腸炎弧菌磷脂醯絲胺酸脫羧酶之特性分析

Abstract

腸炎弧菌(Vibrio parahaemolyticus)細胞膜組成分主要為磷脂醯乙醇胺、心磷脂與磷脂醯甘油，其中以磷脂醯乙醇胺的比例最高。磷脂醯乙醇胺的關鍵酵素為磷脂醯絲胺酸脫羧酶(Psd)，其基因編碼為VP2825，能將磷脂醯絲胺酸(PS)脫羧合成磷脂醯乙醇胺。本研究首先建構psdpET21a表現質體，表現、純化VpPsd蛋白以製備腸炎弧菌Psd多株抗體，並以LC/MSMS證實序列為VpPsd。分析不同來源Psd之胺基酸序列及保守區，VpPsd具有典型的絲胺酸蛋白酶催化三聯體: Asp-His-Ser (D-H-S)及保守的Leu-Gly-Ser-Thr (LGST)模體。VpPsd單體蛋白預測為32.77 kDa，經內源性水解後成為28.03 kDa之β-次單元及4.67 kDa之α-次單元，利用LC/MSMS證實VpPsd之內源性水解位為Ser-251及Gly-252之間。腸炎弧菌於對數期與平衡期的VpPsd蛋白表現量沒有顯著差異。細胞劃分實驗證實VpPsd之β-次單元位於細胞外，原酶及α-次單元位於細胞膜上。Native PAGE分析VpPsd蛋白質應為多聚體。由FPLC膠體過濾層析實驗證實VpPsd可能以八聚體至十二聚體之形式存在。VpPsd具有絲胺酸蛋白酶催化三聯體，但使用PMSF抑制劑對VpPsd之水解無顯著影響。利用定點突變觀察絲胺酸蛋白酶催化三聯體Asp-His-Ser、保守的Leu-Gly-Ser-Thr模體及預測的受質結合位Phe-265，特定胺基酸對VpPsd內源性水解的影響，His-143、Gly-251、Ser-252、Thr-253對VpPsd之成熟重要，Phe-265以Ala、Lys胺基酸取代會減少水解。結果指出特定胺基酸對VpPsd水解重要。

Phosphatidylethanolamine (PE), cardiolipin (CL) and phosphatidylgylcerol (PG) are three major phospholipids in Vibrio parahaemolyticus , in which PE is the most abundant phospholipids. The key enzyme of phosphatidylethanolamine is phosphatidylserine decarboxylase, whose gene code is VP2825. The Psd can catalyze the formation of PE from phosphatidylserine. In this siudy, we first construct psdpET21a expression vector to express and purify VpPsd protein. Then, produce polyclonal anti-Psd antibody, using LC/MSMS to confirm the sequence of VpPsd. Comparison of Psd sequence with other species reveals a conserved Asp-His-Ser (D-H-S) triad and a conserved Leu-Gly-Ser-Thr (LGST) motif. It is predicted that monomer of VpPsd proenzyme is 32.77 kDa. Psd undergoes an endoproteolytic cleavage and split into a 28.03 kDa β-subunit and a 4.67 kDa α-subunit. Using LC/MSMS demonstrate the endoproteolytic site, which is between Gly-251 and Ser-252 in LGST motif. The expression level of VpPsd is no significant difference between log phase and stationary phase in V. parahaemolyticus. β-subunits of VpPsd appears at extracellular fraction, α-subunits and proenzyme of VpPsd locates at membranes. VpPsd appears as multimers in native PAGE. FPLC gel filtration chromatographic analysis demonstrates that VpPsd could exist in the form from octamer to dodecamer. VpPsd contains the conserved triad of serine protease, but PMSF inhibitor is not found to significantly reduce the processing of VpPsd. Site-directed mutagenesis at the LGST motif, D-H-S triad and predicted substrate binding site, Phe-265, demonstrate VpPsd was processed into two subunits and revealed the crucial role played by His-143, Gly-251, Ser-252 and Thr-253 in protein processing. Phe-265 substituted with Ala and Lys would decrease the maturation of VpPsd. The results indicated the crucial role played by specific residues in VpPsd processing.