Lipopolysaccharide (LPS)-binding Proteins BPI and LBP Form Different Types of Complexes with LPS

Abstract

Lipopolysaccharide (LPS)-binding protein (LBP) andbactericidal/permeability-increasing protein (BPI) are closely related LPS-binding proteins whose binding to LPS has markedly different functional consequences. To gain better insight into the possible basis of these functional differences, the physical properties of LBP-LPS and BPI-LPS complexes have been compared in this study by sedimentation, light scattering, and fluorescence analyses. These studies reveal dramatic differences in the physical properties of LPS complexed to LBP versus BPI. They suggest that of the two proteins, only LBP can disperse LPS aggegates. However, BPI can enhance both the sedimentation velocity and apparent size of LPS aggregates while inhibiting LPS-LBP binding even at very low (1:40 to 1:20) BPI:LPS molar ratios.The lipopolysaccharide (LPS)-binding protein 1 (LBP) and the bactericidal/permeability-increasing protein (BPI) are both LPS-interactive mammalian proteins with approximately 45% amino acid sequence identity (1, 2). LPS is considered to be the principal component of Gram-negative bacteria that alerts the host to invading bacteria and triggers defensive responses (3, 4). These responses are usually beneficial and effective but may also become excessive and lead to endotoxic shock (3-5). Both LBP and BPI modulate the bioactivity of LPS (2,3,5). LBP is a plasma protein that catalyzes the transfer of LPS from LPS aggregates to other LPS-binding proteins (3, 6 -9). Prominent among these is CD14, a surface molecule of myeloid cells that is also present in the circulation as a soluble protein. LBP and CD14 together represent the main pathway by which cells recognize low concentrations of LPS and are stimulated to respond to 10,11). In contrast to the LPS-stimulatory properties of LBP, binding of LPS by BPI results in inhibition of the bioactivities of LPS (2). BPI is produced by polymorphonuclear leukocytes and stored in its azurophilic granules (12, 13). It contributes substantially to both the intracellular and extracellular antibacterial activity of polymorphonuclear leukocyte-rich inflammatory exudates toward Gram-negative bacteria (14, 15). The high affinity of BPI for LPS accounts for the target-cell specificity of the antibiotic activity of BPI for Gram-negative bacteria (2, 16). In contrast to BPI, binding of LBP to bacterial envelope LPS does not produce detectable membrane alterations or other antibacterial effects.We have recently described the use of fluorescein-labeled LPS (FITC-LPS) as well as metabolically labeled ([ 3 H]LPS) and sucrose density gradients to characterize the formation and physical properties of complexes of LPS with LBP and the soluble form of CD14 (7). In this report we describe results obtained by studying the binding of FITC-LPS and [ 3 H]LPS to BPI using the techniques described earlier and in addition compare the effects of LBP and BPI on the light scattering properties of LPS. The results obtained reveal striking differences in the physical properties of LBP-LPS and BPI-LPS complexes. Thus, un...

Structure-Function Analysis of CD14 as a Soluble Receptor for Lipopolysaccharide

“…4B). We interpret these data as we have in the past (32,34); sCD14 has dissociated the large LPS aggregates by forming complexes containing one or a few LPS molecules/sCD14. The complexes of LPS with the PQPD, DDED/PQPD, and AVEVE deletion mutants (data not shown) all resembled LPS⅐wild-type CD14 complexes (Fig.…”

“…For example, both LBP and bactericidal/permeability increasing protein bind FITC-LPS with an increase in fluorescence similar to those seen in Fig. 3, but LPS complexed with bactericidal/permeability increasing protein forms aggregates, and LPS complexed with LBP is dispersed (34). To address the aggregation state, sucrose density gradient experiments were done to evaluate the sedimentation velocity of LPS complexes with mutant CD14.…”

“…The estimated apparent K D of the CD14/LPS interaction for the deletion mutant varied from 2 ϫ 10 Ϫ9 (DDED/PQPD) to Ͻ10 Ϫ6 (DPRQY). Although the fluorescence assay allows us to measure affinity of sCD14 for LPS, results obtained with LBP and bactericidal/permeability increasing protein show that the assay does not report an LPS aggregation state (34).…”

Molecular Cloning and Characterization of spurt, a Human Novel Gene That Is Retinoic Acid-inducible and Encodes a Secretory Protein Specific in Upper Respiratory Tracts

“…BPI has been implicated in bactericidal processes (53)(54)(55)(56)(57). The presence of a BPI-like domain in spurt protein suggests an antibacterial role for this novel protein.…”

Identification of a novel left–right asymmetrically expressed gene in the mouse belonging to the BPI/PLUNC superfamily

“…This superfamily consists of seven PLUNC proteins (LPUNC1-4 and SPLUNC1-3) and four plasma lipid transfer/lipopolysaccharide binding proteins, including bactericidal/permeability-increasing protein (BPI), lipopolysaccharide binding protein (LBP), cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP). BPI, LBP, PLTP, and CETP are involved in the response to the bacterial and lipid transport in plasma, through binding and transfer lipopolysaccharides, phospholipids, and cholesteryl esters (Beamer et al, 1997(Beamer et al, , 1999Tobias et al, 1997;Bruce et al, 1998;Yamashita et al, 2001). The function of PLUNC proteins is unclear, but they are hypothesized to be involved in host defense against bacteria, through their BPI-related Nterminal domain (Sung et al, 2002).…”