Identifying the role of specific motifs in the lens fiber cell specific intermediate filament phakosin

Abstract

Purpose: Phakosin and filensin are lens fiber cell-specific intermediate filament (IF) proteins. Unlike every other cytoplasmic IF protein, they assemble into a beaded filament (BF) rather than an IF. Why the lens fiber cell requires two unique IF proteins and why and how they assemble into a structure other than an IF are unknown. In this report we test specific motifs/domains in phakosin to identify changes that that have adapted phakosin to lens-specific structure and function.

Methods: Phakosin shows the highest level of sequence identity to K18, whose natural assembly partner is K8. We therefore exchanged conserved keratin motifs between phakosin and K18 to determine whether phakosin's divergent motifs could redirect the assembly of chimeric K18 and K8. Modified proteins were bacterially expressed and purified. Assembly competence was assessed by electron microscopy.

Results: Substitution of the phakosin helix initiation motif (HIM) into K18 does not alter assembly with K8, establishing that the radical divergence in phakosin HIM is not by itself the mechanism by which IF assembly is redirected to BF assembly. Unexpectedly, K18 bearing phakosin HIM resulted in normal IF assembly, despite the presence of an otherwise disease-causing R-C substitution, and two helix-disrupting glycines. This disproves the widely held belief that mutation of the R is catastrophic to IF assembly. Additional data are presented that suggest normal IF assembly is dependent on sequence-specific interactions between the IF head domain and the HIM.

Conclusions: In the lens fiber cell, two members of the IF family have evolved to produce BFs instead of IFs, a change that presumably adapts the IF to a fiber cell-specific function. The authors establish here that the most striking divergence seen in phakosin is not, as hypothesized, the cause of this altered assembly outcome. The authors further establish that the HIM of IFs is far more tolerant of mutations, such as those that cause some corneal dystrophies and Alexander disease, than previously hypothesized and that normal assembly involves sequence-specific interactions between the head domain and the HIM.

Figure 1

Mutant phakosin and K18 proteins. Left: different protein domains contained in wild-type and mutant phakosin and K18 proteins that were created. Right: name assigned to each protein and filament assembly competency with K8.

Figure 2

SDS-PAGE of bacterially expressed, chromatographically purified K18, phakosin, and K8. Relative molecular weights in kilodal-tons, derived from standards, are indicated.

Figure 3

Comparison of types I and II keratin assembly with phakosin and type II assembly. (a) Type I keratin K18 dialyzed with natural type II keratin partner K8 forms normal 10-nm intermediate filaments. (b) Lens-specific intermediate filament protein phakosin dialyzed with K8 fails to form intermediate filaments.

Figure 4

Correction of phakosin helix initiation and termination motifs. (a) Phakosin with wild-type K18 helix initiation motif LNDR (phakosin-LNDR) dialyzed with K8. (b) Phakosin with wild-type K18 helix termination motif of TYRRLLEDGE (phakosin-TYRR) dialyzed with K8. (c) Phakosin with wild-type K18 helix initiation and termination motifs (phakosin-LNDR-TYRR) dialyzed with K8. All three corrected phakosin mutants failed to assemble with K8 to form intermediate filaments.

Figure 5

Replacement of wild-type cytokeratin HIM with phakosin sequence. (a) Type I keratin K18 with LNDC mutation in the helix initiation motif (K18-LNDC), which eliminates assembly competency with k8. (b) Type I keratin K18 with LGGC helix initiation sequence (K18-LGGC) and normal filament assembly with K8. (c) Type II keratin K8 with LGGC helix initiation sequence replacing wild-type LNNK motif (K8-LGGC) and normal filament assembly with K18.

Figure 6

Phakosin rod domain is assembly incompetent with type II keratins. (a) K18 head domain fused to phakosin rod domain (K18-H/phakosin-RT) and dialyzed with K8. Arrow: some form of higher order structure but incomplete filament assembly. (b) K18 head and tail domain fused to phakosin rod domain (K18-H/phakosin-R/K18-T) and dialyzed with K8. Both proteins containing the phakosin rod domain failed to assemble into intermediate filaments with K8, though some rodlets were detected.

Figure 7

Phakosin head domain assembly competent with K8. (a) Phakosin head domain fused to wild-type K18 rod and tail domain (phakosin-H/K18-RT) and dialyzed with K8 fails to form intermediate filaments. (b) Phakosin head domain fused to K18 rod and tail domain containing a helix initiation mutation of LNDR to LGGC (phakosin-H/K18-RT-LGGC) dialyzed with K8 forms normal intermediate filaments.