked with poor prognosis in patients (P < 0.001). PGLYRP2 overexpression in HCC cells significantly enhanced antitumor immune responses in immune‐competent mice and elevated immune response rates of peripheral blood mononuclear cells against HCC. Mechanistically, DNA methyltransferase 3A–mediated promoter hypermethylation was responsible for the down‐regulation of PGLYRP2 in HCC. PGLYRP2 promoted production of chemokine (C‐C motif) ligand 5 (CCL5) in HCC through binding to the CCL5 promoter, which contributed to the enhanced antitumor immunity.ConclusionsWe provide evidence that tumor‐derived PGLYRP2 acts as a candidate biomarker for adequate immune response against HCC and improved patient outcomes, indicating the importance of hepatic PGLYRP2 in cancer immunosurveillance and in designing immunotherapeutic approaches.

Characterization of gene expression profiles and functional analysis of peptidoglycan recognition protein 2 from rock bream (Oplegnathus fasciatus)

Peptidoglycan recognition protein 2 (PGRP2) is a Zn2+-dependent peptidase that plays important roles in binding to microbial components of the cell membrane, inducing phagocytosis and antimicrobial activity. Rock bream (Oplegnathus fasciatus) PGRP2 (RbPGRP2) was identified in the intestine by next generation sequencing (NGS) analysis. The open reading frame (ORF) the RbPGRP2 cDNA (470 amino acid residues) contains a peptidoglycan recognition protein domain (residues 300 to 446). Alignment analysis revealed that RbPGRP2 shares 37.6–53.5% overall sequence identity with the PGRP2s of other species. Phylogenetic analysis revealed that RbPGRP2 clustered together with PGRP2s from teleosts. In healthy rock bream, RbPGRP2 was found to be ubiquitously expressed in all of the examined tissues, especially in the liver. RbPGRP2 expression was significantly upregulated in all of the examined tissues of rock bream after infection with Edwardsiella piscicida, Streptococcus iniae and red sea bream iridovirus (RSIV) compared with the control. Purified rRbPGRP2 interactions with bacteria and inhibited the growth of bacteria in the presence of Zn2+. These results indicate that RbPGRP2 plays an important role in the innate immune response against bacterial infection.

RNA‐seq analysis identifies novel gene signatures common to SHP knockout mice and human steatosis, fibrosis, NASH and cirrhosis

Chronic hepatitis C virus and non‐alcoholic fatty liver disease are major causes of cirrhosis and hepatocellular carcinoma. It is vital to identify genetic changes fueling liver disease progression in order to better prevent, diagnose and treat chronic liver disease. Nuclear receptor small heterodimer partner (Shp) null mice are an attractive model as they have altered hepatic lipid and bile acid metabolism and develop spontaneous hepatoma. Using next generation RNA sequencing, we identified common genes that were significantly differentially expressed in Shp−/− mice, hepatitis C cirrhosis, and non‐alcoholic steatohepatitis (NASH). Novel gene signatures and their functional significance were validated in human steatosis, fibrosis, NASH, alcohol and hepatitis C cirrhosis as well as in Shp−/− and SHP transgenic mice, liver fibrosis, inflammation and bile acid injury models. The novel genes include peptidoglycan recognition protein 2 (PGLYRP2), monooxygenase, DBH‐like 1 (MOXD1), dual specific phosphatase‐4 (DUSP4), thrombospondin 1 (THBS1), tetraspanin 4 (TSPAN4), SPARC‐related modular calcium binding protein‐2 (SMOC‐2), latent transforming growth factor‐beta binding protein 4 (LTBP4), and family with sequence similarity 198a (FAM198A).ConclusionOur study has identified novel genes implicated in chronic human liver diseases using RNA sequencing and Shp−/− mice as our primary model.This study was supported by the NIH DK080440 (LW) and CA148068 (CHH).

