HASEBE Akira Hokkaido University, School of Dentistry, Instructor, 歯学部, 助手 (90281815)
WATANABE Tsuguo Hokkaido University, School of Dentistry, Professor, 歯学部, 教授 (10064362)
|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1998 : ¥2,000,000 (Direct Cost : ¥2,000,000)
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
-, because it is well known that lipid is bound to SH group of the N-terminal cysteine in lipoproteins from prokaryotes
At 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