Identification of S-(2,3-dihydroxypropyl)cystein in a macrophage-activating lipopeptide from Mycoplasma fermentans.
Identification of S-(2,3-dihydroxypropyl)cystein in a macrophage-activating lipopeptide from Mycoplasma fermentans.
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DOI：
10.1021/bi9602831
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发表时间：
1996-06
期刊：
影响因子：
2.9
通讯作者：
P. Mühlradt;Holger Meyer;Rolf Jansen
中科院分区：
文献类型：
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作者：
P. Mühlradt;Holger Meyer;Rolf Jansen
支原体能够刺激单核细胞和巨噬细胞释放细胞因子、野牡丹素和一氧化氮。本研究的目的是表征先前分离的化合物的化学性质[Mühlradt，P.F.，& Frisch，M. 04 The Story of the Woman（1994）Immun. 62，3801-3807]来自发酵支原体的巨噬细胞刺激物质“MDHM”。支原体脱脂，MDHM活性用辛基葡萄糖苷提取，并通过反相HPLC进一步纯化。通过C3 H/HeJ内毒素低应答小鼠腹腔巨噬细胞释放一氧化氮来监测巨噬细胞刺激活性。在蛋白酶K处理之前和之后，在分析规模RP 18柱上对HPLC纯化的MDHM进行再层析。蛋白酶处理并没有减少生物活性，但转向更高的亲脂性MDHM洗脱，这表明巨噬细胞刺激活性可能驻留在脂蛋白的脂肽部分。将蛋白酶K处理的MDHM水解，氨基进行丹磺酰化，并通过HPLC分离丹磺酰化的物质。丹磺酰化S-（2，3-二羟丙基）半胱氨酸（甘油基半胱氨酸硫醚），典型的布朗氏胞壁脂蛋白，和Dns-Gly和Dns-Thr通过串联质谱鉴定。这些氨基酸是从生物活性组分中分离出来的，而不是从相邻的非活性HPLC组分中分离出来的。来自蛋白酶K处理的、HPLC纯化的MDHM和来自合成脂肽[2，3-双（棕榈酰氧基）-（2-RS）-丙基]-N-棕榈酰-（R）-CysSerSer AsnAla的IR光谱非常相似。这些数据综合起来表明，以前在真细菌中检测到的脂蛋白在M.这些脂蛋白中的至少一种和由其衍生的脂肽构成来自这些支原体的巨噬细胞活化成分MDHM。
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.