Catalytic Properties of Lipopolysaccharide (LPS) Binding Protein: TRANSFER OF LPS TO SOLUBLE CD14

Bo Yu,Samuel D Wright

doi:10.1074/jbc.271.8.4100

Abstract

Lipopolysaccharide (LPS) binding protein (LBP) is a lipid transfer protein that catalyzes transfer of LPS monomers from micelles to a binding site on soluble CD14 (sCD14) and transfer of LPS from LPS.sCD14 complexes to HDL particles. To characterize the first of these two reactions, LPS covalently derivatized with the fluorophore, boron dipyrromethene difluoride (BODIPY), was used to monitor LBP-catalyzed movement of LPS in real time. The fluorescence efficiency of micelles of BODIPY-LPS was low but was strongly increased upon dissolution in detergent or upon binding to sCD14. Spontaneous binding of BODIPY-LPS to sCD14 was very slow but was accelerated by substoichiometric concentration of LBP, and the rate of binding was measured under a variety of conditions. LBP-catalyzed transfer was first order with respect to both sCD14 and LPS concentration, and the apparent Km values were 1 approximately 2 microg/ml for sCD14 and 100 ng/ml for LPS. The maximum turnover number for LBP was approximately 150 molecules of LPS min-1 LBP-1. LBP alone caused a small but measurable increase in the fluorescence of BODIPY-LPS, suggesting that it bound LPS aggregates but did not readily remove LPS monomers. The subsequent addition of sCD14 caused a large fluorescence increase, suggesting transfer of BODIPY-LPS to sCD14. These and other observations suggest that LPS is transferred by an ordered ternary complex reaction mechanism in which LBP transfers LPS monomer from LPS aggregates to sCD14 without dissociating from the LPS aggregate.

Highlights

Lipopolysaccharide (LPS) binding protein (LBP) is a lipid transfer protein that catalyzes transfer of LPS monomers from micelles to a binding site on soluble CD14 and transfer of LPS from LPS1⁄7sCD14 complexes to HDL particles
The Fluorescence Spectrum of boron dipyrromethene difluoride (BODIPY)-LPS Reports Its Aggregation State—BODIPY is an efficient, photostable fluorescent probe whose emission spectrum depends on the local concentration of fluorophore
The emission spectrum of BODIPY-LPS in PD (Fig. 1A) showed a broad peak at 620 nm, indicative of a high local concentration of fluorophore. This observation indicates that the BODIPY in our preparations is aggregated, most likely because it is coupled to LPS, and the BODIPY-LPS exists in micelles or aggregates

Summary

Introduction

Lipopolysaccharide (LPS) binding protein (LBP) is a lipid transfer protein that catalyzes transfer of LPS monomers from micelles to a binding site on soluble CD14 (sCD14) and transfer of LPS from LPS1⁄7sCD14 complexes to HDL particles. The subsequent addition of sCD14 caused a large fluorescence increase, suggesting transfer of BODIPY-LPS to sCD14 These and other observations suggest that LPS is transferred by an ordered ternary complex reaction mechanism in which LBP transfers LPS monomer from LPS aggregates to sCD14 without dissociating from the LPS aggregate. As a first step in obtaining the catalytic constants for the above three reactions, we have prepared a fluorescent derivative of LPS that exhibits a large spectral shift upon movement from a micelle to sCD14, and we have used this compound to measure the turnover number for the LBP-catalyzed binding of LPS to sCD14 as well as a Km for each reactant. Our studies suggest that the transfer reaction proceeds via an ordered ternary complex of LPS, LBP, and sCD14

Methods

Results

Conclusion