Fig. 4: Disruption of alpha globin/eNOS interaction through the deletion of four residues in Hba1.

a A colorized crystallographic model of alpha globin (from PDBid: 1Z8U) showing the residues previously shown to interact with eNOS (blue) surrounding four residues deleted in the Hba1^WT/Δ36–39 mouse model (magenta). The sequences for the proteins encoded by the Hba1^WT and Hba1^Δ36–39 alleles, including the deleted residues (magenta), are shown in the box below. b Using a guide RNA design (blue) targeting the eNOS binding region (green) resulted in an in-frame deletion of the nucleotides boxed in pink. This is confirmed by the chromatogram in c, which shows that the 12-nucleotide deletion scrambles downstream reading of the nucleotide sequence in NGS protocols. d The mutant protein is expressed, as seen in immunoprecipitation-coupled mass spectrometry. A peak corresponding to a protein with a molecular weight ~400 Da less than that of the dominant species is seen in hemoglobin captured from lysed red blood cells. e–g The blood hemoglobin content (e), hematocrit (f), and the number of red blood cells (g) are shown for the Hba1^WT/WT and Hba1^WT/Δ36–39 groups. h Results of fluorescence polarization assays for determining the binding of the mutant allele to eNOS, using an alpha globin mimetic peptide known to bind (top) and the Δ36–39 peptide (bottom). No binding affinity could be calculated for the Δ36–39 peptide. The points are centered on the mean value of three measurements per concentration, and the error bars represent the standard deviation of the triplicate measurements. These results are representative of n > 5 individual experiments. i The proximity ligation assay (PLA) signal (red puncta, marked by arrows) highlights the close localization of alpha globin and eNOS. Nuclei appear blue (with DAPI staining), the autofluorescence of the internal elastic lamina appears green, and the lumen of the vessel is indicated by an asterisk (*). The scale bar represents 15 μm. j Endothelial PLA signals, normalized to the length of the internal elastic lamina, were reduced in the Hba1^WT/Δ36–39 mice. The difference was significant when these mice were compared to the Hba1^WT/WT mice, but not when they were compared to IgG staining controls. For the experiments in e–g, n = 6 for Hba1^WT/WT mice and n = 5 for Hba1^WT/Δ36–39 mice were used. For the experiments in j, n = 3 mice of each genotype were used, and the significance of differences was determined by a one-sided t test with multiple comparisons correction. Bar graphs are centered on mean with error bars denoting standard error. Source data are provided in the Source data file.