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A real‐time cell‐binding assay reveals dynamic features of STxB–Gb3 cointernalization and STxB‐mediated cargo delivery into cancer cells

Crispim Encarnação J, Napolitano V, Opassi G, Danielson HU, Dubin G, Popowicz GM, Munier‐Lehmann H, Buijs J, Andersson K, Björkelund H.

FEBS Letters. 2020. Epub ahead of print.

Summary

This paper investigates Shiga toxin B‐subunit (STxB) and its capacity to carry small molecules and proteins into cells. First, the specificity of STxB to the Gb3 receptor was confirmed with Gb3 positive Daudi cells and Gb3 negative K562 cells. Both cell lines were attached with the suspension cell protocol for LigandTracer, using BAM.

For Daudi cells, as well as Ramos and HT-29 cells, a biphasic behavior of the STxB – Gb3 interaction was observed, with a fast initial binding and a slow linear binding, regardless of if STxB was labeled with FITC or connected to the much larger eGFP. It was found that the linear phase was the result of STxB internalization, which was affected by surrounding temperature. The internalization rate increased with higher STxB concentrations, indicating that more receptors became accessible or available for STxB with higher concentrations. This was also observed in an displacement assay, where the presence of non-labeled STxB did not reduce the uptake of labeled STxB as would have been expected, but rather the opposite: the signal increased because STxB binding induced clustering and subsequent internalization. The cluster effects were further analyzed with quencher conjugated STxB.