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

支原体脂蛋白中分子量为44 kDa （Lp44）的脂蛋白具有诱导ICAM-1表达的活性。Lp44的N端氨基酸序列为CGDPKH-PKSFTEWVA -， N端半胱氨酸氨基是游离的。Lp44的活性对蛋白酶K具有抗性，对脂蛋白脂肪酶敏感，表明Lp44的脂质部分而非蛋白质部分参与了活性的表达。采用反相高效液相色谱法纯化Lp44的脂质部分。用90%左右的异丙醇洗脱活性物。为了获得活性实体的结构信息，收集了具有活性的组分并将其应用于红外光谱仪。红外光谱分析表明，产物中存在脂肪酸烷基链和典型的酯键。因此，推测Lp44的n端脂肽（Lpp44）的结构为S-（2,3 -双酰基氧丙基）- cgdpkhpksftewva . More -，因为众所周知，脂质与原核脂蛋白中n端半胱氨酸的SH基团结合，目前尚不清楚该脂肪酸是什么。为了证实Lpp44是否具有诱导ICAM-1表达的活性，我们合成了一个类似于Lpp44的脂肽S-（2,3 -双棕榈酰基氧丙基）- cgdpkhpksf。发现Lpp44类似物具有诱导ICAM-1表达的活性，并诱导HGF产生IL-6和THP-1细胞（单核细胞系）产生TNF-α。Lpp44类似物诱导ICAM-1表达的活性显著高于大肠杆菌和肠炎沙门氏菌纯化的LPS，但低于后者。重组人TNF-α。因此，本研究证明Lp44是唾液分枝杆菌中参与ICAM-1表达和诱导细胞因子产生活性的脂蛋白之一，其n端脂肽是活性实体。Lpp44与MALP-2， S-（2,3 -双酰基氧丙基）- cgnndesnisfkek的氨基酸序列不同，但结构相似，后者是从M. fermentans中纯化鉴定出来的，能够激活单核/巨噬细胞诱导一氧化氮和TNF-α的产生。已知大肠杆菌小鼠脂蛋白n端脂肽的合成类似物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，原核生物的脂蛋白被推测为巨噬细胞、B细胞和成纤维细胞的有效激活剂。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