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

Mühlradt PF, Kiess M, Meyer H, Süssmuth R, Jung G - J. Exp. Med. (1997)

Bottom Line: The sequence could not be found in either the protein identification resource nor the Swiss Prot data bank.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.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology and Structure Research Groups, Gesellschaft für Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany.

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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Related in: MedlinePlus

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.
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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.

Mentions: Our earlier attempts in isolating macrophage activating material from M. fermentans were often frustrated by batches of mycoplasmas with low stimulatory potential. In the knowledge that (a) mycoplasmal macrophage stimulator is derived from lipoproteins or peptides (29), and of (b) the antigen variability and variation of expression of lipoproteins from mycoplasmas (42), we agar cloned mycoplasmas from their original source, a contaminated HL60 cell line, to isolate highly active clones. In total, 103 clones were picked at random, grown in liquid medium, harvested, and finally extracted with hot octyl glucoside. Macrophage stimulatory activity is extracted in this mild detergent (24). As an assay, we chose nitric oxide production by IFN-γ stimulated macrophages from LPS low responder mice. This can be used for quantitative estimation of macrophage-stimulatory activity and allows one to define a U/ml as that dilution which gives half-maximal NO production (24). A comparison of the activity of some of these clones normalized per milligram mycoplasma protein is shown in Fig. 1 and shows a variation of specific activity from 5,000 to 240,000 U/mg mycoplasma protein, i.e., by a factor of ∼50. Some clones were recloned and retained their good stimulatory properties (see, e.g., clone II 29 in Fig. 1). Since the variation of MSA upon recloning was higher than can be accounted for by experimental error, we speculate that it may be due to clonal variation.


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

Mühlradt PF, Kiess M, Meyer H, Süssmuth R, Jung G - J. Exp. Med. (1997)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196331&req=5

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.
Mentions: Our earlier attempts in isolating macrophage activating material from M. fermentans were often frustrated by batches of mycoplasmas with low stimulatory potential. In the knowledge that (a) mycoplasmal macrophage stimulator is derived from lipoproteins or peptides (29), and of (b) the antigen variability and variation of expression of lipoproteins from mycoplasmas (42), we agar cloned mycoplasmas from their original source, a contaminated HL60 cell line, to isolate highly active clones. In total, 103 clones were picked at random, grown in liquid medium, harvested, and finally extracted with hot octyl glucoside. Macrophage stimulatory activity is extracted in this mild detergent (24). As an assay, we chose nitric oxide production by IFN-γ stimulated macrophages from LPS low responder mice. This can be used for quantitative estimation of macrophage-stimulatory activity and allows one to define a U/ml as that dilution which gives half-maximal NO production (24). A comparison of the activity of some of these clones normalized per milligram mycoplasma protein is shown in Fig. 1 and shows a variation of specific activity from 5,000 to 240,000 U/mg mycoplasma protein, i.e., by a factor of ∼50. Some clones were recloned and retained their good stimulatory properties (see, e.g., clone II 29 in Fig. 1). Since the variation of MSA upon recloning was higher than can be accounted for by experimental error, we speculate that it may be due to clonal variation.

Bottom Line: The sequence could not be found in either the protein identification resource nor the Swiss Prot data bank.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.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology and Structure Research Groups, Gesellschaft für Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany.

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.

Show MeSH
Related in: MedlinePlus