PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria
PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria
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DOI：
10.1074/jbc.m604214200
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发表时间：
2006-09-01
影响因子：
4.8
通讯作者：
Kinoshita, Taroh
中科院分区：
文献类型：
--
作者：
Morita, Yasu S.;Sena, Chubert B. C.;Kinoshita, Taroh
磷脂酰肌醇甘露糖（pim）是所有分枝杆菌中主要的一类糖脂。AcPIM2是一种二甘露糖基PIM，是极性PIM的终产物和前体，如己糖基PIM （AcPIM6）和主要的细胞壁脂聚糖，脂阿拉伯甘露聚糖（LAM）。将AcPIM2转化为AcPIM6或LAM的甘露糖基转移酶依赖于聚戊烯醇-磷酸甘露糖（PPM），但尚未被表征。在这里，我们发现了一个被称为pimE的基因，它存在于所有分枝杆菌中，并且是AcPIM6生物合成所必需的。PimE最初是根据真核猪- m甘露糖基转移酶的同源性鉴定的。缺失pime的耻垢分枝杆菌在AcPIM6合成上存在缺陷，积累了四聚氰胺PIM、AcPIM4。PimE的缺失对细胞生长和活力，以及其他细胞内和细胞壁聚糖的生物合成没有影响。然而，检测到细胞壁疏水性和质膜组织的变化，表明AcPIM6在细胞壁和质膜结构完整性中起作用。这些缺陷通过pimE基因的异位表达得到纠正。代谢脉冲追踪放射性标记和无细胞PIM生物合成实验表明，PimE催化α 1,2-甘露糖醇转移合成AcPIM5。PimE中一个Asp残基的突变导致PimE生物合成活性的丧失，该残基是人类PIG-M活性所必需的，表明PimE是催化成分。最后，PimE定位于AcPIM4-6生物合成富集的不同膜组分。综上所述，PimE代表了第一个参与PIM生物合成的ppm依赖性甘露糖基转移酶，其中它介导第五次甘露糖转移。
Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha 1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyltransferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.