Both invertebrate and vertebrate PGRPs function as pattern-recognition and effector molecules in innate immunity.

Consistent with their role in insect immunity, most insect PGRPs are expressed in immune-competent organs 12202122. Insect PGRP-S and other short PGRPs are present in the hemolymph and cuticle and are constitutively synthesized or induced, mainly in the fat-body cells, and some also in the epidermal cells, in the gut, and to a lesser extent in hemocytes. Long insect PGRPs are expressed mainly in hemocytes, although some are also present in the hemolymph (for example Drosophila PGRP-LE). The expression of several short and long insect PGRPs is upregulated by exposure to bacteria or purified bacterial peptidoglycan, which is an essential cell wall component of virtually all bacteria. Differential induction of expression of different PGRPs by different stimuli suggests specificity of induction and effector function of different PGRPs 2122.

Insect PGRPs have recognition, signaling, and effector functions, all of which are important for antimicrobial innate immunity (Figure 4). Three Drosophila PGRPs - PGRP-SA, PGRP-SD, and PGRP-SC1 - recognize bacterial peptidoglycan and activate proteases that cleave Spaetzle, an extracellular cytokine-like protein present in insect hemolymph, which in turn serves as an endogenous activator of Toll 82324 (Figure 4a). Activation of Toll initiates a signal transduction pathway that results in the activation of the Dorsal and Dif transcription factors (which are similar to mammalian nuclear factor NF-κB), which translocate into the nucleus, bind to the NFκB sites in the genome, and initiate transcription of drosomycin and other antimicrobial peptides, which are mainly active against Gram-positive bacteria and fungi (Figure 4a). This pathway is essential for Drosophila immunity to Gram-positive bacteria: mutations in recognition or signal-transduction molecules for this pathway make the flies highly susceptible to infections with Gram-positive, but not Gram-negative, bacteria 82324.

Peptidoglycan is a polymer of β(1-4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), crosslinked by short peptides containing alternating L- and D-amino acids (Figures 3a, 4d and 5c). In position 3, the peptide has either diaminopimelic acid (DAP-type peptidoglycan, found in all Gram-negative bacteria and in Gram-positive bacilli; Figure 4d) or L-lysine (Lys-type peptidoglycan, found in most other Gram-positive bacteria, Figures 3a and 5c).

The Toll pathway is preferentially triggered by the Lys-type peptidoglycan and only weakly by the DAP-type peptidoglycan 25, although both types of peptidoglycan bind to PGRP-SA 12. The probable reason for the weak Toll-activating capacity of DAP-type peptidoglycan is that this peptidoglycan, but not Lys-type peptidoglycan, is the substrate for the carboxypeptidase activity of PGRP-SA 12 (Figure 4d). Efficient triggering of the Toll pathway by PGRP-SA requires cooperation (and probably formation of a complex) with another pattern-recognition molecule, Gram-negative binding protein (GNBP)-1 2627 (Figure 4a). GNBP-1 digests peptidoglycan and generates free reducing ends of MurNAc, which are then recognized by PGRP-SA 28. Drosophila PGRP-SC1 and PGRP-SD 2324, as well as other pattern-recognition molecules such as GNBP-3, also activate the Toll pathway (Figure 4a). Both PGRP-SA and PGRP-SC1 are required for the activation of Toll pathway, whereas PGRP-SD is not essential but enhances Toll activation. Recognition of bacteria by PGRP-SC1 and PGRP-SA may also trigger phagocytosis by an as yet unidentified mechanism 24.

Activation of Drosophila PGRP-LC by Gram-negative bacteria and Gram-positive bacilli (also called rods) triggers another signal transduction pathway, the Imd pathway 1925293031323334 (Figure 4b). Binding of peptidoglycan to Drosophila PGRP-LC induces its oligomerization and recruitment and activation of the death-domain-containing Imd protein 19. The Imd pathway is Toll-independent and results in the activation of Relish transcription factor (which is also similar to mammalian NF-κB) and induction of transcription of diptericin and other antimicrobial peptides that are active primarily against Gram-negative bacteria 293031. PGRP-LC responds primarily to DAP-type peptidoglycan. It is a transmembrane protein and has three alternative splice forms (LC-A, LC-B, and LC-C), which differ in the extracellular PGRP domains; they probably cooperate with each other and have somewhat different recognition specificities 2529323334. PGRP-LC activates the Imd pathway in cooperation with PGRP-LE 35 and also probably with another, as yet unidentified co-receptor (Figure 4b). Drosophila PGRP-LC may also have a role in phagocytosis of Gram-negative bacteria, because inhibition of PGRP-LC expression in Drosophila S-2 cells diminishes phagocytosis of Escherichia coli, but not of Staphylococcus aureus 31; the mechanism of this phenomenon is still unclear, however.

