Intermediate Filament Assembly Characteristics of Lens CP49 Mutants with Type II cytokeratins

Abstract

The lens-specific Intermediate Filament protein CP49 is a highly divergent Type I cytokeratin. However, unlike other Type I cytokeratins, CP49 fails to co-assemble into 10 nm filaments with Type II keratins. Site directed mutagenesis and motif swapping were used to determine the role of specific CP49 motifs in permitting/restricting assembly with the type II cytokeratin, K8. Mutagenesis revealed that “correction” of highly divergent CP49 helix initiation and termination motifs to conserved type I cytokeratin sequences did not facilitate normal filament assembly. Chimeric CP49 and K18 proteins did however show that large regions of CP49 outside the rod domain remain assembly competent with K8 despite sequence divergence from K18. These experiments also showed that the highly conserved helix initiation motifs of the Type I (LNDR), and Type II (LNNK) proteins can be mutated to the wild-type CP49 sequence LGGC and still form normal intermediate filaments in vitro. This allowable substitution seemingly rescues the deleterious helix initiation R to C mutation that has been previously associated with severe keratinopathies in other type I cytokeratins.