草蝦一氧化氮合成酶之選殖與定性

Abstract

一氧化氮分子在心血管疾病、神經傳導與免疫反應上都扮演不可或缺的角色，而形成的關鍵即為一氧化氮合成酶（nitric oxide synthase, NOS）。脊椎動物中NOS研究得很多，但無脊椎動物或甲殼類的NOS卻所知甚少。 以DAF-2DA螢光染色確定草蝦血球有產生一氧化氮的能力，進一步由NOS專一性抑制劑，L-NMMA，可削弱和減少螢光，顯示血球細胞內NO確為NOS催化所產生。 利用巢式反轉錄聚合酶鏈鎖反應（nested reverse transcription-polymerase chain reaction, nested RT-PCR）與5’端及3’端快速擴增法（5’- and 3’- rapid amplification of cDNA ends, 5’-RACE, 3’-RACE）自草蝦血球中選殖出一氧化氮合成酶全長序列，以PmNOS表示。PmNOS cDNA序列包含249 bp的5’端非轉譯區（untranslated region, UTR）、3582 bp 的譯碼區 （Open Reading Frame, ORF）以及166 bp的3’非轉譯區；並可轉譯出1193個胺基酸的蛋白質。推測分子量為134.7 kDa，預測的等電點為6.7。利用ExPASy的motif scan預測功能區塊，全段序列自N端起迄C端分別為oxygenase domain、calmodulin binding motif以及reductase domain。其中oxygenase domain由heme binding motif與BH4 binding motif組成；reductase domain則由FMN、FAD、NADPH 三個binding motifs組成。 以MEGA 4進行系統分類與分子演化分析，結果顯示與脊椎動物三種NOS的任一種NOS都無法形成群簇，而與無脊椎動物的NOS歸類在同一個群簇。 利用RT-PCR檢測組織分佈結果顯示PmNOS分佈於神經、眼柄、鰓、殼下表皮細胞、中腸及血球等部位。 取草蝦多種組織粗萃取液利用Griess方法對NOS蛋白測定活性，結果發現胸腹神經的NOS活性最高，其次依序為殼下表皮細胞、血球及肌肉。 利用CpG-ODN 2006於體外刺激草蝦血球與LPS注射草蝦，測試各時間點草蝦NOS是否可被誘發；RT-PCR結果顯示胸腹神經、殼下表皮細胞與血球NOS表現量都沒有顯著改變。 以昆蟲細胞-桿狀病毒表現系統合成草蝦NOS重組蛋白，經Griess方法確認重組的NOS具有生化活性。 綜合前述結果，PmNOS轉錄時為非誘發性，較類似於脊椎動物的nNOS與eNOS；又胸腹神經組織萃取液NOS酵素活性最高，暗示PmNOS可能接近脊椎動物的nNOS。然而NO分子出現於顆粒球表示PmNOS功能上較靠近於脊椎動物的iNOS。由於PmNOS從親緣關係分析無法歸為脊椎動物的任一種NOS、轉錄訊息又都表現於血球、殼下表皮與胸腹神經，因此推測PmNOS可能為原生型NOS。

Nitric oxide (NO) is an essential molecule involved in neuron transduction, cardiac disease, and immune response while nitric oxide synthase (NOS) is a critical enzyme that catalyzes it. Although numerous researches have been carried out to understand NOS, its role is still unclear in invertebrates including crustaceans. 4,5-Diaminofluorescein (DAF-2DA) staining indicated that shrimp hemocytes could produce NO. However its production can be blocked by NOS specific inhibitor, NG-monomethyl-L-arginine, monoacetate salt (L-NMMA). This reveals that the production of NO in hemocytes is catalyzed by NOS. We cloned a cDNA of nitric oxide synthase from Penaeus monodon hemocytes (PmNOS) was cloned by nested-reverse transcription-polymerase chain reaction (nested RT-PCR) and 5’- and 3’-rapid amplification of cDNA ends (5’-RACE, 3’-RACE). The full length cDNA of PmNOS includes 249 bp of 5’UTR, 3582 bp of ORF and 166 bp of 3’UTR. The putative peptide is 1193 amino acid residues in length, with an estimated molecular weight of 134.7 kDa and pI 6.7. PmNOS contains oxygenase domain, calmodulin binding motif and reductase domain from N- to C-termini. The oxygenase domain has heme and BH4 binding motifs while the reductase domain contains FMN, FAD and NADPH binding motifs. Phylogenetic analysis via MEGA4 indicated that PmNOS clusters with known invertebrate NOS, but does not cluster with iNOS, eNOS or nNOS found in vertebrates. PmNOS mRNA was expressed in many tissues of P. monodon including the thoracic and ventral ganglia, hemocytes, eyestalk, midgut, gill, and subcuticular epithelium as demonstrated using RT-PCR. The activity of NOS crude extract from different tissue was determined using Griess assay. NOS activities were demonstrated from high to low in that order from different tissues such as thoracic and ventral nerve, subcuticular epithelium, hemocytes and muscle. RT-PCR revealed that CpG-ODN 2006 did not induce NOS mRNA increase in hemocytes in vitro, neither LPS stimulated NOS mRNA increase in thoracic and ventral nerve, subcuticular epithelium and hemocytes in vivo at any time point. The recombinant PmNOS was synthesized using baculovirus-insect cell system and its activity confirmed by Griess assay. PmNOS is noninducible in transcription which is also true with nNOS or eNOS. In addition, the highest NOS activity of thoracic and ventral nerve implies that PmNOS may resemble the nNOS. However, NO production in hemocytes indicates that the function of PmNOS approaches that of iNOS. Since PmNOS has not been categorized in any kind of vertebrate NOS but the transcriptional messages were found expressed in hemocytes, subcuticular epithelium and thoracic and ventral nerve of P. monodon, therefore we suggest that PmNOS may be a prototype of NOS.