Competition between rBPI23, a recombinant fragment of bactericidal/permeability-increasing protein, and lipopolysaccharide (LPS)-binding protein for binding to LPS and gram-negative bacteria

Abstract

Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are two structurally related lipid A-binding proteins with divergent functional activities. LBP mediates activation of macrophage and other proinflammatory cells. In contrast, BPI has potent bactericidal and LPS-neutralizing activities. A recombinant fragment of BPI (rBPI23) retains the potent biological activities of the holo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis, and endotoxemia. For therapeutic effectiveness in many clinical situations, rBPI23 will have to successfully compete with high serum levels of LBP for binding to endotoxin and gram-negative bacteria. The relative binding affinities of rBPI23 and human recombinant LBP (rLBP) for lipid A and gram-negative bacteria were evaluated. The binding of both proteins to lipid A was specific and saturable with apparent Kds of 2.6 nM for rBPI23 and 58 nM for rLBP. rBPI23 was approximately 75-fold more potent than rLBP in inhibiting the binding of '25I-rLBP to lipid A. The binding affinity of rBPI23 (Kd = 70 nM) for Escherichia coli J5 bacteria was also significantly higher than that of rLBP (Kd = 1,050 nM). In addition, rBPI23 at 0.2 ,ug/ml was able to inhibit LPS-induced tumor necrosis factor release from monocytes in the presence of 20 ,ug of rLBP per ml. These results demonstrate that rBPI23 binds more avidly to endotoxin than does rLBP and that, even in the presence of a 100-fold weight excess of rLBP, rBPI23 effectively blocks the proinflammatory response of peripheral blood mononuclear cells to endotoxin.

Catalytic Properties of Lipopolysaccharide (LPS) Binding Protein

“…LBP Does Not Effectively Disaggregate LPS-LBP is known to bind immobilized LPS and stably coat the surface of LPScoated plastic plates (11,12), LPS-coated erythrocytes or whole Gram-negative bacteria (13). To determine if binding of LBP changes the aggregation state of LPS in solution, BODIPY-LPS aggregates were mixed with increasing concentrations of LBP, and fluorescence emission at 518 nm was measured.…”

“…9B). We have drawn this as an ordered ternary complex reaction with LBP binding to LPS micelles before sCD14 since LBP is known to bind LPS aggregates without sCD14 (11,12), but sCD14 does not bind LBP without LPS (13). Several lines of evidence indicate that LBP utilizes principally the ternary complex intermediate to transfer LPS to sCD14.…”

“…We have failed to observe monomeric LPS⅐LBP complexes in native gels of mixtures of LPS and LBP (5). Moreover, LBP binds with high affinity to LPS-coated surfaces (11,12), suggesting that disassociation is energetically unfavorable. The ternary complex model obviates the need for this unfavorable reaction.…”

Towards a rational development of anti‐endotoxin agents: novel approaches to sequestration of bacterial endotoxins with small molecules

“…That the recognition of LPS and subsequent binding by LPS-binding molecules does not necessarily result in inhibition of endotoxic activity can be adduced from the literature. For instance, LBP , an acute phase-reactant plasma protein synthesized by the liver under conditions of stress (Geller et al, 1993), and the neutrophil granule-derived (BPI) are highly homologous in their sequences (Tobias et al, 1988;Beamer et al, 1998), and bind LPS competitively (Gazzano-Santoro et al, 1994) with comparable affinities. Yet, whereas LBP 'opsonizes' LPS (Hailman et al, 1994), and presents it to CD14 (Yu and Wright, 1996;Schumann et al, 1994), initiating LPSinduced cellular activation processes (Gallay et al, 1994), the binding of BPI to LPS results in neutralization of endotoxic activity (Wilde et al, 1994;von der Möhlen et al, 1995).…”

Characterization of the structural elements in lipid A required for binding of a recombinant fragment of bactericidal/permeability-increasing protein rBPI23

“…BPI's potent bioactivities have been shown to reside in the amino-terminal portion of the holo-protein (20,27,28). A recombinant amino-terminal fragment derived from BPI, designated rBPI 23 , displays bactericidal and LPS-neutralizing properties similar to those of the 55-kDa holo-protein both in vitro (3,9,15,24,25,40) and in vivo (1,16,17,19). Furthermore, the binding properties of rBPI 23 and the holo-protein for lipid A and LPS are essentially identical (10), and rBPI 23 binds to an extensive panel of LPS isolated from clinically relevant gram-negative bacteria (2).…”