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Vestnik FEB RAS. 2018. № 6 Supplement
UDC 577.151.45 DOI: 10.25808/08697698.2018.202.6S.034
N.S. BUINOVSKAYA, S.I. BAKHOLDINA, L A. BALABANOVA
Dephosphorylation of lipopolysaccharides by alkaline phosphatase from marine bacterium
Key words: marine bacterium, alkaline phosphatase, lipopolysaccharide, dephosphorylation
The recombinant alkaline phosphatase from the marine bacterium Cobetia amphilecti KMM 269 (CmAP) with a higher activity (> 12,000 U / mg) among the known analogues was previously biochemically characterized [1]. However, the biological role of the extracellular highly active alkaline phosphatase (AP) of the marine bacterium remains unknown. In addition, the C. amphilecti KMM 296 genome has been found the presence of several genes encoding alkaline phosphatases, probably associated with still unexplored functions for the marine life style [1]. The recent discovery of new properties and biological functions of AP from various sources shows the prospect of using them as drugs for various purposes of medicine and biotechnology [1, 2]. Thus, intestinal APs was able to dephosphorylate lipopolysaccharides (LPS, endotoxins) of bacteria that resulted in a decrease of the overall inflammatory process [2]. It is known that LPS are major components of the cell envelope of gram-negative bacteria, which are an important contributing factor to septic shock, in general, and gram-negative septic shock, in particular. The endotoxic properties of LPS depend on the structural features of lipid A, a phosphoglycolipid. Currently, gram-negative sepsis and endotoxic shock are a serious clinical problem. They give a high percentage of deaths even in countries with a developed health system. Modern medicine does not have specific and effective drugs for anti-endotoxin therapy. Currently, gram-negative sepsis and endotoxic shock are a serious clinical problem. They give a high percentage of deaths even in countries with a developed health system. Modern medicine does not have specific and effective drugs for anti-endotoxin therapy.
One of the approaches used to reduce the toxicity of LPS is dephosphorylation of lipid A. The cleavage of a single phosphate group in lipid A by mild acid hydrolysis of LPS has been shown to cause a significant weakening of its pyrogenicity and toxicity [3]. In this work, we first studied the effect of CmAP on the lipopolysaccharides Esherichia coli S-LPS-055: B5 and Ra-LPS-EH100 (Sigma). The total content of phosphorus in the initial solutions of LPS and after their treatment with AP followed by dialysis against water to remove free phosphate groups was determined by the method described [4]. It was found, that CmAP exhibits enzymatic activity against LPS, which largely depends on the aggregate state of the LPS molecules. The greatest activity of the
* BUINOVSKAYA Nina Sergeevna- Graduate student, BAKHOLDINA Svetlana Ivanovna - PhD, Senior Researcher (G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS, Vladivostok, Russia), BALABANOVA Larissa Anatolyevna - PhD, Senior Researcher, SLEPCHENKO Lubov Vasilevna - Junior Researcher (G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB RAS, Vladivostok, Russia; Far Eastern Federal University, Vladivostok, Russia). *E-mail: [email protected]
The work was supported by the program "Far East", № 18-4-051.
enzyme removing up to 90% phosphorus was observed with LPS in the monomelic form. For complete dissolution of LPS, parameters such as sample concentration, buffer composition and pH, as well as the incubation temperature, were selected.
Thus, new data on the dephosphorylating activity of Cm AP against LPS are a promising basis for developing a new therapeutic approach with the use of alkaline phosphatase for neutralizing the effects of bacterial endotoxins (sepsis, endotoxic shock).
REFERENCES:
1. Balabanova L., Podvolotskaya A., Slepchenko L., Eliseikina M., Noskova Yu., Nedashkovskaya O., Son O., Tekutyeva L., Rasskazov V. Nucleolytic enzymes from the marine bacterium Cobetia amphilecti KMM 296 with antibiofilm activity and biopreservative effect on meat products // Food Control. 2017, Vol. 78. p. 270-278.
2. Lalles JP. Intestinal alkaline phosphatase: novel functions and protective effects // Nutrition Reviews. 2013, Vol.72, №2, p. 82-94.
3. Rudbach, J. A., Myers, K. R., Rechtman, D. J., and Ulrich, J. T. Prophylactic use of monophosphoryl lipid A in patients at risk for sepsis // Prog. Clin. Biol. 1994, Res. 388, p. 107-124.
4. Vaskovsky V.E., Kostetsky EY., Vasendin IM. A universal reagent for phospholipid analysis // J. Chromatog. 1975, Vol. 114, №1, p. 129-141.
