MOLECULAR CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF AN AMIDE TYPE PEPTIDOGLYCAN RECOGNITION PROTEIN FROM MYTILUS CORUSCUS
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Abstract
As a pattern recognition receptor, peptidoglycan recognition protein (PGRP) is a key immune molecule in the innate immune response of mussels. While extensively studied in Drosophila and other insects, its role in marine invertebrates, particularly mussels, remains underexplored. Understanding peptidoglycan recognition proteins in mussels can help to understand the immune sensing and recognition mechanisms, providing new scientific basis for the healthy culture of these organisms. In this study, a novel peptidoglycan recognition protein which named MCAT-PGRP was identified in the gill transcriptome of Mytilus coruscus. In order to elucidate the role of peptidoglycan recognition proteins in mussel immunity, the expression level of the MCAT-PGRP gene was analyzed under different microbial challenges (Gram-positive, Gram-negative, and fungal) using quantitative PCR. In addition, prokaryotic recombinant expression technique were used to obtain rMCAT-PGRP, which was analyzed for antimicrobial activity, agglutination activity, and amidase activity, respectively. The MCAT-PGRP gene has an open reading frame of 1317 bp, encoding a protein of 438 amino acid residues without a signal peptide. This protein contains a typical PGRP domain and an Ami_2 amidase domain. Tertiary structure and molecular docking predictions showed that the PGRP domain of MCAT-PGRP consists of three externally located α-helices and multiple internally located β-folded fragments, with the ability to bind and recognize both DAP-type peptidoglycans and Lys-type peptidoglycans. Furthermore, three conserved zinc-binding amidase catalytic sites (His30, Tyr65, and His140) and four conserved PGN-binding sites (Arg48, Arg79, His140, and Thr145) were identified in the MCAT-PGRP sequence. The results of the phylogenetic tree indicated that MCAT-PGRP was most closely related to the PGRP of the thick-shelled mussel and had a high homology with the PGRP of the scallop. The expression analysis from qPCR showed constitutive expression of MCAT-PGRP in tested organs and hemocytes, with the highest expression in the kidney, followed by the gonads, digestive glands, mantle, and gills. After microbial challenges, MCAT-PGRP expression in gills was significantly upregulated, with the fastest response to Gram-positive bacteria and the slowest to Gram-negative bacteria. The peak expression levels were observed at 4h post-Candida albicans challenge, at 2h and 48h post- Staphylococcus aureus challenge, and at 8h post-Vibrio parahaemolyticus challenge. These findings suggest that MCAT-PGRP has distinct immune recognition and signaling mechanisms for different microorganisms. SDS-PAGE analysis showed that rMCAT-PGRP has a molecular weight of 51 kDa and exhibits Zn2+-dependent antimicrobial, agglutination, and amidase activities, capable of hydrolyzing insoluble peptidoglycan. Subcellular localization analysis revealed that MCAT-PGRP localize to the nuclei of HEK 293T cell, indicating MCAT-PGRP is a nuclear PGRP. The above results reveal the important role of MCAT-PGRP in the innate immunity of Mytilus coruscus and its potential mechanisms. The strong bacteria recognition and inhibition by rMCAT-PGRP also provide new ideas for the development of PGRP-based bio-antibiotics and disease control strategies in aquaculture.
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