An alpha II spectrin mutant peptide with unstable scaffold structure and increased sensitivity to calpain cleavage

A spontaneous missense mutation in the alpha II spectrin (aII) gene, replacing a highly conserved arginine 1098 with the glutamine (R1098Q), causes progressive neurodegeneration in heterozygous mutant mice. The molecular mechanism underlying this phenotype is unknown but the accumulation of 150kD aII breakdown products in brains of homozygous mutant embryos suggests an imbalance in the substrate level control of aII cleavage by calpains. This is further supported by in silico simulation predicting unmasked calpain target site and increased spectrin scaffold bending and flexibility of R1098Q mutant peptide. Here, using spectroscopic and in situ enzymatic techniques, we aimed at obtaining direct experimental support for the impact of R1098Q mutation on the aII stability and its propensity for calpain-mediated degradation. Thermal circular dichroism analyses performed on recombinant wildtype and R1098Q mutant aII peptides, composed of spectrin repeat 9-10 revealed that although both had very similar secondary structure contents, thermal stability curve profiles varied and the observed midpoint of the unfolding transition (Tm) was 5.5 degrees C lower for the R1098Q peptide. Yet, the dynamic light scattering profiles of both peptides closely overlapped, implying the same thermal propensity to aggregate. Calpain digestion of plate-bound aII peptides with and without added calmodulin revealed an enhancement of the R1098Q peptide digestion rate relative to WT control. In summary, these results support the unstable scaffold structure of the R1098Q peptide as contributing to its enhanced intrinsic sensitivity to calpain and suggest physiologic relevance of a proper calpain/spectrin balance in preventing neurodegeneration. (c) 2021 Elsevier Inc. All rights reserved.

Automated summary

A spontaneous missense mutation in the alpha II spectrin gene results in neurodegeneration in heterozygous mutant mice. The R1098Q mutation leads to a decrease in stability of aII peptide and increased sensitivity to calpain, highlighting the importance of maintaining a proper calpain/spectrin balance in preventing neurodegeneration.