Figure 3.

Clustered ASPRV1 Mutations Generate a Novel ASPRV1 Auto-cleavage Product and Result in Accumulation of High-Molecular-Weight Forms of Filaggrin

(A) A model of the ASPRV1 active enzyme crystal structure⁹ (ribbon and stick rendering on left, surface rendering on right) shows that all three pathogenic mutation sites (yellow) are tightly clustered within the three-dimensional protein. The catalytic aspartate is shown in green.

(B and C) Human keratinocytes (HK), either nontransduced or transduced with wild-type or mutant ASPRV1, were switched to high calcium (1.2 mM) for 8 days to promote differentiation. Immunoblotting of protein extracts was performed with anti-actin (Sigma SAB4200248, 1:2,000) as a loading control on replicate blots to show equivalent protein loading across all samples. (B) Immunoblotting with an anti-HA antibody (Cell Signaling 3724, 1:500) shows bands at ~37 kDa and ~28 kDa in cells expressing either wild-type or mutant forms of HA-tagged ASPRV1. In addition, cells expressing each of the ASPRV1 mutant forms show a unique ~20 kDa band not seen in nontransduced cells or cells expressing wild-type ASPRV1 (arrow). (C) Immunoblotting with an anti-filaggrin antibody directed against the N-terminal domain (residues 1–261, Thermo Fisher Scientific PA5-79267, 1:500) shows profilaggrin and higher molecular weight filaggrin cleavage products within differentiated keratinocytes. In nontransduced keratinocytes and keratinocytes transduced with wild-type ASPRV1, there is a low quantity of high-molecular-weight filaggrin products, consistent with normal processing of filaggrin to filaggrin repeat monomers. Cells expressing mutant ASPRV1 (encoding p.Lys199Glu, p.Arg311Pro, or p.Pro314Thr) demonstrate an accumulation of high-molecular-weight filaggrin products, suggesting that ASPRV1 mutations impair filaggrin processing.