Fig. 1: Development of chiral-selective antibodies to detect heterochirality in vivo.

a Schematic representation of Pimt enzymatic activity and experimental design to assess the physiological effect of loss of protein homochirality. Animals lacking Pimt activity accumulate heterochiral proteins due to the lack of correction of post-translational chiral errors. For this and all subsequent figures, light blue shading highlights homochiral and light red shading highlights heterochiral animals. b Detection of hetero-neoepitopes in control and Pimtⁿ¹ chiral-deficient 20 days-old animals. The anti-DEVdG, anti-DEVβDG, and anti-DEVβdG antibodies (where d corresponds to d-aspartate, βD to iso-l-aspartate and βd to iso-d-aspartate) were designed to recognise hetero-neoepitopes in the proteome containing d-, β-l-, and β-d-AA. As a control, anti-DEVDG was raised against a homochiral peptide. Dissected adult female guts were stained with anti-DEVDG (blue), anti-DEVdG (red), anti-DEVβDG (green), or anti-DEVβdG (yellow) antibodies. Guts of control animals did not show any immunoreactivity, while Pimtⁿ¹ showed positive staining for the DEVdG-specific and DEVβDG-specific antibodies. c Quantification of normalised signal intensities of the four chiral-specific immunostainings shown in panel b. Experiments were repeated at least three independent times. n number of animals. Comparison of normalised signal intensities were done with One-way ANOVA test. P values for these and all subsequent statistical tests are defined as ****p < 0.0001; 0.0001 < ***p < 0.001; 0.001 < **p < 0.01; 0.01 < *p < 0.05; and not significant (ns) 0.05 < p and ns = 0.9783, *p = 0.02. d Pimt methyltransferase activity was assayed on the homochiral and heterochiral synthetic substrates Ac-KRKGDEVDGVDEVAK-amide and Ac-KRKGDEVβDGVDEVAK-amide (βD corresponds to iso-l-aspartate), respectively. The sequence corresponds to the human PARP1 fragment outlined in the in vivo cleavage assay (Fig. 4a–d). The calculated methyltransferase activity at 37 °C on the linear portion of the curve is 0.06039 μmol/min/ml. Experiments were repeated three independent times. Values are presented as average ± standard deviation (S.D.). Source data are provided as a Source Data file for c and d.