Localization of the Lipopolysaccharide-binding Protein in Phospholipid Membranes by Atomic Force Microscopy

Ulrich Seydel,Thomas Gutsmann,Stefanie Roes,Florian Mumm

doi:10.1074/jbc.m507634200

Ulrich Seydel, Thomas Gutsmann

Open Access

https://doi.org/10.1074/jbc.m507634200

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Abstract

Lipopolysaccharides (LPS; endotoxin) activate immunocompetent cells of the host via a transmembrane signaling process. In this study, we investigated the function of the LPS-binding protein (LBP) in this process. The cytoplasmic membrane of the cells was mimicked by lipid liposomes adsorbed on mica, and the lateral organization of LBP in these membranes and its interaction with LPS aggregates were characterized by atomic force microscopy. Using cantilever tips functionalized with anti-LBP antibodies, single LBP molecules were localized in the membrane at low concentrations. At higher concentrations, LBP formed clusters of several molecules and caused cross-linking of lipid bilayers. The addition of LPS to LBP-containing liposomes led to the formation of LPS domains in the membranes, which could be inhibited by anti-LBP antibodies. Thus, LBP mediates the fusion of lipid membranes and LPS aggregates.

Highlights

The cell wall of Gram-negative bacteria consists of the cytoplasmic and an additional outer membrane
Our group provided evidence for binding of LPS-binding protein (LBP) to and intercalation into lipid membranes composed of negatively charged phosphatidylserine (PS) or zwitterionic phosphatidylcholine (PC) and in LPS aggregates by using fluorescence resonance energy transfer and surface plasmon resonance experiments [10]
It is likely that LBP is present as an acute phase protein in serum and assumes a transmembrane configuration in the cytoplasmic membrane of immune cells and in this configuration acts as fusion protein for LPS aggregates

Summary

Introduction

The cell wall of Gram-negative bacteria consists of the cytoplasmic and an additional outer membrane. When released from the bacterial surface into the blood circulation of the host, LPS plays an important role in the pathogenesis and manifestation of Gram-negative inflammation, in general, and of septic shock, in particular. Our group provided evidence for binding of LBP to and intercalation into lipid membranes composed of negatively charged phosphatidylserine (PS) or zwitterionic phosphatidylcholine (PC) and in LPS aggregates by using fluorescence resonance energy transfer and surface plasmon resonance experiments [10]. The AFM [16] allows us to acquire the surface topography of biological samples under native conditions in buffer with high resolution [17, 18]

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