Date Published: September 7, 2004
Publisher: Public Library of Science
Author(s): Chung-I Chang, Sébastien Pili-Floury, Mireille Hervé, Claudine Parquet, Yogarany Chelliah, Bruno Lemaitre, Dominique Mengin-Lecreulx, Johann Deisenhofer
Abstract: The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-Å resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.
Partial Text: Activation of innate immunity in response to bacterial pathogens requires a group of molecules, known as the pattern recognition receptors, that recognize conserved motifs, present in bacteria but absent in higher eukaryotes, and trigger downstream signaling events. In Drosophila, two distinct signal transduction pathways are involved in the pathogen-specific innate immune response by inducing the expression of a panel of specific antimicrobial peptides (Tzou et al. 2002; Hoffmann 2003). The Toll signaling pathway responds mainly to Gram-positive bacterial or fungal infections, which lead to the proteolytic processing of the cytokine-like polypeptide Spätzle. Binding of the cleaved Spätzle to the transmembrane receptor Toll activates an intracellular signaling cascade that results in the degradation of the IκB-like protein Cactus and the nuclear localization of the NF-κB–like proteins Dif and Dorsal, which induce the transcription of several antimicrobial peptide genes, such as Drosomycin (Lemaitre et al. 1996, 1997; Meng et al. 1999; Rutschmann et al. 2000b; Tauszig-Delamasure et al. 2002; Weber et al. 2003). By contrast, the immune deficiency (Imd) pathway mediates defense reactions against primarily Gram-negative bacteria through different signaling components and regulates the cleavage and activation of another NF-κB–related nuclear factor, Relish, which activates a different set of antimicrobial peptide genes, including Diptericin (Lemaitre et al. 1995; Hedengren et al. 1999; Leulier et al. 2000; Rutschmann et al. 2000a; Vidal et al. 2001).
To facilitate molecular characterization of PG recognition and signal transduction mediated by PGRP-SA, we overexpressed and purified recombinant PGRP-SA (rPGRP-SA) in a baculovirus-insect cell expression system. PGRP-SA is a secreted protein circulating in the hemolymph (the insect blood) of Drosophila. We tested the activity of rPGRP-SA in vivo by injecting the protein into wild-type (wt) and PGRP-SA–deficient (PGRP-SAseml) flies. For this assay we used flies carrying a Drosomycin-GFP reporter transgene, which served as the target gene of the Toll signaling pathway. The wt flies injected with water produced Drosomycin-GFP after challenge by M. luteus, whereas PGRP-SAseml flies failed to express the reporter gene after the same treatment (Figure 1A and 1B). When 112 ng of rPGRP-SA was injected into PGRP-SAseml flies, the recipient flies became capable of producing Drosomycin-GFP after challenge with M. luteus (Figure 1C). As little as 11 ng of rPGRP-SA was sufficient to rescue PGRP-SAseml flies (Figure 1D). Injection of 11 ng of rPGRP-SA in wt and PGRP-SAseml flies without any further microbial challenge could not activate Drosomycin-GFP expression (unpublished data). These results demonstrate that rPGRP-SA expressed in insect cell culture medium is active in vivo.
The atomic coordinates and structure factors have been deposited in the Protein Data Bank (http://www.rcsb.org/pdb/) under accession number 1S2J.