PPubMed5 Sep 2024Cited 2×

Cell-Int: a cell–cell interaction assay to identify native membrane protein interactions Assessing intercellular protein interactions

Thibaud Aymoz-Bressot, Marie Canis, Florian Meurisse, Anne Wijkhuisen, Benoit Favier, Guillaume Mousseau, Anne Dupressoir, Thierry Heidmann, Agathe Bacquin

Abstract

'Anne Wijkhuisen' 'Benoit Favier' 'Guillaume Mousseau' 'Anne Dupressoir' 'Thierry Heidmann' 'Agathe Bacquin'] A cell interaction assay to study membrane protein interactions in quasi-physiological settings, with multiple applications, from deciphering interaction networks to assessing therapeutic molecules.

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Cell-Int is a flow‑cytometry‑based assay that detects native membrane protein interactions on living cells and can evaluate blocking antibodies.

What’s new

i.Development of the Cell-Int cell‑cell interaction assay for membrane protein PPI detection.
ii.Validation of Cell-Int with known partners such as Juno/Izumo1 and PD‑1/PD‑L1.
iii.Application of Cell-Int to screen an ORF library and map HLA‑G isoform interaction networks.
iv.Demonstration that Cell-Int can quantify antibody inhibition using IC50 values.

How they did it

Unmodified membrane proteins are expressed in suspension 293‑F cells transfected with GFP or RFP markers. After co‑incubation, cell aggregates are identified as double‑positive events by flow cytometry. Competitive assays add blocking antibodies to determine IC50 values.

What they found

Juno/Izumo1 interaction Kd = 12.3 ± 0.2 µM (SPR).
PD‑1/PD‑L1 Kd = 8.2 ± 0.1 µM (SPR).
CD80/CTLA‑4 Kd = 0.42 ± 0.06 µM (SPR).
HLA‑G1/LILRB1 Kd = 2.0 ± 0.7 µM (SPR).
Cell‑Int detected significant increases for all tested virus‑receptor pairs (e.g., SARS‑CoV‑2 Spike/ACE2, MeV H/Nectin‑4).

Caveats

✕Background signal observed for Syncytin‑A due to endogenous Ly‑6E expression.
✕Affinity not determined (ND) for several tested pairs (e.g., Syncytin‑2/MFSD2).
✕Assay relies on overexpression, which may not reflect native protein levels.

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