In patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) several groups recently identified mutations in the DES gene. Desmin is a 53 kDa highly conserved muscle-specific intermediate filament (IF) protein. In many cases, these mutations result in a loss of the filamentous network due to abnormal aggregation of desmin. However, little is known about the molecular patho-mechanisms of ARVC-associated desmin mutations. Recently we and others published the ARVC-related DES-mutations p.N116S and p.N342D, which lead to desmin aggregates. Currently it is unknown, if the loss of the asparagine residues or the gains of new functionalities are causative for desmin aggregation. We therefore constructed model variants of the corresponding asparagine-residue to get insight into the molecular side chains, which are responsible for desmin filament formation defects.Methods
We generated the different desmin model mutants (p.N116S/A/D/Q/T and p.N342D/A/Q/E) by side-directed mutagenesis and investigated the IF-formation in different transfected cell lines by fluorescence microscopy.Results
By generation of different model mutants for the ARVC-associated DES-mutations p.N116S and p.N342D we found, that the impact on filament assembly of both asparagine-residues is different. All substitutions at the position 116 caused massive desmin aggregation indicating a key role of the amino acid N116 for proper desmin filament formation. Even the elongation of the side chain (p.N116Q) induced desmin filament formation defects. In contrast, substitution of N342 against alanine, glutamine and glutamate led to filamentous networks in transfected cells. These results indicate specific pathogenic filament assembly defects caused by the aspartate-residue.Conclusion
In summary we found that any substitution of N116, belonging to the IF consensus motif ‘LNDR’ leads to a loss of function mutation with aggregate formation. We suggest that the amide group and its position of N116 are absolutely necessary for IF-formation. In contrast N342 can be replaced by several other amino acids but not by aspartate. Probably, the ARVC-related mutant p.N342D is a gain of function mutation.