Peptidoglycan recognition protein 2(PGLYRP2) is an enzyme (EC 3.5.1.28), N-acetylmuramoyl-L-alanine amidase (NAMLAA), that hydrolyzes bacterial cell wall peptidoglycan and is encoded by the PGLYRP2 gene. [1] [2] [3] [4] [5] [6]
The N-acetylmuramoyl-L-alanine amidase enzymatic activity was first observed in human and mouse serum in 1981 by Branko Ladešić and coworkers. [11] The enzyme (abbreviated NAMLAA) was then purified from human serum by this [12] and other groups. [13] [14] [15] [16] The sequence of 15 N-terminal amino acids of NAMLAA was identified, [15] but the cDNA for the protein was not cloned and the gene encoding NAMLAA was not known.
In 2000, Dan Hultmark and coworkers discovered a family of 12 Peptidoglycan Recognition Protein (PGRP) genes in Drosophilamelanogaster and by homology searches of available human and mouse sequences predicted the presence of long forms of human and mouse PGRPs, which they named PGRP-L by analogy to long forms of insect PGRPs. [17]
In 2001, Roman Dziarski and coworkers discovered and cloned three human PGRPs, named PGRP-L, PGRP-Iα, and PGRP-Iβ (for long and intermediate size transcripts), [1] and established that human genome codes for a family of 4 PGRPs: PGRP-S (short PGRP) [18] and PGRP-L, PGRP-Iα, and PGRP-Iβ. [1] Subsequently, the Human Genome Organization Gene Nomenclature Committee changed the gene symbols of PGRP-S, PGRP-L, PGRP-Iα, and PGRP-Iβ to PGLYRP1 (peptidoglycan recognition protein 1), PGLYRP2 (peptidoglycan recognition protein 2), PGLYRP3 (peptidoglycan recognition protein 3), and PGLYRP4 (peptidoglycan recognition protein 4), respectively, and this nomenclature is currently also used for other mammalian PGRPs. Sergei Kiselev and coworkers also independently cloned mouse PGLYRP2 (which they named TagL). [2] [19]
In 2003 Håkan Steiner and coworkers [3] and Roman Dziarski and coworkers [4] discovered that mouse [3] and human [4] PGLYRP2 (PGRP-L) proteins encoded by the mouse and human PGLYRP2 genes are N-acetylmuramoyl-L-alanine amidases. Recombinant and native human PGLYRP2 proteins were then further shown to be identical with the previously identified and purified serum NAMLAA. [20]
Human and mouse PGLYRP2 is constitutively expressed in the adult and fetal liver, from where it is secreted into the blood. [1] [3] [20] [21] [22] PGLYRP2 (NAMLAA) is present in human plasma at 100 to 200 µg/ml [16] [23] and at lower concentrations in saliva, milk, cerebrospinal fluid, and synovial fluid. [23] PGLYRP2 is also expressed to a much lower level in the colon, lymph nodes, spleen, thymus, heart, and polymorphonuclear leukocyte granules. [1] [24] [25] PGLYRP2 is differentially expressed in the developing brain and this expression is influenced by the intestinal microbiome. [26] Bacteria and cytokines induce low level of PGLYRP2 expression in the skin and gastrointestinal and oral epithelial cells, [22] [27] [28] [29] [30] and also in intestinal intraepithelial T lymphocytes, dendritic cells, NK (natural killer) cells, and inflammatory macrophages. [31] [32] Some mammals, e.g. pigs, express multiple splice forms of PGLYRP2 with differential expression. [33]
Bacteria and cytokines induce expression of PGLYRP2 in epithelial cells through the p38 mitogen activated protein kinase (MAPK) and IRAK1 (interleukin-1 receptor-associated kinase 1) signaling pathways. [27] [30] Constitutive and induced expression of PGLYRP2 is controlled by different transcription factors whose binding sequences are located in different regions of the PGLYRP2 promoter. [22] Constitutive expression of PGLYRP2 in hepatocytes is regulated by transcription factors c-Jun and ATF2 (activating transcription factor 2) through sequences in the proximal region of the promoter. [22] Induced expression of PGLYRP2 in keratinocytes is regulated by transcription factors NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Sp1 (specificity protein 1) through sequences in the distal region of the promoter. [22]
PGLYRP2 has one canonical carboxy-terminal catalytic peptidoglycan-binding type 2 amidase domain (also known as a PGRP domain) with predicted peptidoglycan-binding and catalytic cleft with walls formed by α-helices and the floor by a β-sheet. [1] [3] [34] PGLYRP2 also has a long N-terminal segment that comprises two thirds of the PGLYRP2 sequence, has two hydrophobic regions, is not found in other mammalian PGLYRP1, PGLYRP3, and PGLYRP4 and in invertebrate PGRPs, and is unique with no identifiable functional motifs or domains. [1] [3] [34] The C-terminal segment is also longer than in other mammalian PGLYRPs. [1] [3] [34] PGLYRP2 has two pairs of cysteines in the PGRP domain that are conserved in all human PGRPs and are predicted to form two disulfide bonds. [1] Human PGLYRP2 is glycosylated [13] [15] and secreted, [12] [13] [14] [15] [16] [20] [21] and forms non-disulfide-linked homodimers. [15]
PGLYRP2, similar to all other amidase-active PGRPs (invertebrate and vertebrate), has a conserved Zn2+-binding site in the peptidoglycan-binding cleft, which is also present in bacteriophage type 2 amidases and consists of two histidines, one tyrosine, and one cysteine (His411, Tyr447, His522, Cys530 in human PGLYRP2). [4]
The PGLYRP2 protein plays an important role in the innate immune responses.