Silkworm (Bombyx mori) and mealworm (Tenebrio molitor) PGRP-S are present in the hemolymph and cuticle, bind bacteria and Lys- and DAP-peptidoglycan, and activate the prophenol-oxidase cascade (Figure 4c) 3637. This generates antimicrobial products, such as melanin and reactive oxygen species, surrounds the infection site with melanin, and contains the infection. Drosophila PGRP-LE 35 and beetle (Holotrichia diomphalia) PGRP-1 38 (and probably other PGRPs) also activate the prophenol-oxidase cascade, but H. diomphalia PGRP-1 responds to 1,3-β-D-glucan, a common constituent of fungal cell walls.

Drosophila PGRP-SC1 and PGRP-LB are N-acetylmuramoyl-L-alanine amidases 714, which hydrolyze the amide bond between MurNAc and L-alanine and thus remove stem peptides from peptidoglycan (Figure 4d). Stem peptides are the four to five amino acids directly bound to MurNAc. Digestion of peptidoglycan with amidase reduces or eliminates the ability of polymeric peptidoglycan to stimulate insect cells 14, and thus the function of amidase PGRPs in vivo may be to prevent excessive activation of the immune system by bacteria 3940. On the basis of the conserved structure of the active site of the amidase, several other insect PGRPs are predicted to have amidase activity, whereas several others are not 91415 (Figure 1 and Table 1). One PGRP that is not an amidase, Drosophila PGRP-SA, has an L,D-carboxypeptidase activity with specificity for the bond between DAP and D-Ala of the stem peptide present in peptidoglycan of Gram-negative bacteria and Gram-positive rod bacteria 12 (Figure 4). The biological significance of this carboxypeptidase activity is not certain.

Mammalian PGLYRPs are differentially expressed in various organs and tissues and have two major functions: amidase activity and antibacterial activity. Mammalian PGLYRP-2 (and probably other vertebrate PGLYRP-2s) is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes the lactyl bond between the MurNAc and L-alanine in bacterial peptidoglycan (Figure 5c) 915. PGLYRP-2 is constitutively produced in the liver and is secreted from the liver into the blood 16. This liver PGLYRP-2 and serum N-acetylmuramoyl-L-alanine amidase (which was identified earlier but not cloned) are the same protein, encoded by the PGLYRP2 gene 16. The function of this amidase is probably to eliminate the proinflammatory peptidoglycan and thus to prevent overactivation of the immune system and excessive inflammation.

Mammalian PGLYRP-2 is also expressed in the intestinal follicle-associated epithelial cells 41. PGLYRP-2 is not expressed in healthy human skin, but its expression is induced in keratinocytes and other epithelial cells by exposure to bacteria and cytokines 4243. Some mammals express multiple splice forms of PGLYRP-2 that may have different expression and possibly multiple functions. For example, pigs have two PGLYRP-2 splice forms, short and long. They both have N-acetylmuramoyl-L-alanine amidase activity, and the long form has similar expression to human PGLYRP-2, whereas the short form is constitutively expressed in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin 44.

Mammalian PGLYRP-1 is highly expressed in the bone marrow 13, and the protein is almost exclusively present in the granules of polymorphonuclear leukocytes 4546474849 (Figure 5d). Mammalian PGLYRP-3 and PGLYRP-4 proteins are selectively expressed in the skin epidermis, hair follicles, sebaceous glands and sweat glands; in the eye's ciliary body (which produces aqueous humor that fills the anterior and posterior chambers of the eye); in the eye's corneal epithelium; in the mucus-secreting cells of the main salivary (sub-mandibular) gland and in mucus-secreting glands in the throat (both mucus-secreting glands selectively express PGLYRP-4, but not PGLYRP-3); in the tongue and esophagus in squamous epithelial cells; in the stomach in acid-secreting parietal cells (PGLYRP-3) and glycoprotein-secreting neck mucous cells (PGLYRP-4); and in the small and large intestine in the columnar absorptive cells, but not in mucus-secreting goblet cells and not in Paneth cells in the crypts, which produce antimicrobial peptides 1750 (Figure 5a,b). Bacteria and their products increase the expression of PGLYRP-3 and PGLYRP-4 in keratinocytes 17 and oral epithelial cells 51, probably through activation of the Toll-like receptors TLR2, TLR4, Nod1, and Nod2.

