Biophysical analysis of the interaction of the serum protein human β2GPI with bacterial lipopolysaccharide

Abstract

There are several human serum proteins for which no clear role is yet known. Among these is the abundant serum protein beta2-glycoprotein-I (β2GPI), which is known to bind to negatively charged phospholipids as well as to bacterial lipopolysaccharides (LPS), and was therefore proposed to play a role in the immune response. To understand the details of these interactions, a biophysical analysis of the binding of β2GPI to LPS and phosphatidylserine (PS) was performed. The data indicate only a moderate tendency of the protein (1) to influence the LPS-induced cytokine production in vitro, (2) to react exothermally with LPS in a non-saturable way, and (3) to change its local microenvironment upon LPS association. Additionally, we found that the protein binds more strongly to phosphatidylserine (PS) than to LPS. Furthermore, β2GPI converts the LPS bilayer aggregates into a stronger multilamellar form, and reduces the fluidity of the hydrocarbon moiety of LPS due to a rigidification of the acyl chains. From these data it can be concluded that β2GPI plays a role as an immune-modulating agent, but there is much less evidence for a role in immune defense against bacterial toxins such as LPS.

Keywords: Cytokine production; FRET, fluorescence resonance energy transfer spectroscopy; FTIR, Fourier-transform infrared spectroscopy; HDL, high-density lipoproteins; Human glycoprotein β2GPI; ITC, isothermal titration calorimetry; Immune modulation; LAL test; LAL, Limulus amebocyte lysate; LBP, lipopolysaccharide-binding protein; LDL, low-density lipoproteins; LPS, lipopolysaccharides; Lipopolysaccharide; MNC, mononuclear cells; PC, phosphatidylcholine; PS, phosphatidylserine; SAXS, small-angle X-ray scattering; β2GPI, beta2-glycoprotein-I.

Fig. 1

Secretion of tumor-necrosis-factorα from human mononuclear cells induced by lipopolysaccharide LPS Ra, and LPS:β₂GPI mixtures. The error bar results from the determination of TNFα in an ELISA in duplicate.

Fig. 2

(A) Trp-fluorescence emission spectra of β₂GPI/LPS at a protein concentration of 1.1 μM and in the presence of increasing concentrations of LPS varying from 0 to 40 mol LPS/mol protein. Excitation wavelength was 292 nm. The results are presented in a mesh plot. (B) Changes in the emission maximum of Trp-fluorescence of β₂GPI (1.1 μM) in the presence of lipid vesicles as a function of lipid concentration for LPS (●) and egg-PC (○).

Fig. 3

Quenching of the intrinsic Trp fluorescence of β₂GPI by acrylamide. The protein concentration was set to 1.1 μM. The acrylamide concentration was increased to 0.45 M. The Stern–Volmer plot is shown for β₂GPI in the absence (▴) and in the presence of 20 mol LPS/mol protein (●).

Fig. 4

Gel to liquid crystalline phase transition of the acyl chains of LPS Ra in the absence and presence of β₂GPI at [LPS]:[β₂GPI] 10:1 weight%. Shown is the peak position of the symmetric stretching vibration of the methylene groups ν_s(CH₂) versus temperature. In the gel phase, the position lies around 2850.5, in the liquid crystalline phase at 2852.5–2853 cm⁻¹.

Fig. 5

Infrared vibrational spectra in the range of the amide I band (predominantly CO stretching vibration). The position of the peak maxima can be assigned to different secondary structures due to different water binding, i.e., α-helix between 1650 and 1655 cm⁻¹, β-sheets between 1625 and 1640 cm⁻¹, and a particular helical structure 3₁₀-helix between 1637 and 1643 cm⁻¹.

Fig. 6

Small-angle X-ray scattering (SAXS) of a [LPS]:[β₂GPI] 1:4 weight% dispersion at 20–80 °C. The logarithm of the scattering intensity log I is plotted versus the scattering vector s (=1/d, d = spacings of the reflections).

Fig. 7

Isothermal titration calorimetric experiments of LPS protein mixtures. To a LPS dispersion (1 mM), β₂GPI (2 mg/ml) is titrated in 3 μl portions. Measurements were done at 37 °C. Exothermic processes lead to negative, endothermic processes to positive enthalpy changes ΔH_c.

Fig. 8

Förster resonance energy transfer spectroscopy (FRET) of mixtures from doubly labeled 0.01 mM phosphatidylcholine (A), phosphatidylserine (B) and LPS Ra (C) with β₂GPI (10 μM) added after 50 s. The FRET signal I_D/I_A is a sensitive measure of incorporation of the protein into the lipids.