Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration - PubMed (original) (raw)

Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration

P F Mühlradt et al. J Exp Med. 1997.

Abstract

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.

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Figures

Figure 1

Figure 1

Macrophage-stimulatory activity of clones from M. fermentans. Individual colonies grown on agar were picked and then cultured in liquid medium. Mycoplasmas were harvested and MSA was extracted with hot octyl glucoside and determined in the NO release assay with IFN-γ–treated peritoneal exudate cells from LPS low-responder mice. MSA was calculated as U/mg mycoplasma protein.

Figure 2

Figure 2

Comparison of silver stain and MSA in gel slices after SDS-PAGE of M. fermentans PG 18 type strain and clone II-29/1. Samples were run in 15% gels in the discontinuous buffer system of Lämmli under reducing conditions, applying 10 μg mycoplasma protein per lane for silver staining. 70 μg were subjected to electrophoresis on a neighboring lane. This lane was cut in 3-mm segments which were extracted in 0.3 ml hot octyl glucoside for subsequent determination of MSA.

Figure 2

Figure 2

Comparison of silver stain and MSA in gel slices after SDS-PAGE of M. fermentans PG 18 type strain and clone II-29/1. Samples were run in 15% gels in the discontinuous buffer system of Lämmli under reducing conditions, applying 10 μg mycoplasma protein per lane for silver staining. 70 μg were subjected to electrophoresis on a neighboring lane. This lane was cut in 3-mm segments which were extracted in 0.3 ml hot octyl glucoside for subsequent determination of MSA.

Figure 3

Figure 3

HPLC of octyl glucoside extracted MSA from M. fermentans. MSA was extracted as in Table 1. 2.6 × 107 U were applied to a 10 × 250–mm RP8 reversed phase column and eluted with 2-propanol. (solid line) MSA as determined in the NO release assay; (dotted line) inorganic phosphate to monitor phospholipids. The bar below OG shows where octyl glucoside elutes from the column.

Figure 4

Figure 4

MALDI spectrum of HPLC-purified MALP-2. The lipopeptide gave rise to a [M + H]+ ion at m/z 2164.3 and a [M + Na]+ ion at m/z 2186.0. The signal at m/z 2867.8 is due to the [M + 2H]2+ signal of the internal calibration standard bovine insulin.

Figure 5

Figure 5

Concentration dependency of MALP-2 in the NO release assay. (A) HPLC-purified MALP-2 from M. fermentans clone II-29/1 prediluted in octyl glucoside (solid line) or PBS (dotted line). (B) HPLC-purified MALP-2 (solid line) and synthetic dipalmitoyl MALP-2 (dotted line), both prediluted in octyl glucoside. The diamond on the y-axis shows NO release of control cells with IFN-γ only. Data are the average of triplicate determinations ± standard deviation.

Figure 5

Figure 5

Concentration dependency of MALP-2 in the NO release assay. (A) HPLC-purified MALP-2 from M. fermentans clone II-29/1 prediluted in octyl glucoside (solid line) or PBS (dotted line). (B) HPLC-purified MALP-2 (solid line) and synthetic dipalmitoyl MALP-2 (dotted line), both prediluted in octyl glucoside. The diamond on the y-axis shows NO release of control cells with IFN-γ only. Data are the average of triplicate determinations ± standard deviation.

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