PGRP-LB (AGAP001212) is an Anopheles gambiae peptidoglycan recognition protein with a conserved zinc-dependent amidase domain. Phylogenetic evidence supports cleavage of bacterial peptidoglycan, reducing immunostimulatory ligands and regulating the IMD/REL2 innate immune pathway. Work on the Anopheles coluzzii ortholog shows that gut PGRPLB restrains late systemic antimicrobial responses after blood feeding, with no change in the measured local midgut CEC1 response upon knockdown. Two predicted membrane-associated isoforms differ in their transmembrane regions; their detailed localization and secretion remain unresolved.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005615 extracellular space | IBA GO_REF:0000033 | UNDECIDED | Summary: Extracellular-space localization remains unresolved for the predicted membrane-associated mosquito PGRP-LB products. Reason: GOA traces this IBA to PTN002475783 in PTHR11022. The current PAINT snapshot places extracellular region at this node, whereas the older row specifies extracellular space. PMID:28494453 describes two Anopheles coluzzii PGRPLB isoforms differing in predicted transmembrane domains, but does not experimentally establish secretion, ectodomain release, or complete exclusion from extracellular space. Membrane prediction is insufficient to replace an inherited extracellular annotation categorically. Keep the original location unresolved pending isoform-resolved localization assessment. Propagation Review Root cause: UNRESOLVED Sources checked: PANTHER:PTN002475783 · PTN002475783 UNRESOLVED Current IBD is extracellular region; GOA carries extracellular space. Predicted membrane topology raises a location question but is not a demonstrated loss. Supporting Evidence: PMID:28494453 PGRPLB encodes 2 isoforms, which share the same PGRP domain and differ in their transmembrane domains |
| GO:0006955 immune response | IBA GO_REF:0000033 | ACCEPT | Summary: Immune regulation is central to PGRP-LB function and supports this broad immune-process annotation. Reason: An amidase that removes immune-stimulating peptidoglycan performs a regulatory step in immunity; it need not activate an immune response to participate. The PTHR11022 ancestral immune-response placement at PTN002475783 is consistent with direct PGRPLB knockdown in Anopheles coluzzii, a member of the Anopheles gambiae complex formerly designated its M form. The primary experiment supports restraint of systemic responses, rather than the previous claim of reduced local midgut CEC1 expression. Supporting Evidence: PMID:28494453 PGRPLB silencing did not affect CEC1 expression in the gut but significantly induced CEC1 in the fat body 72 h after the blood meal |
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: This is the correct core molecular function for PGRP-LB. Unlike the non-catalytic PGRP-S1, PGRP-LB retains the conserved zinc-coordinating residues required for N-acetylmuramoyl-L-alanine amidase activity. This enzymatic activity cleaves the amide bond between N-acetylmuramic acid and L-alanine in peptidoglycan stem peptides, thereby degrading immunostimulatory peptidoglycan fragments. The UniProt entry explicitly states PGRP-LB "Belongs to the N-acetylmuramoyl-L-alanine amidase 2 family" and annotates the Amidase_2 domain (Pfam PF01510). Reason: Core molecular function annotation. PGRP-LB contains conserved PGRP and amidase domain residues characteristic of catalytic PGRPs. Multiple sources confirm that amidase-type PGRPs like PGRP-LB enzymatically degrade PGN, reducing IMD/REL2 activation. This contrasts with non-catalytic PGRPs like PGRP-S1 which lack these catalytic residues. Supporting Evidence: file:ANOGA/PGRPLB/PGRPLB-deep-research-falcon.md Amidase PGRPs cleave the amide bond between N-acetylmuramic acid and L-alanine in the PGN stem peptide (N-acetylmuramoyl-L-alanine amidase activity) file:ANOGA/PGRPLB/PGRPLB-deep-research-falcon.md Evidence indicates conserved PGRP domain and amidase residues characteristic of N-acetylmuramoyl-L-alanine amidases in insect PGRP-LB proteins UniProt:F5HMW5 Belongs to the N-acetylmuramoyl-L-alanine amidase 2 family |
| GO:0016019 peptidoglycan immune receptor activity | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PGRP-LB is a peptidoglycan amidase rather than the mosquito peptidoglycan signaling receptor. Reason: The focused OpenScientist report resolved the receptor-vs-substrate-binding ambiguity. F5HMW5 retains a catalytic PGRP-LB active site and is predicted to cleave peptidoglycan, while the Anopheles Imd receptor role belongs to PGRP-LC. GO:0016019 requires peptidoglycan binding coupled to signal transduction that initiates innate immunity; the evidence for PGRP-LB instead supports enzymatic ligand depletion and negative regulation. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN002475783 · PTN002475783 SUPPORTS SOURCE BUT NOT TARGET Current ancestral PGRP placement appears to mix recognition PGRPs with catalytic PGRP-LB amidases. Target-specific evidence supports peptidoglycan catabolism and negative immune regulation, not signal-initiating receptor activity. Supporting Evidence: PMID:28494453 the peptidoglycan receptor PGRPLC activates the immune-deficiency (Imd) pathway limiting both the microbiota load and Plasmodium infection. file:ANOGA/PGRPLB/PGRPLB-hypotheses/immune-receptor-defense-and-secretion/openscientist.md PGRP-LB binds peptidoglycan as a **substrate for catalysis**, not for signal transduction. |
