Although specific immunity has evolved, innate immunity continues to play a greater role in the immunization process for fish (Saurabh & Sahoo, 2008; Yang et al., 2019). As highly conserved molecular structures common on the surface of pathogenic microorganisms, pathogen-associated molecular patterns (PAMPs) are critical targets to be recognized by the innate immune system of fish. In this process, it is the conserved PRR that exerts specific recognition of PAMPs. PRRs, including lectin, Gram-negative bacterial-binding proteins (GNBPs), scavenger receptors (SRs), and PGRPs, are highly conserved germline-encoded genes that recognize PAMPs specifically through different structures on which they can induce a rapid immune response (Lu et al., 2020; Wei et al., 2018).
As members of the PRRs, PGRPs can specifically recognize PGN, which functions as a direct target for innate immune receptors, commonly with a peptidoglycan-binding domain consisting of approximately 165 amino acids (Dziarski, 2004; C. Liu, Xu, Gupta, & Dziarski, 2001; Sharma et al., 2011; Wolf & Underhill, 2018). Concerning the size of the relative molecular mass, PGRPs can be broken into the categories of L-type (above 90 kDa, with transmembrane and intracellular domains) and S-type (usually below 25 kDa, generally small extracellular secretory proteins with signal peptides) (Royet, Gupta, & Dziarski, 2011). Their sequence shares a similarity of 30% with bacteriophage T7 lysozyme and are highly evolutionarily conserved (Guan & Mariuzza, 2007), homologous to N-acetylcytidylic acid-alanine amidase. Notably, PGRP has a Zn2+ binding site consisting of four amino acid residues, which enables it to eradicate invading pathogens greatly enhanced by the action of Zn2+ (Hou et al., 2023; Yang et al., 2019). However, studies in insects have shown that not all PGRPs have amidase activity. PGRPs with amidase activity that have IMD pathway regulation secretion, while those PGRPs that do not exhibit amidase activity act mainly through the Toll pathway (Kurata, 2010; Maillet, Bischoff, Vignal, Hoffmann, & Royet, 2008; Q. Wang et al., 2021; Zaidman-Remy et al., 2006). The situation is the same in mammals, PGLYRP1-4 have only antimicrobial activity, except for PGLYPR2 with nicotinamide and antimicrobial activity (Z. M. Wang et al., 2003). In general, PGRPs are involved in the immune process of different organisms in several forms and play an important role such as recognizing pathogenic bacteria and degrading their peptidoglycans, promoting phagocytosis, and participating in the regulation of Toll and IMD signaling pathways (Kim, Byun, & Oh, 2003; Lemaitre & Hoffmann, 2007; Leulier et al., 2003; Werner et al., 2000). Although about many PGRPs have been identified in teleost fish such as zebrafish (Danio rerio) (Chang & Nie, 2008), greater amberjack (Pseudosciaena crocea) (Mao, Wang, Zhang, Ding, & Su, 2010), rainbow trout (Oncorhynchus mykiss) (Jang, Kim, & Cho, 2017) and American redfish (Sciaenops ocellatus) (M. F. Li, Zhang, Wang, & Sun, 2012), the molecular mechanisms through which the family exerts its immune effects are still needed to be studied in more detail. Fish PGRP2, homologous to mammalian PGLYRP2, has had numerous functions successively confirmed across various species in recent years (Sun et al., 2014). PGRP2 from Ctenopharyngodon idella was characterized as having the ability to specifically bind PGN and possessing an amidase activity (J. H. Li, Chang, Xue, & Nie, 2013). PGRP2 from Oncorhynchus mykiss was demonstrated to modulate the body's inflammatory response by reducing NF-κB activity during bacterial infection (Choi et al., 2018). In Lateolabrax maculatus, Ssb-PGRP-L2 expressed bactericidal effects on Vibrio harveyi, Staphylococcus aureus, and Edwardsiella tarda (X. Li et al., 2020). However, the exact function and molecular mechanism of fish PGRP2 in the course of innate immunity remain ambiguous and require extensive research.
In the present research, we have recombinantly expressed and purified a long-type PGRP from fat greenling, a major economic fish in the Yellow Sea and Bohai Sea of China. To investigate the role of HoPGRP-L2 in the process of Hexagrammos otakii innate immunity and its molecular mechanism, we performed a PGN binding assay, pathogen binding assay, and agglutination assay of HoPGRP-L2. In addition, we also investigated the inhibitory effect of HoPGRP-L2 on the growth of the pathogenic bacterium Aeromonas hydrophila in vitro, and these data may be of interest to further understanding the role of fish PGRP-L2 in the innate immune process.