Polymorphism in PGLYRP-2 gene by PCR-RFLP and its association with somatic cell score and percentage of fat in Chinese Holstein

Peptidoglycan recognition protein 2 (PGLYRP-2), which belongs to the PGRP family, is the only member that has no direct bactericidal activity but has N-acetylmuramoyl-l-alanine amidase activity. This feature of PGLYRP-2 indicates that it may play an important role in eliminating the pathogen associated molecular pattern (PAMP), such as peptidoglycan (PGN), which can reduce leukocytes in blood and lower somatic cell count (SCC) in milk. To investigate whether the PGLYRP-2 gene is associated with mastitis and milk production traits in dairy cattle, the polymorphism of this gene was analyzed by PCR-RFLP in a population of 546 Chinese Holstein cows. A total of five single nucleotide polymorphism (SNP) loci were identified. The association analysis of a single SNP locus showed that the C+4867T locus was significantly associated (P < 0.05) with somatic cell score (SCS). Surprisingly, all loci were significantly associated (P < 0.01 or P < 0.05) with percentage of fat. Association analysis between combined genotypes and SCS and milk production traits indicated that H2H2 was associated with higher percentage of fat (P < 0.05). These findings demonstrated that SNPs in PGLYRP-2 gene were related to mastitis resistance and percentage of fat, and that H2H2 would be a useful genetic marker of combined genotypes for breeding of Chinese Holstein.

Control of intestinal Nod2-mediated peptidoglycan recognition by epithelium-associated lymphocytes.

Innate immune recognition of the bacterial cell wall constituent peptidoglycan by the cytosolic nucleotide-binding oligomerization domain 2 (Nod2) receptor has a pivotal role in the maintenance of intestinal mucosal homeostasis. Whereas peptidoglycan cleavage by gut-derived lysozyme preserves the recognition motif, the N-acetylmuramoyl-L-alanine amidase activity of the peptidoglycan recognition protein 2 (PGLYRP-2) destroys the Nod2-detected muramyl dipeptide structure. PGLYRP-2 green fluorescent protein (GFP) reporter and wild-type mice were studied by flow cytometry and quantitative RT-PCR to identify Pglyrp-2 expression in cells of the intestinal mucosa and reveal a potential regulatory function on epithelial peptidoglycan recognition. CD3(+)/CD11c(+) T lymphocytes revealed significant Pglyrp-2 expression, whereas epithelial cells and intestinal myeloid cells were negative. The mucosal Pglyrp-2-expressing lymphocyte population demonstrated a mixed T-cell receptor (TCR) αβ or γδ phenotype with predominant CD8α and less so CD8β expression, as well as significant staining for the activation markers B220 and CD69, presenting a typical intraepithelial lymphocyte phenotype. Importantly, exposure of peptidoglycan to PGLYRP-2 significantly reduced Nod2/Rip2-mediated epithelial activation. Also, moderate but significant alterations of the intestinal microbiota composition were noted in Pglyrp-2-deficient animals. PGLYRP-2 might thus have a significant role in regulation of the enteric host-microbe homeostasis.

Unexpected expression of peptidoglycan recognition protein 2 in lymphocytes and bone marrow hematopoietic stem cells (87.8)

Abstract Peptidoglycan recognition factors are highly conserved molecules involved in recognition of fragments of bacterial peptidoglycans in innate immune responses. Peptidoglycan recognition protein 2 (PGLYRP2) is a serum N-acetylmuramoyl-alanine amidase secreted by the liver. We used a green fluorescent reporter allele engineered into the gene to label cells in the mouse that endogenously express PGLYRP2. Unexpectedly, PGLYRP2 was expressed constitutively in resting CD4 and CD8 lymphocytes and in NK cells. Reconstitution of PGLYRP2-deficient mice with PGRLYRP2-sufficient T cells led to the accumulation of PGLYRP2 in serum. In assessing bone marrow populations in these mice, we identified constitutive PGLYRP2 expression in a subset of lineage-negative, c-kit+Sca1+ cells that had the properties of hemopoietic stem cells, including the capacity to establish hematopoietic lineages and for self renewal when transferred into irradiate recipient mice. Expression of PGLYRP2 was also confirmed within the conventionally identified hematopoietic stem cell compartment in wildtype mice, confirming the unexpected expression of this enzyme in bone marrow stem cells.

