Identification and characterization of a bovine lipopolysaccharide-binding protein.
Endogenous regulatory mechanisms exist in mammals that enable a rapid response to lipopolysaccharide (LPS, endotoxin) stemming from gram-negative bacterial infections. Serum proteins and cell surface receptors exist that bind LPS, and this interaction may either aid in nonpathogenic removal of LPS from the body or potentiate the effects of LPS. We have used a photoreactive, thiol-cleavable, radiolabeled derivative of E. coli 0111:B4 LPS [LPS-(p-azidosalicylamido)-1,3'-dithiopropionamide; 125I-ASD-LPS], to identify the presence of LPS-binding proteins (LBPs) in bovine serum. Ion exchange chromatography was used to fractionate bovine serum, and eluted protein was subsequently photoaffinity labeled using 125I-ASD-LPS. LBPs were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several LBPs including three with apparent molecular masses of 65, 60, and 50 kDa were variably present within the chromatography pools. A 22-residue NH2-terminal amino acid sequence of the 60-kDa protein showed 77% homology with human LBP and 68% with rabbit LBP within this region. Further purification utilizing high-performance liquid chromatography yielded a protein fraction that contained the 60-kDa protein and was distinctly more active than whole bovine serum in LPS-dependent macrophage activation assays (up to 1600-fold on a weight/volume basis). The LPS-mediated macrophage activation in concert with chromatographically purified serum protein in tissue factor assays was inhibitable using anti-CD14 monoclonal antibodies. The results indicate that an LPS-binding protein exists in samples of pooled bovine serum and that this protein has features in common with human and rabbit LBP.
Khemlani
L S
LS
Department of Pathology, University of Tennessee College of Veterinary Medicine, Knoxville.
Yang
Z
Z
Bochsler
P N
PN
D000209:Acute-Phase Proteins; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000032:analysis / Q000378:metabolism; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli; D005260:Female; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D000345:Affinity Labels; D001798:Blood Proteins; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D000099231:Lipopolysaccharide-Binding Protein