Physiological and pathological roles of adhesion molecules expressed on gingival fibroblasts in response to oral microbes

分枝杆菌脂蛋白中一种分子质量为44 kDa的脂蛋白(Lp44)具有诱导ICAM-1表达的活性。Lp44的N端氨基酸序列为CGDPKH-PKSFTEWVA-，N端半胱氨酸的氨基是自由的。Lp44的活性对蛋白酶K不敏感，对脂蛋白脂酶敏感，提示Lp44活性的表达与脂类部分有关，而与蛋白部分无关。采用反相高效液相色谱分离纯化Lp44的脂类成分。该活性在90%左右的异丙醇中洗脱。为了获得有关活性物质结构的信息，收集了具有活性的级分，并将其应用于红外光谱仪。红外光谱分析表明，馏分中含有脂肪酸烷基链和典型的酯键。因此，推测Lp44(Lpp44)N端脂多肽的结构为S-(2，3-双酰氧丙基)-CGDPKHPKSFTEWVA…此外，由于脂类与原核脂蛋白中N端半胱氨酸的SH基团结合是众所周知的，所以目前还不清楚S所说的脂肪酸是什么。为了确定Lpp44是否具有诱导细胞间黏附分子-1表达的活性，合成了一种类似于Lpp44的脂肽S-(2，3-双棕榈酰氧丙基)-CGDPKHPKSF。Lpp44类似物具有诱导细胞间黏附分子-1表达的活性，并通过肝细胞生长因子和肿瘤坏死因子-α的产生诱导单核细胞系THP-1细胞产生IL-6。Lpp44类似物对ICAM-1的诱导活性明显高于从大肠杆菌和肠炎沙门氏菌中纯化的内毒素，但低于从大肠杆菌和肠炎沙门氏菌中纯化的内毒素。因此，本研究证明Lp44是一种唾液分枝杆菌脂蛋白，具有诱导细胞间黏附分子-1表达和细胞因子产生的活性，其N端脂多肽是其活性实体。Lp44在氨基酸序列上不同于从发酵分枝杆菌中分离鉴定的S-(2，3-双酰氧丙基)-α-CGNNDESNISFKEK，在氨基酸序列上与MALP-2相似，能够激活单核/巨噬细胞产生一氧化氮和肿瘤坏死因子-α。人工合成的大肠杆菌Murein脂蛋白N端脂多肽类似物被认为是有效的巨噬细胞和B细胞激活剂。这些脂肽的结构与MALP-2和Lpp44相似。因此，原核生物中的脂蛋白被认为是巨噬细胞、B细胞和成纤维细胞的有效激活剂。较少

A lipoprotein with a molecular mass of 44 kDa (Lp44) in Mycopiasma lipoproteins possessed the ICAM-1 expression-inducing activity. The N-terminal amino acid sequence of Lp44 was suggested to be CGDPKH-PKSFTEWVA - and the amino group of the N- terminal cysteine is free. The activity of Lp44 was resistant to proteinase K and sensitive to lipoprotein lipase, suggesting that the lipid moiety, but not the proteinous moiety, of Lp44 is involved in the expression of the activity. The lipid moiety of Lp44 was purified by a reverse-phased HPLC. The activity was eluted at around 90% isopropanol. In order to obtain informations on the structure of the active entity, the fractions with the activity were collected and applied to the Infrared (IR) spectrometer. The IR spectra of the fractions revealed the presence of fatty acid alkyl chains and the typical ester bond. Therefore, the structure of the N-terminal lipo-peptide of Lp44 (Lpp44) is speculated to be S-(2,3 -bisacyloxypropyl)-CGDPKHPKSFTEWVA … More -, because it is well known that lipid is bound to SH group of the N-terminal cysteine in lipoproteins from prokaryotesAt present, it remains unknown what the fatty acid(s) is(are). In order to confirm whether Lpp44 possesses the ICAM-1 expression-inducing activity, a lipopeptide, S-(2,3 -bispalmitoyloxypropyl)-CGDPKHPKSF, analogous to Lpp44 was synthesized. The Lpp44 analogue was found to possess the ICAM-1 expression-inducing activity, and to induce IL-6 production by HGF and TNF-α production by THP-1 cells, a monocytic cell line as well. The ICAM-1 expression-inducing activity level of the Lpp44 analogue was **nd to be significantly higher than that of LPS purified from Escherichia coli and Salmonella enteritidis, but lower than that. Of human recombinant TNF-α.Thus, this study demonstrated that Lp44 was one of M. salivarium lipoproteins responsible for the ICAM-1 expression- and cytokine production-inducing activities and its N-terminal lipopeptide was the active entity.Lpp44 is different in the amino acid sequence from, but similar in the structure to MALP-2, S-(2,3 -bisacyloxypropyl)-CGNNDESNISFKEK, which is purified and identified from M. fermentans and is capable of activating monocytes/macrophages to induce production of nitric oxide and TNF-α. Synthetic analogues of N-terminal lipopeptide of E. coli murein lipoproteins are known to be potent macrophage and B cell activators. The structures of these lipopeptides were similar to those of MALP-2 and Lpp44. Therefore, lipoproteins from prokaryotes are speculated to be a potent activator for macrophages, B cells and fibroblasts. Less