Subcellular localization and identification of acyl-CoA: lysophosphatidylethanolamine acyltransferase (LPEAT) in the arachidonic acid-rich green microalga, Myrmecia incisa Reisigl
Subcellular localization and identification of acyl-CoA: lysophosphatidylethanolamine acyltransferase (LPEAT) in the arachidonic acid-rich green microalga, Myrmecia incisa Reisigl
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酰基辅酶 A 的亚细胞定位和鉴定：富含花生四烯酸的绿色微藻 Myrmecia incisa Reisigl 中的溶血磷脂酰乙醇胺酰基转移酶 (LPEAT)
DOI：
10.1007/s10811-021-02681-z
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发表时间：
2022-01-27
影响因子：
3.3
通讯作者：
Zhou，Zhi-Gang
中科院分区：
文献类型：
--
作者：
Liu，Yi;Bao，Hong;Zhou，Zhi-Gang
溶血磷脂酰乙醇胺酰基转移酶（LPEAT）通过Lands循环参与磷脂的酰基重塑，从而改变三酰甘油（TAG）中的脂肪酸组成。本研究从一种富含花生四烯酸（ArA）的绿色微藻桃金娘（Myrmartinincisa）中克隆了MiLPEAT基因。其cDNA全长为1,303 bp，含有一个981 bp的开放阅读框，编码326个氨基酸。将其cDNA与相应的克隆DNA序列进行比较，发现MiLPEAT含有6个内含子。生物信息学分析表明，MiLPEAT的C端存在一个磷酸酰基转移酶结构域PlsC，由NH（x）4D、GCxYbR、FPEGT和PVxPVx 4个典型基序组成，N端存在两个（至少一个）跨膜结构域。由此将对应于C-末端224-残基的cDNA独立地亚克隆到载体pET-28 a和pMAL-c2 X中，用于产生重组MiLPEAT（rMiLPEAT）。将纯化的可溶性rMiLPEAT与麦芽糖结合蛋白融合用于酶活性测定，催化产物的薄层层析图谱证明rMiLPEAT可将溶血磷脂酰乙醇胺酰化为磷脂酰乙醇胺，从而鉴定了MiLPEAT的功能。纯化的rMiLPEAT融合6×His标签，制备抗MiLPEAT多克隆抗体。免疫电镜结果表明，MiLPEAT定位于切丝分枝杆菌质膜上，免疫细胞化学结果进一步证实了这一点。在C-末端存在的二赖氨酸基序暗示MiLPEAT是内质网居民，因此讨论了MiLPEAT如何被运输到微藻质膜。本研究为进一步了解切割分枝杆菌如何利用ArA合成TAG奠定了基础。
Lysophosphatidylethanolamine acyltransferase (LPEAT) plays an important role in acyl remodeling of phospholipid via the Lands’ cycle, and consequently alters fatty acid compositions in triacylglycerol (TAG). In the present study,MiLPEATwas cloned from an arachidonic acid (ArA)-rich green microalgaMyrmecia incisa. Its full-length cDNA was 1,303 bp containing a 981-bp open reading frame that encoded a 326-amino acid protein. Comparing the cDNA to its corresponding cloned DNA sequence showed thatMiLPEATpossessed 6 introns. Bioinformatics analysis of LPEAT indicated that a phosphate acyltransferase domain, PlsC, consisting of 4 typical motifs, NH(x)4D, GCxYVxR, FPEGT, and PVxPVx, was present at the C-terminus of MiLPEAT while two (at least one) transmembrane domains at the N-terminus. The cDNA corresponding to C-terminal 224-residues was thereby subcloned into the vectors pET-28a and pMAL-c2X independently for production of recombinant MiLPEAT (rMiLPEAT). The purified soluble rMiLPEAT fused with maltose-binding protein was used for enzyme assay, and thin-layer chromatography profiles of the catalytic products demonstrated that rMiLPEAT could acylate lysophosphatidylethanolamine to phosphatidylethanolamine, thus functionally identifyingMiLPEAT. Anti-MiLPEAT polyclonal antibody was generated against the purified rMiLPEAT fused with 6×His tag. Immuno-electron microscopic results with this polyclonal antibody illustrated that MiLPEAT was localized onM.incisaplasma membrane, and this was further supported by immunocytochemical observations. A di-lysine motif present at the C-terminus implying that MiLPEAT was an endoplasmic reticulum resident, how MiLPEAT was transported to the microalgal plasma membrane was therefore discussed. This study will lay a foundation to understand that howM.incisauses ArA to synthesize TAG.