CHARACTERIZATION OF THE STRUCTURAL ELEMENTS IN LIPID-A REQUIRED FOR BINDING OF A RECOMBINANT FRAGMENT OF BACTERICIDAL/PERMEABILITY-INCREASING PROTEIN RBPI(23)

Both human bactericidal/permeability-increasing protein (BPI) and a recombinant amino-terminal fragment of BPI (rBPI(23)) have been shown to bind with high affinity to the lipid A region of lipopolysaccharide (LPS) (H. Gazzano-Santoro, J. B. Parent, L. Grinna, A. Horwitz, T. Parsons, G. Theofan, P. Elsbach, J. Weiss, and P. J. Conlon, Infect. Immun. 60:4754-4761, 1992). In the present study, lipid A preparations derived from bacterial LPS as well as synthetic lipid A's and various lipid A analogs were used to determine the structural elements required for rBPI(23) binding. rBPI(23) bound in vitro to a variety of synthetic and natural lipid A preparations (both mono- and diphosphoryl forms), including lipid A's prepared from Escherichia coli and Salmonella, Neisseria, and Rhizobium species. Binding does not require that the origin of negative charge be phosphate, since rBPI(23) bound with high affinity to lipid A's isolated from Rhizobium species that contain carboxylate (Rhizobium trifolii) or sulfate (Rhizobium meliloti) anionic groups and lack phosphate. Lipid A acyl chains are important, since rBPI(23) did not bind to four synthetic variants of the beta(1-6)-linked D-glucosamine disaccharide lipid A head group, all devoid of acyl chains. rBPI(23) also bound weakly to lipid X, a monosaccharide lipid precursor of LPS corresponding to the reducing half of lipid A. Lipid IVA, a precursor identical to E. coli lipid A except that it lacks the 2' and 3' acyl chains, was the simplest structure identified in this study that rBPI(23) bound with high affinity. These results demonstrate that rBPI(23) has a binding specificity for the lipid A region of LPS and binding involves both electrostatic and hydrophobic components.