仿刺参新型分泌型肽聚糖识别蛋白AjPGRP-S1在固有免疫中的功能研究

Abstract

Peptidoglycan recognition proteins (PGRPs) comprise a family of pattern recognition receptors (PRRs) with an important role in innate immunity. Here, we amplified the full-length cDNA of a novel PGRP from the sea cucumber A. japonicus (named AjPGRP-S1) using RACE. Subsequently, we prepared a codon-optimized recombinant AjPGRP-S1 (rAjPGRP-S1) protein and raised a polyclonal antibody against rAjPGRP-S1 by immunizing rabbits. At the transcriptional level, the tissue expression pattern of AjPGRP-S1 was determined by in situ hybridization and RT-qPCR, while the gene expression of AjPGRP-S1 after microbial polysaccharide challenge was also evaluated by RT-qPCR. Amidase activity was measured using the microplate method; the pathogen recognition characteristics were determined by ELISA and indirect immunofluorescence assay; and the immune factors that interact with AjPGRP-S1 were screened and identified via pull-down assay combined with LC-MS/MS. The AjPGRP-S1 protein was predicted to contain a signal peptide , a PGRP domain, and a molecular mass of 24.6 kDa, but lacked a cysteine residue that is indispensable for the formation of a Zn 2+ -binding site and amidase activity. Indeed, no amidase activity was detected from rAjPGRP-S1. These results indicated that AjPGRP-S1 might be a secretory, short-type, and recognition-type PGRP. AjPGRP-S1 transcripts were relatively highly abundant in coelomocytes and were markedly induced by challenge with diaminopimelic acid (DAP)-type peptidoglycan (PGN), suggesting that AjPGRP-S1 is mainly expressed by coelomocytes and might play a crucial role in the defense against Gram-negative bacteria. rAjPGRP-S1 was determined to be capable of binding to DAP-type PGN, lysine (Lys)-type PGN, β-1,3-glucan, Vibrio splendidus , Pseudoalteromonas nigrifaciens , Shewanella baltica , Bacillus cereus , Escherichia coli , and Staphylococcus aureus , but not lipopolysaccharide or mannan, implying that AjPGRP-S1 may have a broad-spectr