Human PGLYRP-1, PGLYRP-3, PGLYRP-4, the heterodimer formed by PGLYRP-3 and PGLYRP-4, (PGLYRP-3:4), and bovine PGLYRP-1 are bactericidal for many pathogenic and nonpathogenic Gram-positive and Gram-negative bacteria 174647 (Figure 5a,b,d). PGLYRP-1, PGLYRP-3, and PGLYRP-4 from other mammalian species are also likely to have similar bactericidal activity. Bovine PGLYRP-1 also has some microbicidal activity against a fungus, Cryptococcus neoformans 4647. This broader spectrum of microbicidal activity of bovine PGLYRP-1 could reflect a true difference between the human and bovine orthologs, or it might simply reflect a difference in the protein purification methods and assay conditions.

Crystallographic analysis of human PGLYRP-1 and the carboxy-terminal PGRP domain of PGLYRP-3, as well as insect PGRP-LB, -SA, -LC and -LE, show that all these PGRPs have a ligand-binding groove that binds peptidoglycan and is specific for MurNAc bound to three peptide-bonded amino acids (muramyl-tripeptide), which is the minimum peptidoglycan fragment hydrolyzed by PGLYRP-2 791011121352535455. It can accommodate a larger structure, such as GlcNAc-MurNAc-tetrapeptide or MurNAc-pentapeptide (Figure 3), but it does not bind muramyl-dipeptide or a peptide without MurNAc 565758. These results are consistent with the specificity of human PGLYRP-2 for muramyl-tripeptide and with the specificity and high affinity K_d = 13 nM) of murine PGLYRP-1 for uncrosslinked polymeric peptidoglycan but not muramyl-dipeptide or pentapeptide 45. The high-affinity binding of peptidoglycan to PGLYRP is achieved by burying both the peptide and MurNAc portions of peptidoglycan in a deep cleft that completely excludes solvent 52.

Human PGLYRP-1 and a carboxy-terminal fragment of PGLYRP-3 bind muramyl-tetrapeptide and muramyl-pentapeptide with higher affinity than muramyl-tripeptide 5658. Moreover, binding of muramyl-pentapeptide (but not muramyl-tripeptide) to the carboxy-terminal fragment of PGLYRP-3 induces a conformational change in the PGLYRP-3 molecule that locks the ligand in the binding groove (Figure 3) 58. Some PGRPs (such as a carboxy-terminal fragment of human PGLYRP-3) have a preference for binding the Lys-type over the DAP-type peptidoglycan, whereas others (such as human PGLYRP-1 or Drosophila PGRP-LCx and PGRP-LE) bind DAP-type peptidoglycan with higher affinity than Lys-type peptidoglycan 54555657. The only difference between Lys and DAP is the presence of an additional carboxylate at carbon 1 of DAP. Discrimination between Lys- and DAP-type peptidoglycan is based on three amino acids in the peptidoglycan-binding groove, corresponding to Asn236, Phe237, and Val256 in human PGLYRP-3 for binding Lys, or Gly68, Trp69, and Arg88 in human PGLYRP-1 in the same position for binding DAP, or Gly234, Trp235 and Arg254 in Drosophila PGRP-LE for binding DAP 54555657. The importance of these Asn and Phe or Gly and Trp for binding Lys and DAP is verified by mutations in these positions that can change the specificity of the binding from Lys to DAP or DAP to Lys 57. This allows prediction of binding specificity of various PGRP domains for Lys- or DAP-type peptidoglycan. Moreover, both human and insect PGRPs have a dual strategy for discrimination among different types of peptidoglycan, using detection of Lys or DAP in the stem peptide together with the type of peptide crossbridge 57. Detection of peptide-crosslinked peptidoglycan would require engagement of two peptidoglycan-binding sites in two PGRP domains, which could be accomplished by PGRPs with two PGRP domains and/or by dimeric PGRPs, which is consistent with recent demonstration of dimeric PGRPs in mammals 17 and insects 1819.

