Lipid a 4′ kinase

Details Lipid a 4′ kinase Lipid a 4′ kinase

1998-05-18

2002-10-15

Ketter, James (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023700

Reexamination Certificate

active

06465631

ABSTRACT:

TECHNICAL FIELD
The present invention relates, in general, to lipid A 4′ kinase and, in particular, to a nucleic acid encoding lipid A 4′ kinase and to a method of producing lipid A 4′ kinase recombinantly using same. The invention further relates to methods of producing 4′ phosphorylated lipid A analogs using the recombinantly produced lipid A 4′ kinase.
BACKGROUND
Lipopolysaccharide (LPS) is the major glycolipid of the outer membrane of gram-negative bacteria. Lipid A, or endotoxin, is the hydrophobic anchor of LPS, and it is a potent immunostimulant. It appears to be responsible for many of the features of septic shock that can accompany severe gram-negative infections. Lipid A is a disaccharide of glucosamine that is phosphorylated at the 1 and 4′ positions and is acylated with R-3 hydroxymyristate at the 2, 3, 2′, and 3′ positions. In
E. coli
, two additional fatty acyl chains are also esterified to the 2′ and 3′ R-3 hydroxymyristate groups to form acyloxyacyl units.
Lipid A biosynthesis begins with the acyl-ACP dependent acylation of UDP-N-acetylglucosamine (Anderson et al, J. Biol. Chem. 260:15536 (1985), Anderson et al, J. Biol. Chem. 262:5159 (1987), Anderson et al, J. Biol. Chem. 268:19858 (1993), Williamson et al, J. Bacteriol. 173:3591 (1991), Raetz et al, Science 270:997 (1995)). Nine enzymes are required for the complete synthesis of Kdo
2
-lipid A (Raetz, J. Bacteriol. 175:5745 (1993), Raetz,
Escherichia coli
and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, Second Ed., pp. 1035-1063, American Society for Microbiology, Washington, D.C. (1996), Raetz et al, J. Biol. Chem. 265:1235 (1990)). Seven of the nine structural genes coding for the enzymes of lipid A biosynthesis in
E. coli
have been identified, however, the lipid A 4′ kinase gene has remained elusive (Raetz,
Escherichia coli
and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, Second Ed., pp. 1035-1063, American Society for Microbiology, Washington, D.C. (1996)). The 4′ kinase catalyzes the sixth step of the pathway (
FIG. 2
) (Ray et al, J. Biol. Chem. 262:1122 (1987)). It phosphorylates the 4′ position of a tetraacyldisaccharide-1-phosphate intermediate (termed DS-1-P) to form tetraacyldisaccharide 1, 4′ bis-phosphate, also known as lipid IVA (
FIG. 2
) (Ray et al, J. Biol. Chem. 262:1122 (1987), Raetz et al, J. Biol. Chem. 260:16080 (1985), Strain et al, J. Biol. Chem. 260:16089 (1985)).
Identification of the 4′ kinase gene has been hampered because mutants lacking the 4′ kinase have not been identified (Raetz,
Escherichia coli
and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, Second Ed., pp. 1035-1063, American Society for Microbiology, Washington, D.C. (1996)). Attempts to purify the kinase to homogeneity have been thwarted by the protein's association with membranes and its instability in the presence of detergents (Ray et al, J. Biol. Chem. 262:1122 (1987), Hampton et al, Methods in Enzymology 209:466 (1992)).
The lipid A 4′ kinase can be used to make 4′-
32
P labeled lipid A precursors, such as [4′-
32
P]-lipid IVA and Kdo
2
-[4′-
32
P]-lipid IVA, for biochemical analyses of late pathway reactions. The 4′ kinase activity found in wild type
E. coli
membranes, however, is relatively inefficient and unstable, especially in the presence of low chemical concentrations of ATP. The inability to achieve high levels of
32
P transfer makes it virtually impossible to use the 4′ kinase for phosphorylating DS-1-P analogs that are utilized less rapidly. Identification and overexpression of the 4′ kinase gene would facilitate the synthesis of 4′ phosphorylated lipid A analogs with activity as endotoxin antagonists or mimetics (Raetz, J. Bacteriol. 175:5745 (1993), Raetz,
Escherichia coli
and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed) Vol. 1, Second Ed., pp. 1035-1063, American Society for Microbiology, Washington, D.C. (1996)). The present invention provides a nucleic acid encoding lipid A 4′ kinase and a method of producing 4′ kinase using same.
OBJECTS AND SUMMARY OF THE INVENTION
It is a general object of the invention to provide a nucleic acid encoding lipid A 4′ kinase.
It is another object of the invention to provide a method of producing lipid A 4′ kinase recombinantly.
It is a further object of the invention to provide a recombinantly produced lipid A 4′ kinase.
It is a further object of the invention to provide a method of producing lipid A analogs suitable for use as endotoxin mimetics or endotoxin antagonists, using lipid A 4′ kinase.
The foregoing objects are met by the present invention which relates to a nucleic acid encoding 4′ kinase, to an expression construct comprising that nucleic acid and to a host cell into which the construct has been introduced. The invention further relates to a method of producing 4′ kinase using such host cells and to the 4′ kinase produced thereby. In addition, the invention relates to the production of 4′ phosphorylated lipid A analogs using the recombinantly produced 4′ kinase.
Further objects and advantages of the invention will be clear from the description that follows.


REFERENCES:
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R. Ingalls et al.; Membrane Expression of Soluble Endotoxin-binding Proteins Permits Lipopolysaccharide Signaling in Chinese Hamster Ovary Fibroblasts Independently of CD14; May 1999, The Journal of Biological Chemistry, vol. 274, 13993-13998.*
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C. Lam et.al; Immunostimulatory, but not Antiendotoxin, Activity of Lipid X is due to Small Amounts of Contaminating N, O-Acylated Disccharide-1-Phosphate: In Vitro and In Vivo Reevaluation of the Biological Activity of Synthetic Lipid X; Jul. 1991 Infecti.*
Ray et al, “The Biosynthesis of Gram-negative Endotoxin: A Novel Kinase InEscherichia ColiMembranes That Incorporates The 4′-Phosphate of Lipid A”, The Journal of Biological Chemistry 202(3):1122-1128 (1987).
Hampton et al, “Lipid A 4′-Kinase formEscherichia coli: Enzyme Assay and Preparation of 4′-32P-Labeled Probes of High Specific Radioactivity”, Methods in Enzymology 209:466-475 (1992).
Garrett et al, “Identification of the Gene Encoding theEscherichia coliLipid A 4′-Kinase”, The Journal of Biological Chemistry 272(35):2185521864 (1997).
Karow et al, “The essentialEscherichia coli msbAgene, a multicopy suppressor of null mutations in thehtrBgene, is related to the univesally conserved family of ATP-dependent translocators”, Molecular Microbiology 7(1):69-79 (1993).

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