title: Identification of S-(2,3-dihydroxypropyl)cystein in a macrophage-activating lipopeptide from Mycoplasma fermentans.
abstract: Mycoplasmas are capable of stimulating monocytes and macrophages to release cytokines, prostaglandins, and nitric oxide. The aim of this study was to characterize the chemical nature of the previously isolated [Mühlradt, P. F., & Frisch, M. (1994) Infect. Immun. 62, 3801-3807] macrophage-stimulating material "MDHM" from Mycoplasma fermentans. Mycoplasmas were delipidated, and MDHM activity was extracted with octyl glucoside and further purified by reversed-phase HPLC. Macrophage-stimulating activity was monitored by nitric oxide release from peritoneal macrophages from C3H/HeJ endotoxin low responder mice. HPLC-purified MDHM was rechromatographed on an analytic scale RP 18 column before and after proteinase K treatment. Proteinase treatment did not diminish biological activity but shifted MDHM elution toward higher lipophilicity, suggesting that the macrophage-stimulating activity might reside in the lipopeptide moiety of a lipoprotein. Proteinase K-treated MDHM was hydrolyzed, amino groups were dansylated, and the dansylated material was isolated by HPLC. Dansylated S-(2,3-dihydroxypropyl)cystein (glycerylcystein thioether), typical for Braun's murein lipoprotein, and Dns-Gly and Dns-Thr were identified by tandem mass spectrometry. These amino acids were isolated from biologically active but not from the neighboring inactive HPLC fractions. IR spectra from proteinase K-treated, HPLC-purified MDHM and those from the synthetic lipopeptide [2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-CysSerSer AsnAla were very similar. The data, taken together, indicate that lipoproteins of a nature previously detected in eubacteria are expressed in M. fermentans and that at least one of these lipoproteins and a lipopeptide derived from it constitute the macrophage-activating principle MDHM from these mycoplasmas.
authors.affiliation: GBF, Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany.
authors.forename: P F
authors.initials: PF
authors.lastname: Mühlradt
authors.forename: H
authors.initials: H
authors.lastname: Meyer
authors.forename: R
authors.initials: R
authors.lastname: Jansen
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D003619:Dansyl Compounds; D019286:Endopeptidase K; D005260:Female; D008074:Lipoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D051379:Mice; D008809:Mice, Inbred C3H; D016858:Mycoplasma fermentans / Q000737:chemistry* / Q000254:growth & development; D012697:Serine Endopeptidases; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013055:Spectrophotometry, Infrared
chemical_list: D003619:Dansyl Compounds; D008074:Lipoproteins; C031164:glycerylcysteine; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D003545:Cysteine