The three-dimensional structure and recognition mechanism of Manduca sexta peptidoglycan recognition protein-1
Graphical abstract
Introduction
Recognition of microbe-associated molecular patterns is critically important for a successful innate immune response against pathogen invasion. Pattern recognition receptors have evolved in insects to specifically bind peptidoglycans, β-1,3-glucans and other surface components of bacteria and fungi (Jiang et al., 2010; Kurata, 2014). Clustering of pattern recognition receptors on the microbial surface triggers a serine protease system that activates cytokines (e.g. Spätzle) and phenoloxidase (PO) to induce antimicrobial peptide (AMP) synthesis, stimulate cellular responses, and kill the infectious agents (Kanost and Jiang, 2015; Lemaitre and Hoffmann, 2007; Strand, 2008).
Peptidoglycans (PGs) are unique and essential components of walled bacteria, with repetitive structures eliciting innate immune responses of vertebrate and invertebrate hosts (Guan and Mariuzza, 2007). Their glycan strands of alternating β-1,4-linked N-acetylglucosamine and N-acetylmuramate (NAM) are attached with peptide stems of 3–5 amino acids via the lactyl group on some NAM residues. The adjacent stems are cross-linked either directly or through a short peptide bridge to form a mesh-like PG layer (Vollmer et al., 2008). The polysaccharide chain is conserved in all bacteria, but the stem peptides vary in amino acid composition as well as degree of cross-linking. Gram-negative bacteria and Gram-positive bacteria in the genera of Bacillus and Clostridium contain meso-diaminopimelate (DAP) as the third residue of the stems, whereas other Gram-positive bacteria have a Lys at the same position (Vollmer et al., 2008).
Peptidoglycan recognition proteins (PGRPs) bind to PGs and regulate antimicrobial responses ranging broadly from arthropods to mammals (Dziarski, 2004; Dziarski and Gupta, 2006). All PGRP family members adopt a conserved protein architecture similar to that from bacteriophage T7 lysozyme, containing an N-terminal segment with varying lengths and a C-terminal PG-binding domain of about 165 amino acid residues. Similar to T7 lysozyme (Cheng et al., 1994), some PGRPs possess a Zn-dependent amidase activity by hydrolyzing the bond between lactyl and Ala at position-1 of the stems on PGs and generate non-immunogenic fragments. These PGRPs are hence classified as catalytic PGRPs (Gelius et al., 2003; Kim et al., 2003; Mellroth et al., 2003; Wang et al., 2003). However, most PGRPs lack the amidase activity and only act as receptors for ligand-dependent signaling (Werner et al., 2000). Some of the Drosophila and human PGRP structures contain monomeric DAP- or Lys-PG, indicating that several residues may be involved in differential recognition of DAP- and Lys-PGs (Chang et al., 2005, 2006; Cho et al., 2007; Guan et al., 2004, 2006; Kim et al., 2003; Leone et al., 2008; Lim et al., 2006; Reiser et al., 2004).
In the tobacco hornworm Manduca sexta, 5 PGRPs are up-regulated upon microbial challenge (Zhang et al., 2015). PGRP1 acts as a sensor of the prophenoloxidase (proPO) activation system, which binds to soluble DAP-PG of Escherichia coli and insoluble PGs from various Gram-negative and certain Gram-positive bacteria, but not to soluble Lys-PG of Staphylococcus aureus (Sumathipala and Jiang, 2010). The differential recognition of DAP- and Lys-type PGs is in fact common across the PGRP family (Swaminathan et al., 2006). Our recent study showed that PGRP1 along with microbe binding protein (MBP) interacts with PGs, which lead to the autoactivation of hemolymph protease-14 precursor (proHP14) to yield active HP14 that initiates the proPO activation system in a Ca2+-dependent manner (Wang and Jiang, 2017). The proPO activation in response to specific recognition of bacterial PGs is remarkably sensitive. In fact, this phenomenon has led to the development of a commercial kit for detecting bacterial contamination of human platelet units, by using M. sexta hemolymph as the key component (Heaton et al., 2014).
To understand the mechanism of PG recognition by PGRP1, we expressed and purified M. sexta PGRP1 from Sf9 insect cells and determined its crystal structure to 2.1 Å resolution, which represents the first PGRP structure from Lepidoptera. Through structural comparison with other known PGRP structures, we have identified unique structural features of its PG-binding pocket, providing insights into the recognition mechanism of PGRPs.
Section snippets
Expression and purification of M. sexta PGRP1
As described previously (Sumathipala and Jiang, 2010), a recombinant baculovirus stock (1−2 × 108 pfu/ml) was prepared from the PGRP1 bacmid for infecting Sf9 cells at 2.4 × 106 cells/ml in 1000 ml of Sf-900™ III serum-free medium at a multiplicity of infection of 5–8. At 72 h after infection, the cell culture was centrifuged at 2500×g for 20 min, diluted with 1.0 l of 1.0 mM benzamidine, and centrifuged at 20,000×g rpm for 30 min. The supernatant was loaded onto a dextran sulfate-Sepharose
Binding properties of the recombinant M. sexta PGRP1
We chemically synthesized the monomeric Lys- and DAP-PGs to study their interactions with M. sexta PGRP1 (Fig. 1). As determined by MALDI-TOF mass spectrometry, the observed Mr's (784.7832 and 828.7411 Da) were identical to the theoretical values of Lys-MPP (C31H55N9O13Na [M+Na]: 784.8308 Da) and DAP-MPP (C32H55N9O15Na [M+Na]: 828.8398 Da). SPR was used to examine the binding affinity and kinetics of recombinant M. sexta PGRP1 with the ligands. PGRP1 was immobilized on a CM5 sensor chip whereas
Data deposition
Atomic coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) with PDB ID of 6CKH.
Acknowledgments
We gratefully acknowledge the staff of beam-line 19ID at the Advanced Photon Source for their support. This work was supported by National Institutes of Health Grants AI112662 and GM58634. Mass spectrometry analyses were performed in the DNA/Protein Resource Facility at Oklahoma State University. This article was approved for publication by the Director of the Oklahoma Agricultural Experiment Station and supported in part under projects OKL03054 and OKL03060.
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