There is likely, however, to be considerable variation in the fine specificity of different PGRPs, because the residues in and around the peptidoglycan-binding groove are relatively variable; they are less than 50% conserved among PGRPs 71152. This structural variation may correspond to different ligand specificities of different PGRPs. Mammalian PGLYRPs bind to both Gram-positive and Gram-negative bacteria and also some fungi 1747, and some insect PGRPs (such as H. diomphalia PGRP-1) bind fungal β-glucan 38. Therefore, binding to peptidoglycan is not always responsible for PGRP binding, and even with bacteria there are indications that some PGRPs may also bind to other polymers, such as lipoteichoic acid and lipopolysaccharide 174547. Human and mouse PGLYRPs have the highest affinity for peptidoglycan, however, and much lower affinities for lipoteichoic acid and lipopolysaccharide 1745, whereas bovine PGLYRP-1 seems to have high affinity for lipoteichoic acid and lipopolysaccharide 47. It is not clear, however, whether these other ligands bind to the peptidoglycan-binding groove or to another portion of the PGLYRP molecule, such as the hydrophobic region on the opposite side of the molecule. Binding of peptidoglycan outside the peptidoglycan-binding groove was recently shown, which contributes to the formation of PGRP-LE oligomers 54 or PGRP-LCx:PGRP-LCa dimers 55.

The diversity of PGRP specificities is also increased by duplication of PGRP domains and dimerization. PGLYRP-3 and PGLYRP-4 both have two PGRP domains, and each PGRP domain has one ligand-binding site 52. Thus, whereas PGLYRP-1 monomers and dimers have one and two identical ligand-binding sites, respectively, PGLYRP-3 and PGLYRP-4 monomers and dimers have two and four ligand-binding sites, respectively (Figure 5). Because these PGRP domains in PGLYRP-3 and PGLYRP-4 are not identical (they have 37-43% identity), however, the fine binding specificity or affinity of each PGRP domain in these PGLYRP molecules is probably different. For example, the carboxy-terminal and amino-terminal PGRP domains in human PGLYRP-3 are specific for DAP-type and Lys-type peptidoglycan, respectively 57. The diversification of PGLYRP specificities is then further increased by formation of PGLYRP-3:4 heterodimers, which have four different binding sites. In this way, the host can fine-tune the specificities of PGLYRPs by expressing PGLYRP-3 and PGLYRP-4 either in the same or in separate cells, to form hetero- or homodimers, respectively. In addition, PGRPs have hydrophobic domains on the opposite side of the molecule from the ligand-binding groove, which were previously hypothesized to interact with signal transduction molecules 7. In mammalian PGLYRPs, however, these hydrophobic domains may either have a role in the interaction of PGLYRPs with bacteria, or in the formation of dimers.

Mammalian PGLYRP-1, PGLYRP-3, and PGLYRP-4 form a new class of bactericidal proteins that have a different structure, mechanism of action, and expression from those of currently known mammalian antimicrobial peptides 617. PGLYRPs are much larger than all currently known vertebrate antibacterial peptides: PGLYRP-1, PGLYRP-3, PGLYRP-3:4, and PGLYRP-4 proteins are disulfide-linked glycosylated 44 kDa, 89 kDa, 98 kDa, and 115 kDa dimers 17, and vertebrate antimicrobial peptides are typically 3 kDa to 15 kDa. PGLYRPs require divalent cations and N-glycosylation for bactericidal activity, which are not usually required by membrane-permeabilizing antibacterial peptides, such as defensins or magainin 17. Mammalian PGLYRPs also differ from antimicrobial peptides in their mechanism of bactericidal activity: they kill bacteria by interacting with cell-wall peptidoglycan, whereas antimicrobial peptides do so by permeabilizing bacterial membranes 17. Furthermore, the expression patterns of mammalian PGLYRPs and antimicrobial peptides are different, and some cells that produce large amounts of these peptides, such as Paneth cells (which produce defensins, phospholipase A₂, and lysozyme), do not express PGLYRPs 17.