Biomedical Chemistry: Research and Methods 2023, 6(4), e00206

β1-Adrenergic Receptor Solubilized in the Form of Nanodiscs: Screening of Various Amphipatic Polymers

M.M. Peclo, L.N. Lipatova, N.M. Kashirina, T.V. Sharf, I.B. Kuznetzova, E.E. Efremov, E.V. Yanushevskaya, P.N. Rutkevich, I.N. Rybalkin, T.N.Vlasik*

National Medical Research Center of Cardiology named after Academician E.I.Chazov , 15a Academician Chazov str., Moscow, 121552 Russia; *e-mail: tanya.vlasik@gmail.com

Keywords:autoantibodies to β1-adrenergic receptor; nanodiscs; amphipatic polymers

DOI:10.18097/BMCRM00206

The whole version of this paper is available in Russian.

The development of a reliable and easily used diagnostic test for measuring autoantibodies to β1-adrenergic receptor (β1ADR Ab) in patient blood is an unmet clinical need. The enzyme-linked immunosorbent assay (ELISA) is considered as the most appropriate method for this task. In ELISA, the use of peptides corresponding to various fragments of amino acid sequence of β1ADR as antigens leads to inadequate results as β1ADR Ab appear to recognize conformationally dependent epitopes that are generated during the formation of unique tertiary structure of the receptor. Isolation of β1ADR preserving the native conformation and functional characteristics is a quite challenging task. A promising approach to address this task is the use of amphipatic polymers capable of forming nanodiscs, which permits to successfully solubilize membrane proteins. Here, we describe the results of testing 17 various amphipatic polymers with the aim to obtain the preparations of solubilized β1ADR that can be used as antigens in ELISA. The best relative solubilization value (RSV) were demonstrated by UltrasoluteTM Amphipol 17 (87%) and 18 (62%), as well as by AASTY 11-45 (76%), 11-50 (77%) and 6-50 (78.5%).
Figure 1. Binding of specific antibodies  hAB2367 to β1ADR as measured by optical density (OD) at 450 nm versus the amount of MF (membrane fraction) and MF+Polymer preparations added to a well (see Procedure, Enzyme-linked immunosorbent assay). A, B, C, D stand for MF+Polymer preparations with polymer concentrations 0.5%, 0.25%, 0.125% and 0.0625%, respectively. MF stands for a preparation of membrane fraction. The results of ELISA are provided for polymers with high (UltrasoluteTM Amphipol 17), moderate (AASTY 11-55) and low (DIBMA 10) relative solubilization value (RSV).

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Table 1. Relative solubilization value (RSV) at various polymer concentrations. Ultr Amph – UltrasoluteTM Amphipole.

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Table 2. Maximum RSV as function of molecular weight (MW) and structural features of polymers.  S:MA – styrene:maleic acid ratio. S:MALEI – styrene:maleimide ratio. DIB:MA – diisobutylene:maleic acid ratio. AA:STY – acrylic acid:styrene  ratio. X:Y – ratio of hydrophobic to hydrophylic units in polymer. Ultr Amphipole – UltrasoluteTM Amphipole.

FUNDING

This work is financed by the Ministry of Health of the Russian Federation (state assignment on the subject No. 152 “The development of the enzyme-linked immunosorbent assay for measuring autoantibodies to β1-adrenergic receptor in cardiac patients with a purified recombinant β1-adrenergic receptor in native conformation as an antigen”, Ref. No. NIOKTR 122020400212-0) and “Mona Ltd.” (Moscow, OGRN 5087746251896).

Supplementary materials are available at http://dx.doi.org/10.18097/BMCRM00206

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