Defining the properties of the nonhelical tail domain in type II keratin 5: insight from a bullous disease-causing mutation

Abstract

Inherited mutations in the intermediate filament (IF) proteins keratin 5 (K5) or keratin 14 (K14) cause epidermolysis bullosa simplex (EBS), in which basal layer keratinocytes rupture upon trauma to the epidermis. Most mutations are missense alleles affecting amino acids located in the central alpha-helical rod domain of K5 and K14. Here, we study the properties of an unusual EBS-causing mutation in which a nucleotide deletion (1649delG) alters the last 41 amino acids and adds 35 residues to the C terminus of K5. Relative to wild type, filaments coassembled in vitro from purified K5-1649delG and K14 proteins are shorter and exhibit weak viscoelastic properties when placed under strain. Loss of the C-terminal 41 residues contributes to these alterations. When transfected in cultured epithelial cells, K5-1649delG incorporates into preexisting keratin IFs and also forms multiple small aggregates that often colocalize with hsp70 in the cytoplasm. Aggregation is purely a function of the K5-1649delG tail domain; in contrast, the cloned 109 residue-long tail domain from wild type K5 is distributed throughout the cytoplasm and colocalizes partly with keratin IFs. These data provide a mechanistic basis for the cell fragility seen in individuals bearing the K5-1649delG allele, and point to the role of the C-terminal 41 residues in determining K5's assembly properties.

Figure 1.

Schematic representation of the mutants tested and expression levels of wild-type and mutant K5 proteins in transiently transfected PtK2 cells. (A) Schematic representation of the tripartite domain structure shared by K5 and all other keratins and intermediate filament proteins. A central α-helical “rod” domain acts as the major determinant of self-assembly and is flanked by nonhelical “head” and “tail” domains at the N and C termini, respectively. The rod domain is segmented into subdomain 1A, 1B, 2A, and 2B separated by three short nonhelical linkers, L1, L12, and L2. The extremities of this rod domain feature 15–20 residue-long regions (dark-shaded boxes) that are highly conserved among all intermediate filaments. For transfection studies only, a Myc or GFP epitope tag was fused in frame to the N terminus of K5. The location and nature of the mutants tested in this study are shown. “ΔX” refers to deletion of X amino acids. The 1649delG mutation induces a frameshift that results in a lengthening of the open reading frame (see black box). Below the full-length K5 diagram is a representation of the Myc-T5 and Myc-T5-1649delG mutant constructs. (B) PtK2 cells (10-cm dishes) were transfected with Myc-tagged K5, wild type and mutant, as described under Materials and Methods. At 48 h posttransfection, cells were harvested, processed for total protein extraction. Proteins were resolved by SDS-PAGE and analyzed by Western blot by using a Myc antibody to detect transfected proteins and a rabbit anti-K18 polyclonal antibody to detect the corresponding endogenous keratin. Bound primary antibodies were detected by enhanced chemiluminescence.

Figure 2.

Assembly properties of wild-type and mutant K5 proteins. Purified recombinant proteins were copolymerized with partner K14 and assessed by negative staining and electron microscopy (A–C, E–G) and a high-speed filament sedimentation assay (D). (A–D) Polymerization assays were conducted under standard assembly condition at pH 7.4. (E–G) Polymerization was conducted in pH 7.0 buffer, a condition that favors the formation of large keratin filament bundles (see inset, Frame E) (Yamada et al., 2002). The type I–type II composition of polymers is given in the lower left corner of each micrograph. In frame D, s refers to supernatant, and p refers to pellet.

Figure 3.

Impact of wild-type and mutant K5 expression on keratin filament organization in transfected cells. PtK2 kidney epithelial cells were transiently transfected with Myc-wtK5 (A–A″), Myc-K5E477K (B–B″), Myc-K5D328H (C–C″), Myc-K5CΔ111 (D–D″), or Myc-K5CΔ41 (E–E″) expression constructs. At 48 h posttransfection, cells were fixed and processed for double immunofluorescence staining by using antibodies to Myc, to detect the transfected protein, and K18, to visualize the endogenous keratin IF network. The identity of the labeled antigen is given in the lower left corner of each micrograph. See Table 2 for a quantitation of the outcome of these experiments.

Figure 4.

Impact of K5 tail domain mutant expression on keratin filament organization in transfected cells. PtK2 kidney epithelial cells (A, B, C, and E) or 308 mouse epidermal keratinocytes (D) were transiently transfected with Myc-K5-1649delG alone (A–A″, B–B″, D–D″, and E and E′) or along with K14 (C–C″). At 48 h posttransfection, cells were fixed and processed for double immunofluorescence staining by using antibodies to Myc, to detect the transfected protein, and K18, to visualize the endogenous keratin IF network (A–D), or hsp70 (E). The identity of the labeled antigen is given in the lower left corner of each micrograph. All micrographs except A–A″ were taken using a confocal microscope. See Table 2 for a quantitation of the outcome of these experiments.

Figure 5.

Fate of isolated K5 tail domain in transfected epithelial cells. PtK2 kidney epithelial cells were singly transfected with Myc-wtT5 (A–A″), Myc-T5-1649delG (B–B″), GFP-wtK5 (C–C″), GFP-K5-1649delG (D–D″), or doubly transfected with Myc-T5-1649delG and GFP-K5-1649delG (E–E″) expression constructs. At 48 h posttransfection, cells were fixed and processed for double immunofluorescence staining by using antibodies to Myc, to detect the transfected protein, and K18, to visualize the endogenous keratin IF network. GFP fusion proteins were detected using GFP-s intrinsic fluorescence. The identity of the labeled antigen is given in the lower left corner of each micrograph. See text for details.

Figure 6.

Biochemical and Immunological analyses of transfected proteins in PtK2 epithelial cells. (A) PtK2 kidney epithelial cells were transfected with Myc-wtT5 or Myc-T5-1649delG. At 48 h post-transfection, they were sequentially extracted to assess the distribution of transfected protein though SDS-PAGE and Western immunoblotting. Lanes are as follows: L, low-salt fraction; H, high-salt fraction; and U, final pellet solubilized in urea buffer. (B) PtK2 kidney epithelial cells were transfected with Myc-wtK5 or Myc-K5-1649delG, and 48 h later, were extracted with Empigen BB-containing buffer and subjected to immunoprecipitation by using rabbit immune serum to K18 or normal rabbit serum used as control (c). Immunoprecipitates were analyzed by Western blotting by using antibodies directed against Myc (top blot) and K18 (bottom blot. C) Cosedimentation assay in vitro. Reconstituted K5-K14 (10, 5, and 2.5 μm) or vimentin filaments (10 μm) were mixed with purified His-T5 (10 μm) and subjected to high-speed centrifugation (see Materials and Methods). Filaments were omitted in a tube acting as control. Pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE and Coomassie Blue staining.