Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans...

Macrophages are typically stimulated by components of microbial cell walls. Surprisingly, cell wall-less mycoplasmas can also very efficiently stimulate macrophages. We showed recently that mycoplasma-derived lipopeptides constitute the active principle. We have now isolated a clone of Mycoplasma fermentans expressing mainly one macrophage-stimulating lipopeptide. This lipopeptide was detergent-extracted and isolated by reversed-phase high-performance liquid chromotography, using nitric oxide release from C3H/HeJ mouse macrophages as bioassay for detection. In contrast to "conventional" bacterial lipoproteins, this lipopeptide had a free NH2 terminus. Amino acid composition, sequence, and the molecular weight of 2,163. 3 are consistent with the following structure: S-(2, 3-bisacyloxypropyl)cysteine-GNNDESNISFKEK with one mole C16:0, and a further mole of a mixture of C18:0 and C18:1 fatty acid per lipopeptide molecule. The sequence could not be found in either the protein identification resource nor the Swiss Prot data bank. We named this 2-kD lipopeptide, macrophage-activating lipopeptide-2 (MALP-2). Synthetic dipalmitoyl MALP-2 and mycoplasma-derived MALP-2 were compared with the bioassay. Both lipopeptides showed an identical dose dependency with a half-maximal response at 10(-11) M concentration. MALP-2 may be one of the most potent natural macrophage stimulators besides endotoxin.