PGLYRP2 is an enzyme (EC 3.5.1.28), N-acetylmuramoyl-L-alanine amidase, that binds and hydrolyzes bacterial cell wall peptidoglycan. [1] [3] [4] [11] [12] [13] [14] [15] [16] [35] Peptidoglycan is the main component of bacterial cell wall and is a polymer of β(1-4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) with MurNAc-attached short peptides, typically composed of alternating L and D amino acids, that cross-link the adjacent polysaccharide chains.
PGLYRP2 hydrolyzes the amide bond between the MurNAc and L-Ala, the first amino acid in the stem peptide. [3] [4] [11] [12] This hydrolysis separates the crosslinking peptides from the polysaccharide chains and solubilizes cross-linked bacterial peptidoglycan into uncross-linked polysaccharide chains. [4] The minimal peptidoglycan fragment hydrolyzed by PGLYRP2 is MurNAc-tripeptide. [4]
The peptidoglycan-binding site, which is also the amidase catalytic domain, is located in the C-terminal PGRP domain. This PGRP domain is sufficient for the enzymatic activity of PGLYRP2, although this activity of the isolated C-terminal fragment is diminished compared with the entire PGLYRP2 molecule. [4] Zn2+ and Zn2+-binding amino acids (His411, Tyr447, and Cys530 in human PGLYRP2) are required for the amidase activity. [4] Cys419 in human PGLYRP2, which is broadly conserved in invertebrate and vertebrate PRGPs, forms a disulfide bond with Cys425 (in human PGLYRP2) and is required for the amidase activity, as this disulfide bond is essential for the structural integrity of the PGRP domain. [4] Cys530 is conserved in all amidase-active vertebrate and invertebrate PGRPs, whereas non-catalytic PGRPs (including mammalian PGLYRP1, PGLYRP3, and PGLYRP4) have serine in this position, [1] and thus the presence of Cys or Ser in this position can be used to predict amidase activity of PGRPs. [4] However, Cys530 and seven other amino acids that are all required for the amidase activity of PGRPs are not sufficient for the amidase activity, which requires additional so far unidentified amino acids. [4]
PGLYRP2 plays a limited role in host defense against infections. PGLYRP2-deficient mice are more sensitive to Pseudomonas aeruginosa -induced keratitis [36] and Streptococcus pneumoniae -induced pneumonia and sepsis. [37] However, PGLYRP2-deficient mice did not show a changed susceptibility to systemic Escherichia coli , Staphylococcus aureus , and Candidaalbicans infections [21] or intestinal Salmonella enterica infection, [32] although the latter was accompanied by increased inflammation in the cecum. [31]
Although PGLYRP2 is not directly bacteriolytic, [4] it has antibacterial activity against both Gram-positive and Gram-negative bacteria and Chlamydiatrachomatis. [38]
Mouse PGLYRP2 plays a role in maintaining healthy microbiome, as PGLYRP2-deficient mice have significant changes in the composition of their intestinal microbiome, which affect their sensitivity to colitis. [39] [40]
PGLYRP2 directly and indirectly affects inflammation and plays a role in maintaining anti- and pro-inflammatory homeostasis in the intestine, skin, joints, and brain.
