分枝杆菌脂聚糖的分离、纯化及其结构和功能分析

Isolation, purification and characterization of lipoglycans from mycobacteria

摘要:

【目的】建立分离、纯化分枝杆菌脂聚糖的方法，初步比较分析不同菌株来源的脂阿拉伯甘露聚糖(Lipoarabinomannan，LAM)和脂甘露聚糖(Lipomannan，LM)的结构差异及研究脂聚糖刺激对巨噬细胞环氧合酶-2 (Cyclooxygenase-2，COX-2)蛋白表达的影响。【方法】应用Triton X-114液相法提取脂聚糖，电洗脱法分离纯化，基质辅助激光解析电离串联飞行时间质谱(MALDI-TOF/TOF-MS)进行分子量鉴定；基于特异性识别非还原性末端α-D-甘露糖基的刀豆球蛋白(Concanavalin A，Con A)分析新诺分枝杆菌JDM601、结核分枝杆菌H37Rv标准株和耻垢分枝杆菌mc2155脂聚糖的结构差异；进一步用Western blot检测脂聚糖刺激的RAW 264.7巨噬细胞COX-2蛋白的表达。【结果】通过电洗脱法成功纯化出3种菌株脂聚糖；MALDI-TOF/TOF-MS鉴定发现，分子量从小到大依次为新诺分枝杆菌JDM601、耻垢分枝杆菌mc2155和结核分枝杆菌H37Rv来源的脂聚糖。Western blot显示，Con A能与结核分枝杆菌H37Rv标准株来源的LAM相互作用，而不能与新诺分枝杆菌JDM601和耻垢分枝杆菌来源的LAM相互作用；并且发现Con A与新诺分枝杆菌JDM601来源的LM有很强的反应，然而与其余两种来源的LM反应很弱。3种菌株来源的脂聚糖均能刺激RAW 264.7巨噬细胞COX-2蛋白的表达。【结论】首次成功对来源于中国临床分枝杆菌分离株的脂聚糖进行了分离纯化，初步探讨了不同菌株来源分枝杆菌脂聚糖的结构差异，并表明LAM和LM均能刺激巨噬细胞诱导COX-2蛋白的表达，为进一步研究其对宿主的毒力和免疫机制奠定了基础。

Abstract:

[Objective] Lipoglycans were purified and characterized from mycobacteria, and the structure of lipoarabinomannan (LAM), lipomannan (LM) from diverse mycobacterial strains were compared, the effects of lipoglycan trigged cyclooxygenase-2 (COX-2) protein expression in murine macrophages were studied. [Methods] Lipoglycans were extracted using the Triton X-114 phase partition, and then purified by electroelution. MALDI-TOF/TOF-MS was used to analyze the molecular weight of lipoglycans. We analyzed the structure of lipoglycans from different strains by concanavalin A (ConA), which specifically reacted with the α-D-mannosyl domain at the non-reducing end of the molecule. Western blot was used to determine the expression of COX-2 in RAW 264.7 macrophages. [Results] Lipoglycans were successfully obtained from M. sinov JDM601, M. tuberculosis H37Rv and M. smegmatis mc2155 by using electroelution. Furthermore, using MALDI-TOF/TOF-MS, we found the ranking of molecular mass from small to large are M. sinov JDM601, M. smegmatis mc2155, M. tuberculosis H37Rv, respectively. Con A interacted with LAM from M. tuberculosis H37Rv but not with those of M. sinov JDM601 and M. smegmatis mc2155; LM from M. sinov JDM601 reacted strongly with Con A compared to LM from the other strains. In addition, all of the lipoglycans enhanced COX-2 expression in RAW 264.7 macrophages. [Conclusion] This is the first study to purify the lipoglycans from Chinese clinical mycobacterial isolate, and the result showed that the lipoglycans have different structures in comparison with the standard stains and indicated that LAM and LM can enhance COX-2 expression in macrophages. The study also laid the foundation for further researches on their virulence and immune mechanism on hosts.