Fig. 2: In vitro effect of the Nbs on GCase activity and thermal stability (Tm).
From: Developing nanobodies as allosteric molecular chaperones of glucocerebrosidase function
A Schematic representation of the protocol for the in vitro 4-MU GCase activity assay; B Velaglucerase activity in the presence of each of the 20 Nbs showed that a subset of Nbs is able to significantly improve in vitro GCase enzymatic activity. Isofagomine (IFG, 25 µM) was used as a negative control (n = 6 replicates in three independent experiments, data represented as mean ± SEM, statistical analysis was performed using an Ordinary One Way Anova multiple comparison test, DF Nbs = 21, DF residual = 110, F value = 18.08); C GCase activity assay in cellular lysates in the presence of each of the 20 Nbs showed that a subset of Nbs is able to significantly improve in vitro GCase enzymatic activity in the cellular lysates, quite coherently with the impact of Nbs on the Velaglucerase activity. Isofagomine (IFG, 25 µM) was used as a negative control (n = 5 replicates in three independent experiments, data represented as mean ± SEM, statistical analysis was performed using an Ordinary One Way Anova multiple comparison test, DF Nbs = 21, DF residual = 88, F value = 47.54); D Thermal unfolding curves of GCase, obtained using a thermal shift assay at pH 7.0 (black line) and pH 5.2 (red line) in absence or presence of Nb1. E Overview of the results of the TSA assay for the full set of 20 Nbs. The plotted variation of Tm (ΔTm) is the difference between the Tm of GCase and GCase + Nb. The covalent inhibitor conduritol-β-epoxide (CBE) was used as positive control, and an irrelevant nanobody (Irr Nb) as negative control. Each TSA signal is the result of three independent experiments, shown as mean values (bars) with standard deviations (error bars). Source data are deposited as Source Data files on Zenodo. GCase, glucocerebrosidase; Nb, nanobody.
