Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis
Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis
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DOI：
10.1128/jb.178.9.2498-2506.1996
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发表时间：
1996-05-01
影响因子：
3.2
通讯作者：
Belisle, JT
中科院分区：
文献类型：
--
作者：
Dobos, KM;Khoo, KH;Belisle, JT
最近在结核分枝杆菌（Mycobacterium tuberculosis）的45-kDa MPT 32分泌蛋白的背景下提供了分枝杆菌蛋白质的真正糖基化的化学证据（K.多博斯湾Swiderek，K. H. Khoo，P.J. Brennan和J.T.贝丽尔感染Immun. 63：2846-2853，1995），然而，糖基化的完整程度和性质以及糖基化氨基酸的位置仍然不确定。首先，为了检查共价连接的糖的性质，从用D-[U-C-14]葡萄糖代谢标记的细胞中获得45-kDa蛋白质，并进行组成分析，其显示甘露糖是唯一的共价结合糖。用内切蛋白酶枯草杆菌蛋白酶消化蛋白质，并基于证明中性损失己糖（m/z 162）或戊糖（m/z 132）的片段通过液相色谱-电喷雾-质谱法分析产物，在总共50种肽中揭示了5种糖肽，S-7、S-18、S-22、S-29和S-41，所有这些仅产生m/z 162碎片离子缺失。快速原子轰击质谱，N-末端氨基酸测序，和半乳糖苷酶消化证明了通用的O糖基化的Thr残基与一个单一的α-D-Man，甘露二糖，或甘露三糖单位。甘露二糖和甘露三糖内的键均为α 1-2，如通过β-消除释放的寡糖的气相色谱质谱所证明，许多糖基化和非糖基化肽的总序列与关于整个45-kDa蛋白质的推导氨基酸序列的公开信息相结合，证明糖基化位点位于Pro-1中。在多肽骨架的N末端和C末端附近的丰富结构域。具体地，位置10和18处的Thr残基被α-D-Manp（1->2）-α-D-Manp取代，位置27处的Thr残基被单个α-n-Manp取代，并且Thr-277被α-D-Manp、α-D-Manp（1->2）-α-D-Manp或α-D-Manp（1-> 2）-α-D-Manp（1-> 2）-α-D-Manp取代。本报告进一步证实了真正的原核糖蛋白的存在，定义了分枝杆菌甘露糖蛋白的完整结构和第一个甘露糖基化分枝杆菌蛋白的完整结构，并为其他分枝杆菌糖蛋白的研究建立了原则。
Chemical evidence for the true glycosylation of mycobacterial proteins was recently provided in the context of the 45-kDa MPT 32 secreted protein of Mycobacterium tuberculosis (K. Dobos, K. Swiderek, K.-H. Khoo, P.J. Brennan, and J.T. Belisle, Infect. Immun. 63:2846-2853, 1995), However, the full extent and nature of glycosylation as well as the location of glycosylated amino acids remained undefined. First, to examine the nature of the covalently attached sugars, the 45-kDa protein was obtained from cells metabolically labeled with D-[U-C-14]glucose and subjected to compositional analysis, which revealed mannose as the only covalently bound sugar. Digestion of the protein with the endoproteinase subtilisin and analysis of products by liquid chromatography-electrospray-mass spectrometry on the basis of fragments demonstrating neutral losses of hexose (m/z 162) or pentose (m/z 132) revealed five glycopeptides, S-7, S-18, S-22, S-29, and S-41, among a total of 50 peptides, all of which produced only m/z 162 fragmentation ion deletions. Fast atom bombardment-mass spectrometry, N-terminal amino acid sequencing, and cy mannosidase digestion demonstrated universal O glycosylation of Thr residues with a single alpha-D-Man, mannobiose, or mannotriose unit. Linkages within the mannobiose and mannotriose were all alpha 1-2, as proven by gas chromatography mass spectrometry of oligosaccharides released by beta-elimination, Total sequences of many of the glycosylated and nonglycosylated peptides combined with published information on the deduced amino acid sequence of the entire 45-kDa protein demonstrated that the sites of glycosylation were located in Pro-rich domains near the N terminus and C terminus of the polypeptide backbone. Specifically, the Thr residues at positions 10 and 18 were substituted with alpha-D-Manp(1-->2)-alpha-D-Manp, the Thr residue at position 27 was substituted with a single alpha-n-Manp, and Thr-277 was substituted with either alpha-D-Manp, alpha-D-Manp(1-->2)-alpha-D-Manp, or alpha-D-Manp(1-->2)-alpha-D-Manp (1-->2)-alpha-D-Manp. This report further corroborates the existence of true prokaryotic glycoproteins, defines the complete structure of a mycobacterial mannoprotein and the first complete structure of a mannosylated mycobacterial protein, and establishes the principles for the study of other mycobacterial glycoproteins.