皱纹盘鲍肽聚糖识别蛋白在免疫防御中的作用

A peptidoglycan recognition protein (PGRP) from Haliotis discus hannai: Possible roles in antibacterial properties

中文关键词: 皱纹盘鲍  肽聚糖识别蛋白  免疫防御

英文关键词: Haliotis discus hannai  Peptidoglycan recognition protein  Immune response

中文摘要:

      本研究从皱纹盘鲍(Haliotis discus hannai)中鉴定并克隆了一种肽聚糖识别蛋白(PGRP)，命名为HdPGRP。HdPGRP的cDNA全长为1467 bp，共编码354个氨基酸，其中含有1个信号肽(1~18氨基酸)、1个SH3b结构域(93~160氨基酸)、1个PGRP结构域(179~322氨基酸)和1个Ami_2结构域(191~332氨基酸)。此外，在HdPGRP序列中发现了4个保守的Zn2+结合位点(H209、Y255、H318和C330)以及5个保守的酰胺酶催化位点(H209、Y255、H318、T328和C330)。经多序列比对和系统发育树分析，表明HdPGRP属于短型PGRP家族成员。在健康鲍鱼中，hdpgrp主要在肝胰腺中表达，其次依次在血细胞、外套膜和鳃中。在鳗弧菌(Vibrio anguillarum)刺激后，血细胞中的hdpgrp表达量在72 h内呈现先上升后下降的趋势，在24 h表达量达到最高。SDS-PAGE结果显示，重组HdPGRP (rHdPGRP)的分子量为30 kDa。rHdPGRP表现为Zn2+依赖酰胺酶活性，可催化降解不溶性肽聚糖。此外，rHdPGRP对革兰氏阳性菌藤黄微球菌(Micrococcus luteus)具有显著的抑制作用，且这种抑制作用可能与其酰胺酶活性有关。本研究表明，HdPGRP在机体抵御入侵细菌等免疫防御中起重要作用。

英文摘要:

      In this study, peptide PGRP (designated HdPGRP) was identified and characterized from the abalone Haliotis discus hannai. Multiple alignments and phylogenetic analyses strongly suggested that HdPGRP is a new member of the PGRP superfamily and belongs to the short PGRP family, similar to peptides from other marine mollusks. The full length of HdPGRP is 1467 bp, encoding a polypeptide of 354 amino acids (aa) with a signal peptide (1~18 aa), an SH3b domain (93~160 aa), a typical PGRP domain (179~322 aa), and an Ami_2 domain (191~322 aa). In addition, four conserved Zn2+-binding sites (H209, Y255, H318, and C330) and five conserved amide-catalysis sites (H209, Y255, H318, T328, and C330) were found in the HdPGRP sequence. In abalone, hdpgrp exhibited different tissue expression patterns, and was strongly expressed in the hepatopancreas, moderately expressed in hemocytes, mantle, and gills, and slightly expressed in muscle. Vibrio anguillarum is one of the main pathogens of H. discus hannai; after V. anguillarum infection, expression of hdpgrp in hemocytes showed a trend of first increasing and then decreasing, reaching a maximum at 24 h. Subsequently, expression of HdPGRP decreased, and there was no significant difference compared with the control group at 72 h, demonstrating that expression of HdPGRP had returned to normal levels. SDS-PAGE results showed that recombinant HdPGRP (rHdPGRP) has a molecular mass of 30 kDa, which is in line with the value predicted for HdPGRP. PGRPs usually have amidase activity, degrading peptidoglycan by hydrolyzing the amide bond that links peptide units to muramic acid residues of glycan strands. rHdPGRP exhibited Zn2+-dependent amidase activity and catalyzed the degradation of insoluble peptidoglycan. In addition, rHdPGRP exhibited significant antibacterial activity against the gram-positive bacterium Micrococcus luteus in the logarithmic phase in the presence of Zn2+, indicating that the antibacterial activity of HdPGRP might be dependent on amidase activity. In summary, HdPGRP plays an important role in PGRP-mediated antibacterial mechanisms, especially for eliminating invading bacteria.