BINDING OF BACTERIAL PEPTIDOGLYCAN TO CD14

The hypothesis that soluble peptidoglycan (sPGN, a macrophage-activato r from Gram-positive bacteria) binds to CD14 (a lipopolysaccharide (LP S) receptor) was tested. sPGN specifically bound to CD14 in the follow ing three assays: binding of soluble P-32-CD14 (sCD14) to agarose-immo bilized sPGN, enzyme-linked immunosorbent assay, and photoaffinity cro ss-linking. sCD14 also specifically bound to agarose-immobilized muram yl dipeptide or GlcNAc-muramyl dipeptide but not to PGN pentapeptide. Binding of sCD14 to both sPGN and ReLPS (where ReLPS is LPS from Salmo nella minnesota Re 595) was competitively inhibited by unlabeled sCD14 , 1-152 N-terminal fragment of sCD14, sPGN, smooth LPS, ReLPS, lipid A , and lipoteichoic acid but not by dextran, dextran sulfate, heparin, ribitol teichoic acid, or soluble low molecular weight PGN fragments. Binding of sCD14 to sPGN was slower than to ReLPS but of higher affini ty (K-D = 25 nM versus 41 nM). LPS-binding protein (LBP) increased the binding of sCD14 to sPGN by adding another lower affinity K-D and ano ther higher B-max, but for ReLPS, LBP increased the affinity of bindin g by yielding two K-D with significantly higher affinity (7.1 and 27 n M). LBP also enhanced inhibition of sCD14 binding by LPS, ReLPS, and l ipid A. Binding of sCD14 to both sPGN and ReLPS was inhibited by anti- CD14 MEM-18 mAb, but other anti-CD14 mAbs showed differential inhibiti on, suggesting conformational binding sites on CD14 for sPGN and LPS, that are partially identical and partially different.