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Periodic bilayer organization in the complexes of Beta-2 Glycoprotein I with anionic lipid membranes

https://doi.org/10.1016/j.colsurfb.2021.112118Get rights and content

Highlights

  • LUVs- β2 glycoprotein aggregates had Bragg diffraction peaks in SAXS experiment.

  • Contact zone were two bilayers and a small fraction of the total membrane area.

  • Contact zones between membranes have reduced fluctuations.

  • Data are consistent with β2 glycoprotein I making a bridge between two bilayers.

  • Bragg peaks were observed in membranes in the liquid phase and low ionic strength.

Abstract

β2 glycoprotein I (β2GPI) is a soluble protein that participates in blood coagulation, clearance of apoptotic bodies and generation of antigens in antiphospholipid syndrome among many other functions. We studied the aggregates formed by β2GPI with the anionic phospholipids palmitoyloleoylphosphatidyl glycerol, dimyristoylphosphatidyl glycerol, dipalmitoylphosphatidyl glycerol and cardiolipin using small angle X-ray scattering. The complexes obtained in a medium containing 0.01 M NaCl showed Bragg peaks up to the sixth order in a well-defined integer sequence indicating the presence of a lamellar stacking with a periodicity of 17.8 nm and with largely reduced membrane fluctuations. Modeling the complex signal allowed us to conclude that the coherence length was only two bilayers and that about 15% of the total surface was actually stacked. The space between bilayers allows accommodating an extended β2GPI molecule making a bridge between the interacting bilayers. The interactions between membranes mediated by β2GPI was favored when the membranes were in the liquid crystalline state.

Introduction

β2-Glycoprotein I (β2GPI, PDB ID 1QUB [1] and 1C1Z [2]) is a glycoprotein present in large amounts in human plasma at a concentration of about 0.2 mg/mL. It is a 54 kDa single-chain glycoprotein composed of four equal “sushi” domains or short consensus repeat (SCR) domains of 60 aminoacids (SCR domains I to IV) with the characteristic fold of the family and a fifth C-terminal domain, (domain V) of 82 aminoacids with a similar fold including an extra flexible and positively charged loop [1], [2].

β2GPI participates in the generation of the antigen in the anti-phospholipid autoimmune disease [3], [4] and in the regulation of blood coagulation, both as pro- and anticoagulant [4], [5]. It has been proposed that besides of these well-known activities, β2GPI participates in many other processes described in more than 2000 publications since the discovery in 1990 of its functions as antigen in the autoimmune disease [4]. Most of these functions and effects are closely related to the capacities of β2GPI to bind anionic molecules and interfaces and to produce aggregation of cells and of lipid vesicles [6], [7], [8], [9], [10], [11], [12]. Several studies have addressed basic questions about the aggregation of lipid vesicles and cells mediated by β2GPI or by the homolog protein from other mammals. Phase contrast microscopy of giant unilamellar vesicles have shown that a planar contact area is generated between vesicles aggregated by β2GPI [13]. The contact area and the contact angle increase with protein concentration and decreases with the ionic strength [14]. Necessarily, the flattening of the contact area must occur with deformation of the vesicles. Then, as expected, there is a relationship between the strength of protein binding, the deformability of the membrane and the final shape acquired by the aggregates. A quantitative relationship between the modulus of membrane deformability and binding of β2GPI was developed in the works by Kovačič et al. [15] and Šuštar et al. [16].

In a previous work we studied the aggregation of large unilamellar vesicles (LUVs) of anionic lipids by β2GPI. Macroscopic aggregates precipitate from the solution when β2GPI is added to anionic LUVs both in low ionic strength solution (absence of added salt) or in the presence of 0.1 M NaCl [17]. We have also shown that thermal unfolding of the membrane-bound β2GPI above 65 °C produces the disassembling of the aggregates yielding dispersed LUVs with unfolded membrane-bound protein. After cooling, these samples precipitate again and the membrane-bound protein recovers the native fold as shown by FTIR spectroscopy [17]. To further understand the long-range organization of the LUVs aggregates, in the present work we measured their small angle X-ray scattering (SAXS) in the search of repetitive patterns. We studied the β2GPI-mediated aggregates of anionic LUVs of dimyristoylphosphatidyl glycerol (DMPG), palmitoyloleoylphosphatidyl glycerol (POPG), dipalmitoylphosphatidyl glycerol (DPPG) and cardiolipin (CL) at several temperatures to study the organization of these aggregates.

Section snippets

Chemicals and sample preparation

β2GPI was purified from human plasma kindly donated by Banco de Sangre, Universidad Nacional de Córdoba. A precipitation with perchloric acid (70%, v/v) was followed by purification in heparin column according to the method of Polz et al. [18] as we described in [17].

POPG, DMPG, DPPG and CL were from Avanti Polar Lipids and used without further purification. Phospholipid membranes were prepared in 0.01 M phosphate buffer complemented with 0.01 M NaCl (low ionic strength) or 0.1 M NaCl (high

Interactions with POPG

Fig. 1 shows the SAXS pattern of extruded vesicles (LUVs, 100 nm pore size filter) of POPG 1 mM. The same results were obtained with membranes extruded in 400 and 1000 nm pore size filters. The results are expressed as I(q) (intensity) as a function of the scattering vector modulus q, (see above). The slope at low q and the bumps at higher q values, both consistently indicated lamellar structures. The lack of clear diffraction peak (as compared with Fig. 2, see below) indicated that the

Conclusions

We showed that the aggregation of anionic lipid membranes by β2GPI occurs with apposition of membranes that can be detected by diffraction patterns in a SAXS experiment. The contacts between correlated membranes that produce a diffraction pattern corresponds mainly to a periodic structure with only two bilayers compromising only a small proportion of the total lipid area. These structures are largely favored in conditions of low ionic strength and lipids in the liquid crystalline phase. The

CRediT authorship contribution statement

Oliveira RG: performed and designed SAXS experiments, performed calculations; interpreted and discussed results, wrote the paper.

Paolorossi M: prepared protein samples.

Cavalcanti L: conducted SAXS experimets, supported the fine working of SAXS line.

Malfatti-Gasperini A: Interpreted and discussed SAXS results, performed fitting calculations.

Montich GG: prepared samples, performed experiments, discussed results, wrote the paper.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported with grants from Secretaría de Ciencia y Técnica UNC (SECyT UNC), Argentina, Fondo para la Investigación Cientícfica y Tecnológica, (ANPCyT) Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas Consejo Nacional de Investigaciones Científicas y Técnicas CONICET, Argentina (CONICET). We thanks to Laboratorio Nacional de Luz Sincrotron (beamline SAXS2), Campinas, Brazil.

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