Isolation, purification and characterization of lipoglycans from mycobacteria

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.

Key words:Mycobacterium, Lipoarabinomannan, Lipomannan, MALDI-TOF/TOF-MS