Identification of lipopolysaccharide-binding proteins in 70Z/3 cells by photoaffinity cross-linking.
A radioiodinated, photoactivatable derivative of Salmonella minnesota Re595 lipopolysaccharide (LPS) was used to label LPS-binding proteins in 70Z/3 cells. The labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. 125I-Labeled-2-(p-azidosalycylamido)1,3'-dithiopropionamide S. minnesota Re595 LPS (125I-ASD-Re595) labeled a limited number of proteins. The most prominent of these had a apparent molecular mass of 18 kDa. Less prominent labeling of 25- and 28-kDa proteins was also seen. Labeling was saturated by 5 micrograms/ml 125I-ASD-Re595 and was inhibited by a 10-100-fold excess of unlabeled LPS or lipid A. Labeling was maximal within 30 min at 37 degrees C; much less labeling occurred at lower temperatures. The proteins labeled with 125I-ASD-Re595 appear to be on the surface of the cell, since they can be digested by trypsin and were found in the membrane fraction of the cell but not in the cytosol. Studies with competitive inhibitors suggested that the proteins bind to the lipid A region of the LPS molecule. Biologically inactive lipid A analogs were poor inhibitors of labeling, suggesting that the LPS-binding proteins could discriminate between active lipid A and inactive analogs. These studies suggest that the 18- and 25-kDa proteins bind specifically to the lipid A region of the LPS molecule and should be considered as candidates for a functional LPS receptor.
Kirkland
T N
TN
Department of Pathology, University of California, San Diego.
Virca
G D
GD
Kuus-Reichel
T
T
Multer
F K
FK
Kim
S Y
SY
Ulevitch
R J
RJ
Tobias
P S
PS
D000345:Affinity Labels; D001386:Azides; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D003432:Cross-Linking Reagents; D003600:Cytosol / Q000032:analysis; D007700:Kinetics; D008050:Lipid A / Q000378:metabolism / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000378:metabolism*; D008970:Molecular Weight; D010777:Photochemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D012475:Salmonella; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
C047258:2-(4-azidosalicylamido)-1,3'-dithiopropionate-lipopolysaccharide; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D008050:Lipid A; D008070:Lipopolysaccharides; D011480:Protease Inhibitors; D014357:Trypsin