Differential Expression of Peptidoglycan Recognition Protein 2 in the Skin and Liver Requires Different Transcription Factors

Human peptidoglycan recognition protein 2 (PGLYRP2) is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and is differentially expressed in the two major organs in the human body, liver and skin. PGLYRP2 has a high constitutive expression in the liver but is not expressed in healthy human skin. PGLYRP2 mRNA is also not expressed in cultured human keratinocytes but is highly induced upon exposure to bacteria. In this study we identified the transcription start site for pglyrp2 and demonstrated that the differential expression of PGLYRP2 in hepatocytes and keratinocytes is regulated by different transcription factors whose binding sequences are located in different regions of the pglyrp2 promoter. Induction of pglyrp2 in keratinocytes is regulated by sequences in the distal region of the promoter and requires transcription factors NF-kappaB and Sp1, whereas constitutive expression of pglyrp2 in a hepatocyte cell line is regulated by sequences in the proximal region of the promoter and requires transcription factors c-Jun and ATF2. Regulation of constitutive and inducible expression of pglyrp2 is important for systemic and local innate immune responses to bacterial infections.

Peptidoglycan recognition protein 2 (N-acetylmuramoyl-L-Ala amidase) is induced in keratinocytes by bacteria through the p38 kinase pathway.

Human peptidoglycan recognition protein 2 (PGLYRP2) is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes bacterial peptidoglycan and is constitutively produced in the liver and secreted into the blood. Here we demonstrate that PGLYRP2 was not expressed in healthy human skin and had low expression in the eye. However, upon exposure to gram-positive and gram-negative bacteria or cytokines, PGLYRP2 expression was highly induced in keratinocytes and to a lower level in corneal epithelial cells. Expression of PGLYRP2 was not induced in nonepithelial cells. Exposure of keratinocytes to bacteria induced keratinocyte differentiation and stress response and inhibited activation of signal transduction molecules involved in cell proliferation. Induction of PGLYRP2 expression correlated with expression of differentiation markers (cytokeratins and transglutaminase). Bacteria induced activation of p38 mitogen-activated protein kinase (MAPK) in keratinocytes, which was required for the induction of PGLYRP2 expression, because induction of PGLYRP2 transcription by bacteria was inhibited by SB203580 (a specific inhibitor of p38 MAPK) and by a dominant-negative p38 construct. Induction of PGLYRP2 expression by bacteria (in contrast to expression of human beta-defensin-2) was not mediated by Toll-like receptor 2 or 4. PGLYRP2 may function in the skin and the eyes as an inducible scavenger of proinflammatory peptidoglycan.

Identification of serum N-acetylmuramoyl- l-alanine amidase as liver peptidoglycan recognition protein 2

N-acetylmuramoyl- l-alanine amidase (NAMLAA) hydrolyzes bacterial peptidoglycan and is present in human serum. A peptidoglycan-recognition protein 2 (PGLYRP2) is expressed in human liver and has N-acetylmuramoyl- l-alanine amidase activity. Here, we determined the amino acid sequences of human serum NAMLAA and liver PGLYRP2 and tested the hypothesis that serum NAMLAA and PGLYRP2 are the same protein. Liver PGLYRP2 and serum NAMLAA had the same mass determined by mass spectrometry and polyacrylamide gel electrophoresis, and both proteins and recombinant PGLYRP2 reacted with polyclonal anti-NAMLAA and anti-PGLYRP2 antibodies, and with monoclonal anti-NAMLAA antibodies. Digestion of serum NAMLAA with trypsin, chymotrypsin, or trypsin plus V8 protease, or with CNBr yielded, respectively, 37, 40, and 3 overlapping peptides that matched 100% and covered 81% of the deduced amino acid sequence of mature PGLYRP2. These peptides overlapped all exon–intron junctions indicating no alternative splice forms. Digestion of liver PGLYRP2 with trypsin yielded 23 peptides that matched 100% and covered 44% of the deduced amino acid sequence of mature PGLYRP2. Serum NAMLAA had a C398–C404 disulfide, partial phosphorylation of S218, and deamidation of N253 and N301. These results indicate that serum NAMLAA and liver PGLYRP2 are the same protein encoded by the pglyrp2 gene.