Study On The Immune Defense Functions Of Peptidoglycan Recognition Protein In Razor Clam (Solen Grandis)

Razor clam （Solen grandis） is a member of Lamellibranchia widelydistributed in coastal areas of China. S. grandis is an important economicmariculture species with many advantages, such as large size, highcutability, delicious flesh, rich nutrition, great market prospects, andsignificant economic value. Similar to other marine invertebrates, S.grandis lacks the acquired immune system and mainly depends on theinnate immune system to defense against the invasion of micorbialpathogens.Peptidoglycan recognition protein （PGRP） is an important patternrecognition receptor in the innate immune system of invertebrates. PGRPcan specifically recognize bacterial peptidoglycan （PGN）, playing animportant role in antigen recognition and immune response. To date,studies on S. grandis PGRP regarding antigen recognition and immuneresponse have been limited, and the mechanism through which S. grandisdefenses against the invasion of external pathogens remain unclear.In this study, PGRP-mediated immune defense mechanisms in S. grandis were studied using techniques of molecular biology andimmuno-biology, aiming to further our understanding about themechanisms of antigen recognition and immune response in S. grandis.Section1: Sequence analysisTwo short S. grandis PGRPs （SgPGRPs） were identified, designatedas SgPGRP-S1and SgPGRP-S2. Their full-length cDNAs were1672bpand1285bp, encoding a polypeptide of270and141amino acids,respectively. The isoelectric points of SgPGRP-S1and SgPGRP-S2were7.82and5.46, with predicted molecular weight of28.4and14.7kDa,respectively.BLAST analysis revealed that the deduced amino acid sequences ofSgPGRP-S1and SgPGRP-S2shared51%similarity with each other,which showed high similarities with PGRPs from Biomphalaria glabrata（ABO40829,57%） and Saccoglossus kowalevskii （XP₀02734591,56%）,respectively.Multiple sequence alignments showed that both SgPGRPs containedhighly conserved Zn2+-binding sites （H135, H244, and C252inSgPGRP-S1; H115and C123in SgPGRP-S2） and amidase catalytic sites（H135, Y170, H244, T250and C252in SgPGRP-S1; H115, T121andC123in SgPGRP-S2）.A phylogenetic tree was constructed using neighbor-joining method with1000bootstrap test based on the multiple alignments of SgPGRP-S1,SgPGRP-S2and PGRPs from reference organisms （Crassostrea gigas,Chlamys farreri, Argopecten irradians, Drosophila melanogaster andHomo sapiens）. S. grandis showed high homologies to other molluscs andformed a sister group with PGPRs from mollusc C. gigas.All these resultsdemonstrate that both SgPGRP-S1and SgPGRP-S2are members of thePGRP superfamily.Section2: SgPGRP mRNA transcription detected by real-time PCRSYBR Green real-time PCR analysis was performed to detectSgPGRP mRNA transcription in various organs of S. grandis. In healthyindividuals, the mRNA transcripts of SgPGRP-S1and SgPGRP-S2wereconstitutively expressed at varying levels in a wide range of tissues,including mantle, gill, gonad, hemocyte, muscle, and hepatopancreas.SgPGRP-S1mRNA was expressed at the highest level in muscle andhepatopancreas followed by gill and mantle, and the lowest level wasdetected in gonad and hemocyte. SgPGRP-S2mRNA was highlyexpressed in gill and mantle, with the lowest expression level in muscle.Real-time PCR analysis was also performed to study SgPGRP mRNAexpression in hemocytes of S. grandis after challenged by LPS, PGN, andglucan. LPS stimulation did not induce significant fluctuations inSgPGRP-S1mRNA expression. By comparison, PGN challenge induced acute up-regulation of SgPGRP-S1mRNA expression. SgPGRP-S1transcripts were significantly increased to14.3-fold of that in the blankgroup at3h post PGN injection, with a burst of increase to7472.0-foldthat in the blank group at6h after PGN stimulation, followed by relativedecreases down to13.9-and5.7-fold that in the blank group at24and48h, respectively. After β-1,3-glucan challenge, SgPGRP-S1expression wassignificantly up-regulated to22.4-,11.9-and89.7-fold that in the blankgroup at12,24, and48h, respectively.Compared with SgPGRP-S1, SgPGRP-S2exhibited mitigatoryregulation patterns toward external challenges to PAMPs. After LPSchallenge, SgPGRP-S2mRNA expression was significantly upregulated,and peaked at6h,4.1-fold that in the blank group; as time elapsed, it wassignificantly down-regulated to0.1-and0.3-fold that of the blank level at24and48h, respectively. After PGN challenge SgPGRP-S2exhibitedsimilar mRNA expression patterns as SgPGRP-S1, with the highest leveldetected at6h post challenge, followed by relative decreases at12h andsubsequent increase to4.4-fold of that in the control group at24h. Afterβ-1,3-glucan challenge, SgPGRP-S2mRNA expression was alsosignificantly upregulated, with the highest level detected at12h（23.1-fold that of the blank group） and subsequent decrease to the originallevel gradually. Section3: activity assaies of rSgPGRP-S1PAMPs binding assay revealed that recombiant SgPGRP-S1（peptidoglycan recognition protein） exhibited high affinity to PGN and itsbinding ability varied in a concentration-dependent manner. However,rSgPGRP-S1exhibited no affinity to LPS and β-1,3-glucan, suggestingthat S. grandis could specifically recognize PGN. With a broad spectrumof bacterial binding activity, rSgPGRP-S1exhibited strong agglutinationactivity to Gram-negative Escherichia coli and Vibrio anguillarum as wellas Gram-positive Micrococcus luteus. The results preliminarily revealedthat SgPGRP-S1is involved in the immune response process in S. grandisagainst microbial pathogens, especially Gram-positive bacteria.Further, amidase activity assay showed that rSgPGRP-S1exhibitedZn2+-dependent amidase activity and catalyzed the degradation ofinsoluble PGN via the hydrolysis of peptide bond between N-acetylmuramyl acid and L-alanine. Plate diffusion method was used to detect theinhibitory activity of rSgPGRP-S1towards Gram-negative E. coli andGram-positive Staphylococcus aureus. Growth inhibition zone appeared inthe presence of Zn2+.Additionally, the optical density of bacterial culture liquid wasmeasured to detect bacterial growth for verifying the inhibitory effect ofrSgPGRP-S1. The combined protein exhibited significant antibacteral activity in logarithmic phase and bactericidal activity in stationary phase.This inhibitory effect of rSgPGRP-S1might be attributed to its amidebactericidal activity.In summary, this study investigated the function of S. grandis PGRP aspattern recognition receptor, which mediated antigen recognition andimmune response against micorbial pathogens. The results provided afundamental basis for further investigating the mechanism of immuneresponse in invertebrates such as molluscs. The work also enriched anddeveloped research theory regarding the mechanism of immune defense inmarine invertebrates.