Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis
Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis
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DOI：
10.1007/s10719-008-9223-8
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发表时间：
2009-08-01
影响因子：
3
通讯作者：
Mandal, Chitra
中科院分区：
文献类型：
--
作者：
Ghoshal, Angana;Mukhopadhyay, Sumi;Mandal, Chitra
我们报告的发现和表征的糖基化细菌ABC型磷酸盐转运体分离的外周血单核细胞（PBMC）部分内脏利什曼病（VL）患者。通过SDS-PAGE和等电聚焦鉴定了3种与疾病相关的9-O-乙酰化唾液酸糖蛋白（9-O-AcSGPs），其分子量分别为19、56和65 kDa，并确定了它们的纯度、表观质量和等电点。蛋白质印迹分析表明，9-O-乙酰化唾液酸通过α 2 -> 6键与亚末端N-乙酰半乳糖胺连接。对于56 kDa的蛋白质，N-以及O-糖基化被证明由特定的糖苷酶处理，并发现占超过9 kDa的蛋白质质量。通过薄层色谱法、荧光HPLC法和电喷雾电离质谱法进一步确认唾液酸的存在。通过质谱和5个胰蛋白酶片段的从头测序鉴定该蛋白质为铜绿假单胞菌的周质ABC型磷酸盐转运蛋白。所分配的肽的氨基酸序列与NCBI中假单胞菌转运蛋白的条目具有83-100%的同一性。基于最近报道的人类磷酸盐结合蛋白的X射线结构，我们预测了一个3D结构模型的56 kDa的蛋白质使用同源性和线程的方法。最可能的N-和O-糖基化位点的序列基序搜索生物信息学工具，溶剂可及性计算，结构环境分析和质谱数据的组合进行了鉴定。这是第一次报道的糖基化以及唾液酸化的周质组分的ABC型磷酸转运蛋白和少数确定的细菌糖蛋白之一。
We report the discovery and characterization of a glycosylated bacterial ABC-type phosphate transporter isolated from the peripheral blood mononuclear cell (PBMC) fraction of patients with visceral leishmaniasis (VL). Three disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) of 19, 56 and 65 kDa, respectively, had been identified and their purity, apparent mass and pI established by SDS-PAGE and isoelectric focusing. Western blot analyses showed that the 9-O-acetylated sialic acid is linked via alpha 2 -> 6 linkage to a subterminal N-acetylgalactosamine. For the 56 kDa protein, N- as well as O-glycosylations were demonstrated by specific glycosidase treatment and found to account for more than 9 kDa of the protein mass. The presence of sialic acids was further confirmed through thin layer chromatography, fluorimetric HPLC and electrospray ionization-mass spectrometry. The protein was identified by mass spectrometry and de novo sequencing of five tryptic fragments as a periplasmic ABC-type phosphate transporter of Pseudomonas aeruginosa. The amino acid sequences of the assigned peptides had 83-100% identity with the NCBI entry for a Pseudomonas transporter protein. Based on the recently reported X-ray structure of a human phosphate-binding protein, we predicted a 3D structural model for the 56 kDa protein using homology and threading methods. The most probable N- and O-glycosylation sites were identified by combinations of sequence motif-searching bioinformatics tools, solvent accessibility calculations, structural environment analyses and mass spectrometric data. This is the first reported glycosylation as well as sialylation of the periplasmic component of an ABC-type phosphate transporter protein and of one of few identified bacterial glycoproteins.