Hydrolysis of peptidoglycan by PGLYRP2 diminishes peptidoglycan's pro-inflammatory activity. [31] [41] This effect is likely due to amidase activity of PGLYRP2, which separates the stem peptide from MurNAc in peptidoglycan and destroys the motif required for the peptidoglycan-induced activation of NOD2 (nucleotide-binding oligomerization domain-containing protein 2), one of the proinflammatory peptidoglycan receptors. [31]
PGLYRP2-deficient mice are more susceptible than wild type mice to dextran sodium sulfate (DSS)-induced colitis, which indicates that PGLYRP2 protects mice from DSS-induced colitis. [39] Intestinal microbiome is important for this protection, because this increased sensitivity to colitis could be transferred to wild type germ-free mice by microbiome transplant from PGLYRP2-deficient mice. [39]
PGLYRP2-deficient mice are more susceptible than wild type mice to the development of experimentally induced psoriasis-like inflammation, [42] which indicates that PGLYRP2 is anti-inflammatory and protects mice from this type of skin inflammation. This pro-inflammatory effect in PGLYRP2-deficient mice is due to the increased numbers and activity of T helper 17 (Th17) cells and decreased numbers of T regulatory (Treg) cells. [42] PGLYRP2-deficient mice are more susceptible than wild type mice to S. enterica-induced intestinal inflammation, [32] which indicates that PGLYRP2 also has anti-inflammatory effect in the intestinal tract.
However, PGLYRP2 also has opposite effects. PGLYRP2-deficient mice are more resistant than wild type mice to the development of arthritis induced by systemic administration of peptidoglycan or MurNAc-L-Ala-D-isoGln peptidoglycan fragment (muramyl dipeptide, MDP). [43] In this model, PGLYRP2 is required for the production of chemokines and cytokines that attract neutrophils to the arthritic joints. [43] PGLYRP2-deficient mice are also more resistant than wild type mice to bacterially induced keratitis [36] and inflammation in S. pneumoniae-induced lung infection. [37] Moreover, PGLYRP2-deficient mice are more resistant to weight loss in a model of chemotherapy-induced gastrointestinal toxicity, which indicates that in wild type mice PGLYRP2 contributes to the chemotherapy-induced weight loss. [44] These results indicate that under certain conditions PGLYRP2 has pro-inflammatory effects. [36] [37] [43]
PGLYRP2-deficient mice also show higher sociability and decreased levels of anxiety-like behaviors compared with wild type mice, which indicate that PGLYRP2 affects behavior in mice. [26] [45]
Genetic PGLYRP2 variants or changed expression of PGLYRP2 are associated with some diseases. Patients with inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, have significantly more frequent missense variants in PGLYRP2 gene (and also in the other three PGLYRP genes) than healthy controls. [34] These results suggest that PGLYRP2 protects humans from these inflammatory diseases, and that mutations in PGLYRP2 gene are among the genetic factors predisposing to these diseases. PGLYRP2 variants are also associated with esophageal squamous cell carcinoma [46] and Parkinson's disease. [47] [48] [49]
Increased serum PGLYRP2 levels are present in patients with systemic lupus erythematosus and correlate with disease activity index, renal damage, and abnormal lipid profile. [50]
Decreased expression of PGLYRP2 is found in HIV-associated tuberculosis, [51] drug-sensitive tuberculosis, [52] Lyme disease, [53] hepatocellular carcinoma, [54] and myocardial infarction. [55]
Autoantibodies to PGLYRP2 are significantly increased in patients with rheumatoid arthritis. [56]
Peptidoglycan or murein is a unique large macromolecule, a polysaccharide, consisting of sugars and amino acids that forms a mesh-like peptidoglycan layer (sacculus) that surrounds the bacterial cytoplasmic membrane. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Attached to the N-acetylmuramic acid is an oligopeptide chain made of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer. Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm. This repetitive linking results in a dense peptidoglycan layer which is critical for maintaining cell form and withstanding high osmotic pressures, and it is regularly replaced by peptidoglycan production. Peptidoglycan hydrolysis and synthesis are two processes that must occur in order for cells to grow and multiply, a technique carried out in three stages: clipping of current material, insertion of new material, and re-crosslinking of existing material to new material.
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Peptidoglycan recognition proteins (PGRPs) are a group of highly conserved pattern recognition receptors with at least one peptidoglycan recognition domain capable of recognizing the peptidoglycan component of the cell wall of bacteria. They are present in insects, mollusks, echinoderms and chordates. The mechanism of action of PGRPs varies between taxa. In insects, PGRPs kill bacteria indirectly by activating one of four unique effector pathways: prophenoloxidase cascade, Toll pathway, IMD pathway, and induction of phagocytosis. In mammals, PGRPs either kill bacteria directly by interacting with their cell wall or outer membrane, or hydrolyze peptidoglycan. They also modulate inflammation and microbiome and interact with host receptors.
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Peptidoglycan recognition protein 3 is an antibacterial and anti-inflammatory innate immunity protein that in humans is encoded by the PGLYRP3 gene.
Peptidoglycan recognition protein 4 is an antibacterial and anti-inflammatory innate immunity protein that in humans is encoded by the PGLYRP4 gene.
Roman Dziarski is a Polish-born American immunologist and microbiologist. He is best known for his research on innate immunity and bacterial peptidoglycan, for discovering the family of human peptidoglycan recognition proteins, which comprises PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4, and for defining the functions of these proteins.
