Identification of a Lipid A Binding Site in the Acute Phase Reactant Lipopolysaccharide Binding Protein

P S Tobias,K Soldau,R J Ulevitch

doi:10.1016/s0021-9258(18)81700-8

P S Tobias, K Soldau + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(18)81700-8

Copy DOI

Journal: The Journal of biological chemistry	Publication Date: Jun 1, 1989
Citations: 397	License type: cc-by

Affiliation: Scripps Clinic

Abstract

Lipopolysaccharide (LPS) binding protein (LBP), a recently discovered 60-kDa acute phase protein, is present in the acute phase serum of many species including human, rabbits, mice, and rats. Using either highly purified LBP from acute phase rabbit serum or unfractionated acute phase rabbit serum as a source of LBP, we examined the binding of LBP to LPS immobilized on plastic microtiter plates and to LPS electrotransferred to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of LBP bound to LPS was detected with goat anti-rabbit LBP and peroxidase-conjugated rabbit anti-goat IgG. LBP was found to bind to a variety of LPS types from both rough and smooth strains of Gram-negative bacteria, to lipid A, and to the tetraacyl glucosamine disaccharide diphosphate precursor IVA, but bound very poorly to the diacyl glucosamine phosphate, lipid X. No binding to 3-deoxyoctulosonic acid was observed. Binding affinities for LPS are near 10(9) M-1. The data presented here support the concept that LBP contains a binding site for lipid A.

Highlights

Have discovered, purified, characterized, and named lipopolysaccharide binding protein (LBP) [8].LBP is a 60-kDa glycoprotein present in acute phase rabbit serum at 10-50 pg/ml and is estimated to be at less than 100 ng/ml in normal rabbit serum
Inhibition of LBP bindingto the plates was studiedby premixing the compound under study at the desired concentration with1pg/ml purified LBP for 10 min at 37 "C before adding the mixture to the microtiter plate.We found that there were significant differences in
Binding Specificity of LBP-To study the bindingspecificity of LBP, we examined a battery of test substances that electrophoresed in a 15%acrylamide gel containing SDS [13]

Summary

Introduction

Have discovered, purified, characterized, and named lipopolysaccharide binding protein (LBP) [8].LBP is a 60-kDa glycoprotein present in acute phase rabbit serum at 10-50 pg/ml and is estimated to be at less than 100 ng/ml in normal rabbit serum. There are no studies documenting aprotein that hasa specific binding site for lipid A, the biologically active structure of the LPS molecule.

Results

Conclusion