| GO:0050830 defense response to Gram-positive bacterium | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Retain the inherited Gram-positive defense context as non-core pending the focused mechanistic assessment. Reason: PTN002475783 places defense response to Gram-positive bacterium in the ancestral PGRP clade. The previous replacement with Gram-negative defense treated DAP-type peptidoglycan as exclusive to Gram-negative bacteria; some Gram-positive bacteria also contain this chemotype. No target-specific loss of the inherited defense role was established. Anopheles coluzzii PGRPLB directly modulates systemic immunity, although that study does not isolate a Gram-positive challenge. Retain the IBA as an inference, without claiming a mosquito-specific Gram-positive experiment. Supporting Evidence: PMID:28494453 PGRPLB silencing did not affect CEC1 expression in the gut but significantly induced CEC1 in the fat body 72 h after the blood meal |
| GO:0002376 immune system process | IEA GO_REF:0000043 | ACCEPT | Summary: Immune regulation is central to PGRP-LB function and supports this broad immune-process annotation. Reason: An amidase that removes immune-stimulating peptidoglycan performs a regulatory step in immunity; it need not activate an immune response to participate. The PTHR11022 ancestral immune-response placement at PTN002475783 is consistent with direct PGRPLB knockdown in Anopheles coluzzii, a member of the Anopheles gambiae complex formerly designated its M form. The primary experiment supports restraint of systemic responses, rather than the previous claim of reduced local midgut CEC1 expression. Supporting Evidence: PMID:28494453 PGRPLB silencing did not affect CEC1 expression in the gut but significantly induced CEC1 in the fat body 72 h after the blood meal |
| GO:0008270 zinc ion binding | IEA GO_REF:0000002 | ACCEPT | Summary: This annotation is appropriate for PGRP-LB. Unlike the non-catalytic PGRP-S1, PGRP-LB retains the conserved zinc-coordinating residues required for amidase catalytic activity. The zinc ion is essential for the N-acetylmuramoyl-L-alanine amidase reaction mechanism. The deep research confirms PGRP-LB has "conserved PGRP domain and amidase residues characteristic of N-acetylmuramoyl-L-alanine amidases." Reason: Correct annotation. PGRP-LB is a catalytic amidase that requires zinc for its enzymatic activity. The InterPro-based annotation correctly identifies PGRP-LB as a zinc-dependent enzyme, in contrast to non-catalytic PGRPs like PGRP-S1 which have lost the zinc-binding residues. Supporting Evidence: file:ANOGA/PGRPLB/PGRPLB-deep-research-falcon.md conserved PGRP domain and amidase residues characteristic of N-acetylmuramoyl-L-alanine amidases in insect PGRP-LB proteins UniProt:F5HMW5 Belongs to the N-acetylmuramoyl-L-alanine amidase 2 family |
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IEA GO_REF:0000002 | ACCEPT | Summary: Duplicate annotation with the IBA annotation above. Both annotations are correct for PGRP-LB, which is a catalytic amidase PGRP. The InterPro domains (IPR002502, IPR006619, IPR036505) correctly identify this as an amidase-type PGRP with retained catalytic residues. Reason: Correct annotation, duplicates the IBA annotation. PGRP-LB retains the catalytic residues required for amidase activity, unlike non-catalytic PGRPs like PGRP-S1. The IEA annotation from InterPro is well-supported by domain analysis. Supporting Evidence: file:ANOGA/PGRPLB/PGRPLB-deep-research-falcon.md AgPGRP-LB is an amidase-type PGRP predicted to catalyze N-acetylmuramoyl-L-alanine hydrolysis within bacterial PGN |
| GO:0009253 peptidoglycan catabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: This annotation is correct for PGRP-LB. As a catalytic amidase that cleaves peptidoglycan, PGRP-LB directly participates in peptidoglycan catabolism. This contrasts with non-catalytic PGRPs like PGRP-S1 which bind but do not degrade peptidoglycan. Reason: Correct annotation. PGRP-LB enzymatically degrades peptidoglycan through its N-acetylmuramoyl-L-alanine amidase activity. This is a core biological process annotation that accurately reflects PGRP-LB's catalytic function. Supporting Evidence: file:ANOGA/PGRPLB/PGRPLB-deep-research-falcon.md amidase activity: enzymatic cleavage of peptidoglycan (N-acetylmuramoyl-L-alanine amidase) that reduces peptidoglycan ligands for IMD/REL2 activation file:ANOGA/PGRPLB/PGRPLB-deep-research-falcon.md Amidase PGRPs cleave the amide bond between N-acetylmuramic acid and L-alanine in the PGN stem peptide (N-acetylmuramoyl-L-alanine amidase activity), thereby reducing immunostimulatory PGN ligands |
