The Re and Rd chemotypes of lipopolysaccharides (LPS) obtained from Escherichia coli and Salmonella minnesota will be derivatized to the methyl esters and purified to structural homogeneity by utilizing silica gel chromatography and the recently developed 50-nm pore size polyvinylbenzene gel permeation and divinylbenzene gel reverse-phase columns for high performance liquid chromatograhy (HPLC). These highly purified and structurally homogeneous methyl LPS will then be analyzed by chemical means, fast atom bombardment mass spectrometry, california plasma deserption mass spectrometry, 1-H-nuclear magnetic resonance (NMR) spectroscopy, and 31-p-NMR spectroscopy. Using these results, we shall attempt to establish the complete structures of the native LPS and determine the nature of their apparent microheterogeneit. The purified methyl LPS will be subjected to selective biological assays in order to study the LPS structure-to-biological-activity relationship. We shall examine how the biological activities of the lipid A moiety of purified methyl LPS containing 2-keto-3-deoxyoctonate might be modulated by the presence of one or more of the following polar groups: pyrophosphate, ethanolamine, aminoarabinose, and L-glycero-D-mannoheptose. These assays include tests for toxicity and antitumor activities. Lipid A from the following sources will be methylated and purified to homogeneity by HPLC: nontoxic diphosphoryl lipid A from Rhodopseudomonas sphaeroides, phosphatefree lipid A from Rhodomicrobium vannielii, and lipid A from Brucella abortus and Neisseria gonorrhoeae. Complete structures of these lipid A's will be determined and the underivatized samples will be tested for certain biological activities. The long-term objective of this research is to progress to the more complex Rc, Rb, and Ra LPS chemotypes in our structural studies and ultimately to be able to examine the structures of the highly purified smooth strain LPS at the intact level. Knowledge gained from this study might be applied to cancer immunotherapy, the development of an effective adjuvant, protection against X-irradiation, and protection against bacterial and viral infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036054-04
Application #
3289639
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1985-12-01
Project End
1989-11-30
Budget Start
1988-12-01
Budget End
1989-11-30
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Earth Sciences/Resources
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Qureshi, N; Takayama, K; Sievert, T R et al. (1995) Novel method for the purification and characterization of lipopolysaccharide from Escherichia coli D31m3. Prog Clin Biol Res 392:151-60
Kirikae, F; Kirikae, T; Qureshi, N et al. (1995) CD14 is not involved in Rhodobacter sphaeroides diphosphoryl lipid A inhibition of tumor necrosis factor alpha and nitric oxide induction by taxol in murine macrophages. Infect Immun 63:486-97
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Takayama, K; Mitchell, D H; Din, Z Z et al. (1994) Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages. J Biol Chem 269:2241-4
Kirikae, T; Schade, F U; Kirikae, F et al. (1994) Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells. FEMS Immunol Med Microbiol 9:237-43
Din, Z Z; Mukerjee, P; Kastowsky, M et al. (1993) Effect of pH on solubility and ionic state of lipopolysaccharide obtained from the deep rough mutant of Escherichia coli. Biochemistry 32:4579-86
Datta, A K; Takayama, K (1993) Biosynthesis of a novel 3-oxo-2-tetradecyloctadecanoate-containing phospholipid by a cell-free extract of Corynebacterium diphtheriae. Biochim Biophys Acta 1169:135-45
Datta, A K; Takayama, K (1993) Isolation and purification of trehalose 6-mono- and 6,6'-di-corynomycolates from Corynebacterium matruchotii. Structural characterization by 1H NMR. Carbohydr Res 245:151-8
Lei, M G; Qureshi, N; Morrison, D C (1993) Lipopolysaccharide (LPS) binding to 73-kDa and 38-kDa surface proteins on lymphoreticular cells: preferential inhibition of LPS binding to the former by Rhodopseudomonas sphaeroides lipid A. Immunol Lett 36:245-50

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