High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-terminal fragment to the lipid A region of lipopolysaccharide

Abstract

Bactericidal/permeability-increasing protein (BPI) is a 55-kDa cationic protein (nBPI,,) elaborated by polymorphonuclear neutrophils (PMN). BPI has potent bactericidal activity against a wide variety of gram-negative organisms and neutralizes endotoxin activities. An N-terminal fragment of nBPI55 exhibits the bactericidal and antiendotoxin properties of the holoprotein. To further characterize the biological activities of the N-terminal fragment, a recombinant protein (rBPI23) corresponding to the first 199 amino acids of human BPI was produced and purified. rBPI23 had antibacterial activity equivalent to that of nBPI55 against Escherichia coli J5. Furthermore, both rBPI23 and nBPI55 bound identically to a broad range of Rand S-form lipopolysaccharides (LPS) and to natural and synthetic lipid A. Binding of radiolabeled nBPI55 to LPS was inhibited in an identical fashion by either nBPI,, or rBPI23. The binding of both proteins to immobilized E. coli J5 lipid A was inhibited in a comparable fashion by longor short-chain LPS or lipid A. The binding of both rBPI23 and nBPI55 was specific, saturable, and of high affinity, with an apparent Kd of approximately 2 to 5 nM for all ligands tested. These results demonstrate that BPI recognizes the highly conserved lipid A region of bacterial LPS via residues contained within the amino-terminal portion of the BPI molecule.

The bactericidal/permeability-increasing protein (BPI) in antibacterial host defense

“…Among the many known antimicrobial polypeptides, BPI stands out in several ways: (1) at nanomolar concentrations BPI displays highly selective cytotoxicity toward a wide range of gram-negative bacterial (GNB) species (2). This target cell selectivity reflects high-affinity attraction (apparent K d Յ5 nM [24]) to the lipid A moiety of the lipopolysaccharides (LPS) that occupy the outer layer of the outer membrane of the GNB envelope [25]. Binding of BPI to live bacteria results in: (i) a discrete increase in the permeability of the outer membrane [21]; (ii) hydrolysis by bacterial phospholipase and some host phospholipases A 2 of bacterial phospholipids that are refractory to phospholipase action in unperturbed bacteria [26][27][28][29]; and (iii) interruption of cell division.…”

Potential applications of N-terminal recombinant fragments of bactericidal/permeability-increasing protein in liver surgery

“…Evaluation of the BPI binding to many different forms of endotoxin, isolated from clinically relevant gram-negative bacterial species, and to natural and synthetic lipid A has shown that the presence of long O-specific polysaccharide chains on purified endotoxin have little or no effect on BPI reactivity. 6 The minimal structure required for high-affinity binding is the lipid A precursor lipid IVA, a natural intermediate in the biosynthesis of lipid A. Furthermore, the presence of two negatively charged groups on lipid A appears to be important for BPI binding.…”

“…4 The selectivity of BPI against gram-negative bacteria has been attributed to the strong attraction of BPI for this LPS in the bacterial envelope, especially the high affinity for its lipid A component. 5,6 It has become evident that the LPS structure primarily determines bacterial sensitivity. Strains bearing LPS with short polysaccharide chains in their outer membrane are the most sensitive.…”

“…Long polysaccharide chains sterically hinder the access of BPI to the binding sites in the inner core and the lipid A regions of LPS, thereby reducing the affinity of BPI for the envelope LPS, necessitating higher ambient BPI concentrations to achieve the required amount of bound BPI to produce its biological effects. 3,6,7 The cytotoxic effect of BPI on gram-negative bacteria can be divided into two stages. When BPI binds to the outer membrane, several immediate effects are manifest, including arrest of bacterial growth, discrete changes in the permeability of the outer membrane, and selective activation of several envelope enzymes, which degrade phospholipids and peptidoglycans.…”

Apoptotic Cell Death Induced by ofLBP6A, Lipopolysaccharide Binding Protein Model Peptide, Derived from Paralichthy olivaceus on MKN‐28 Cells

“…LBP is comprises two domain, N-terminal and C-terminal domains. Several researches have reported that N-terminal domain of LBP has antibacterial effects via bind to LPS [Gazzano-Santoro et al, 1992;Weiss et al, 1992;Fenton and Golenbock, 1998]. In a previous report, we have synthesized five peptides to investigate the function of the C-terminal domain of LBP.…”