Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
PLoS One. 2013 Jul 26;8(7):e69158. doi: 10.1371/journal.pone.0069158. Print 2013.
Nitric oxide synthase (NOS) is responsible for synthesizing nitric oxide (NO) from L-arginine, and involved in multiple physiological functions. However, its immunological role in mollusc was seldom reported.
In the present study, an NOS (CfNOS) gene was identified from the scallop Chlamys farreri encoding a polypeptide of 1486 amino acids. Its amino acid sequence shared 50.054.7, 40.747.0 and 42.5~44.5% similarities with vertebrate neuronal (n), endothelial (e) and inducible (i) NOSs, respectively. CfNOS contained PDZ, oxygenase and reductase domains, which resembled those in nNOS. The CfNOS mRNA transcripts expressed in all embryos and larvae after the 2-cell embryo stage, and were detectable in all tested tissues with the highest level in the gonad, and with the immune tissues hepatopancreas and haemocytes included. Moreover, the immunoreactive area of CfNOS distributed over the haemocyte cytoplasm and cell membrane. After LPS, β-glucan and PGN stimulation, the expression level of CfNOS mRNA in haemocytes increased significantly at 3 h (4.0-, 4.8- and 2.7-fold, respectively, P < 0.01), and reached the peak at 12 h (15.3- and 27.6-fold for LPS and β-glucan respectively, P < 0.01) and 24 h (17.3-fold for PGN, P < 0.01). In addition, TNF-α also induced the expression of CfNOS, which started to increase at 1 h (5.2-fold, P < 0.05) and peaked at 6 h (19.9-fold, P < 0.01). The catalytic activity of the native CfNOS protein was 30.3 ± 0.3 U mgprot(-1), and it decreased significantly after the addition of the selective inhibitors of nNOS and iNOS (26.9 ± 0.4 and 29.3 ± 0.1 U mgprot(-1), respectively, P < 0.01).
These results suggested that CfNOS, with identical structure with nNOS and similar enzymatic characteristics to nNOS and iNOS, played the immunological role of iNOS to be involved in the scallop immune defense against PAMPs and TNF-α.
一氧化氮合酶（NOS）负责从 L-精氨酸合成一氧化氮（NO），并参与多种生理功能。然而，其在软体动物中的免疫作用很少有报道。
本研究从扇贝 Chlamys farreri 中鉴定出一种 NOS（CfNOS）基因，该基因编码一个由 1486 个氨基酸组成的多肽。其氨基酸序列与脊椎动物神经元（n）、内皮（e）和诱导型（i）NOS 分别具有 50.054.7、40.747.0 和 42.5~44.5%的相似性。CfNOS 含有 PDZ、加氧酶和还原酶结构域，与 nNOS 中的结构域相似。CfNOS mRNA 转录物在 2 细胞胚胎后所有胚胎和幼虫中表达，并在所有测试组织中均可检测到，其中在性腺中表达水平最高，包括免疫组织肝胰腺和血细胞。此外，CfNOS 的免疫反应区域分布在血细胞的细胞质和细胞膜上。在 LPS、β-葡聚糖和 PGN 刺激后，血细胞中 CfNOS mRNA 的表达水平在 3 h 时显著增加（分别为 4.0-、4.8-和 2.7 倍，P<0.01），并在 12 h 时达到峰值（LPS 和 β-葡聚糖分别为 15.3-和 27.6 倍，P<0.01）和 24 h（PGN 为 17.3 倍，P<0.01）。此外，TNF-α也诱导 CfNOS 的表达，其在 1 h 时开始增加（5.2 倍，P<0.05），并在 6 h 时达到峰值（19.9 倍，P<0.01）。天然 CfNOS 蛋白的催化活性为 30.3±0.3 U mgprot(-1)，加入 nNOS 和 iNOS 的选择性抑制剂后显著降低（分别为 26.9±0.4 和 29.3±0.1 U mgprot(-1)，P<0.01）。
这些结果表明，CfNOS 与 nNOS 具有相同的结构，与 nNOS 和 iNOS 具有相似的酶学特征，发挥 iNOS 的免疫作用，参与扇贝对 PAMPs 和 TNF-α 的免疫防御。