| GO:0042834 peptidoglycan binding | IEA GO_REF:0000002 | ACCEPT | Summary: This annotation is correct. PGRP-LB contains a PGRP domain that enables binding to peptidoglycan, which is a prerequisite for its amidase enzymatic activity. The protein must bind its substrate (peptidoglycan) before catalyzing hydrolysis. Reason: Correct annotation. PGRP-LB retains the peptidoglycan binding function through its PGRP domain. Substrate binding is essential for its catalytic amidase activity. The IEA annotation from InterPro is appropriate. Supporting Evidence: file:ANOGA/PGRPLB/PGRPLB-deep-research-falcon.md PGRP-LB is an amidase-type PGRP that recognizes bacterial peptidoglycan (PGN) and enzymatically degrades it |
| GO:0045087 innate immune response | IEA GO_REF:0000120 | ACCEPT | Summary: This annotation is accurate. PGRP-LB is a component of the insect innate immune system, specifically functioning to modulate (negatively regulate) the IMD/REL2 pathway. It acts by degrading peptidoglycan to prevent immune overactivation. Reason: Appropriate annotation for PGRP-LB's role in innate immunity. PGRPs are key components of the insect innate immune system. PGRP-LB specifically functions as a negative regulator by degrading peptidoglycan to maintain immune homeostasis. Supporting Evidence: file:ANOGA/PGRPLB/PGRPLB-deep-research-falcon.md PGRP-LB function is situated within gut/midgut immunity and microbiota homeostasis; Anopheles midgut studies integrating PGRP family functions link PGRP-LB to IMD/REL2 regulation |
| GO:0061060 negative regulation of peptidoglycan recognition protein signaling pathway | ISS PMID:28494453 The Peptidoglycan Recognition Proteins PGRPLA and PGRPLB Reg... | NEW | Summary: PGRP-LB suppresses peptidoglycan-triggered immune signaling through its conserved amidase role. Reason: Retain the pre-existing proposal for negative regulation of peptidoglycan recognition protein signaling. The PGRPLB ortholog in Anopheles coluzzii directly restrains systemic CEC1 induction after blood feeding. The protein itself is inferred to perform the ligand-cleavage step, satisfying participation rather than merely serving as a substrate. The direct mosquito result and conserved amidase mechanism support the existing proposal; the old reviewer-authored IBA metadata has been corrected to ISS with the close-ortholog primary reference, rather than claiming that this proposed term was found on PTN002475783. Supporting Evidence: PMID:28494453 PGRPLB silencing did not affect CEC1 expression in the gut but significantly induced CEC1 in the fat body 72 h after the blood meal |
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Download this section (compressed HTML)Q: What is the precise substrate specificity of Anopheles PGRP-LB? Does it preferentially degrade DAP-type peptidoglycan (Gram-negative) over Lys-type (Gram-positive)?
Suggested experts: George K. Christophides, Elena A. Levashina
Q: Does PGRP-LB localize to the apical membrane of midgut epithelial cells where it would encounter microbial peptidoglycan, or does it have a different topology?
Suggested experts: George K. Christophides
Q: How does PGRP-LB coordinate with PGRP-LC and other pattern recognition receptors to tune the magnitude of IMD pathway activation?
Suggested experts: Elena A. Levashina
Experiment: RNAi knockdown of PGRP-LB in Anopheles gambiae followed by blood meal or bacterial challenge. Measure IMD pathway activation via expression of IMD-regulated antimicrobial peptides (e.g., cecropin, attacin, defensin) compared to controls. Expect increased AMP expression in PGRP-LB knockdown.
Hypothesis: PGRP-LB knockdown leads to IMD pathway hyperactivation and elevated AMP expression
Type: RNAi knockdown with immune challenge
Experiment: Express and purify recombinant PGRP-LB. Perform in vitro amidase assays using purified DAP-type and Lys-type peptidoglycan substrates. Measure peptidoglycan degradation products by HPLC or mass spectrometry. Compare activity to known amidase PGRPs (positive control) and catalytically inactive mutants.
Hypothesis: PGRP-LB has catalytic amidase activity that degrades peptidoglycan
Type: Biochemical enzyme assay
Experiment: Knockdown PGRP-LB and assess gut microbiota composition and abundance using 16S rRNA sequencing. Expect altered microbiota composition and potentially reduced bacterial load due to uncontrolled immune activation.
Hypothesis: PGRP-LB maintains gut microbiota homeostasis
Type: RNAi knockdown with microbiome analysis
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