Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum
Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum
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DOI：
10.1046/j.1365-2958.2003.03822.x
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发表时间：
2004-01-01
影响因子：
3.6
通讯作者：
Vial, H
中科院分区：
文献类型：
--
作者：
Baunaure, F;Eldin, P;Vial, H
在寻找疟原虫磷脂代谢的关键酶，我们证明了寄生虫编码的磷脂酰丝氨酸脱羧酶（PSD）的存在下，恶性疟原虫感染的红细胞的膜部分。从滋养体红内期cDNA文库中筛选出编码磷脂酰丝氨酸脱羧酶（PfPSD）的cDNA。PfPSD基因位于恶性疟原虫第9号染色体上，含有一个938个核苷酸的内含子，转录成一个3.7kb的mRNA。PfPSD cDNA编码362个氨基酸的推定蛋白质，预测分子量为42.6 kDa，显然属于I型PSD家族。只有一个35 kDa的多肽中检测到的寄生虫使用特定的兔抗血清。PfPSD在其羧基末端附近具有314 VGSS 317序列，该序列与大肠杆菌、酵母和人LGST基序相关，该基序是酶原加工的位点。PSD酶在E.在K-M为63+/-19 μ M和V-MAX为680+/-49 nmol的磷脂酰乙醇胺的大肠杆菌中形成h（-1）mg（-1）蛋白。VGSS活性位点的定点突变表明PfPSD酶原被加工成两个不同的亚基（α和β），并揭示了每个残基在酶加工和活性中所起的关键作用。使用间接免疫荧光，PfPSD标记共定位与内质网标记，但不与线粒体活体染料。恶性疟原虫PSD是第一个在内质网室中鉴定的I型PSD。
In search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite-encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum-infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase (PfPSD), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum, contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl-terminus that is related to the Escherichia coli, yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a K-M of 63+/-19 muM and a V-MAX of 680+/-49 nmol of phosphatidylethanolamine formed h(-1) mg(-1) protein. Site-directed mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non-identical subunits (alpha and beta) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co-localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.