Figure 7
R1098Q spectrin exhibits reduced in vitro CaM affinity and enhanced in vivo calpain cleavage. (a) Surface plasmon resonance detected the binding of Ca2+/CaM to recombinant GST-spectrin peptides representing repeats 8–11 (as shown in Fig. 1). The concentration of CaM used for each curve is as indicated. The binding curves fit well to a 1:1 Langmuir binding model over the indicated range of ligand concentrations (dotted lines, generated by BIAevaluation 3.1, https://www.biacore.com/lifesciences/service/downloads/software_licenses/biaevaluation ). The derived rate constants for (ka) and (kd) and their confidence limits as reported by the BIAevaluation software were both reduced for the R1098Q peptide relative to the WT peptide. The derived KD for the mutant peptide of 3.8 ± 0.3E−09 represents a significant 19% reduction in its affinity for CaM. (b) To determine if spectrin was being excessively targeted by calpain in vivo, we examined the cerebellum of 26 week old mice with an antibody raised to the unique epitope created by calpain cleavage of spectrin at Y11769. This antibody only detects calpain-cleaved spectrin. No staining was detected in the cerebellum of either of two WT animals. Conversely, their respective R1098Q heterozygous littermates (two animals) at 26 weeks of age displayed calpain-cleaved spectrin in 5–18% of their PC soma and throughout the apical dendrite. Cleavage product was also detected in the granular cell layer (arrows); the origin of this staining in the granular layer (whether cells or synapses with sBDPs) is undetermined. (c) Embryos (14.5 d) were harvested in-utero and analysed by Western blotting with anti-αII spectrin antibodies to gauge the level of spectrin proteolysis. While break-down was minimal or absent in both the WT and heterozygous embryos, a single sBDP at ≈150 kDa was prominent only in the homozygotes. This sBDP is the major product generated by calpain9. The characteristic cleavage fragment generated by caspase at ≈ 120 kDa during apoptosis74 was notably absent. These results indicate that the R1098Q spectrin is uniquely sensitive to calpain attack in the homozygous embryo (N = 2 WT; 2 het; and 3 homozygotes).
