Gibson Kevin J C, Gilleron Martine, Constant Patricia, Brando Thérèse, Puzo Germain, Besra Gurdyal S, Nigou Jérôme
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
J Biol Chem. 2004 May 28;279(22):22973-82. doi: 10.1074/jbc.M310906200. Epub 2004 Mar 18.
The genus Tsukamurella is a member of the phylogenetic group nocardioform actinomycetes and is closely related to the genus Mycobacterium. The mycobacterial cell envelope contains lipoglycans, and of particular interest is lipoarabinomannan, one of the most potent mycobacterial immunomodulatory molecules. We have investigated the presence of lipoglycans in Tsukamurella paurometabola and report here the isolation and structural characterization of a new lipoarabinomannan variant, designated TpaLAM. Matrix-assisted laser desorption ionization-mass spectrometric analysis revealed that TpaLAM had an average molecular mass of 12.5 kDa and consequently was slightly smaller than Mycobacterium tuberculosis lipoarabinomannan. Using a range of chemical degradations, NMR experiments, capillary electrophoresis, and mass spectrometry analyses, TpaLAM revealed an original carbohydrate structure. Indeed, TpaLAM contained a mannosylphosphatidyl-myo-inositol (MPI) anchor glycosylated by a linear (alpha1-->6)-Manp mannan domain, which is further substituted by an (alpha1-->5)-Araf chain. Half of the Araf units are further substituted at the O-2 position by a Manp-(alpha1-->2)-Manp-(alpha1--> dimannoside motif. Altogether, TpaLAM appears to be the most elaborated non-mycobacterial LAM molecule identified to date. TpaLAM was found to induce the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha when tested with either human or murine monocyte/macrophage cell lines. This induction was completely abrogated in the presence of an anti-toll-like receptor-2 (TLR-2) antibody, suggesting that TLR-2 participates in the mediation of TNF-alpha production in response to TpaLAM. Moreover, we established that the lipomannan core of TpaLAM is the primary moiety responsible for the observed TNF-alpha-inducing activity. This conclusively demonstrates that a linear (alpha1-->6)-Manp chain, linked to the MPI anchor, is sufficient in providing pro-inflammatory activity.
冢村菌属是系统发育类诺卡氏放线菌的成员，与分枝杆菌属密切相关。分枝杆菌的细胞包膜含有脂多糖，特别值得关注的是脂阿拉伯甘露聚糖，它是最有效的分枝杆菌免疫调节分子之一。我们研究了迟缓冢村菌中脂多糖的存在情况，并在此报告了一种新的脂阿拉伯甘露聚糖变体（命名为TpaLAM）的分离和结构表征。基质辅助激光解吸电离质谱分析表明，TpaLAM的平均分子量为12.5 kDa，因此略小于结核分枝杆菌脂阿拉伯甘露聚糖。通过一系列化学降解、核磁共振实验、毛细管电泳和质谱分析，TpaLAM显示出一种独特的碳水化合物结构。实际上，TpaLAM包含一个由线性（α1→6）-甘露糖基（Manp）甘露聚糖结构域糖基化的甘露糖基磷脂酰-肌醇（MPI）锚定基团，该结构域进一步被一个（α1→5）-阿拉伯糖基（Araf）链取代。一半的Araf单元在O-2位置进一步被一个甘露糖基-（α1→2）-甘露糖基-（α1→二甘露糖苷基序取代。总体而言，TpaLAM似乎是迄今为止鉴定出的最复杂的非分枝杆菌LAM分子。在用人类或小鼠单核细胞/巨噬细胞系进行测试时，发现TpaLAM可诱导促炎细胞因子肿瘤坏死因子（TNF）-α。在存在抗Toll样受体2（TLR-2）抗体的情况下，这种诱导作用完全被消除，表明TLR-2参与了对TpaLAM产生反应时TNF-α的产生介导。此外，我们确定TpaLAM的脂甘露聚糖核心是观察到的TNF-α诱导活性的主要部分。这最终证明，与MPI锚定基团相连的线性（α1→6）-Manp链足以提供促炎活性。
