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First published online April 16, 2004
doi: 10.1242/10.1242/jcs.01043

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Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients

D. W. Owens¹, N. J. Wilson¹, A. J. M. Hill¹, E. L. Rugg^1,*, R. M. Porter¹, A. M. Hutcheson², R. A. Quinlan^2,**, D. van Heel^3,, M. Parkes^3,, D. P. Jewell³, S. S. Campbell^4,¶, S. Ghosh^4,, J. Satsangi⁴ and E. B. Lane^1,

¹ Cancer Research UK Cell Structure Research Group, School of Life Science, University of Dundee, Dundee, DD1 5EH, UK
² Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK
³ Gastroenterology Unit, Nuffield Department of Medicine, The Radcliffe Infirmary, Woodstock Road, Oxford, OX2 6HE, UK
⁴ Gastrointestinal Unit, Department of Medical Science, Western General Hospital, Edinburgh, EH4 2XU, UK

View larger version (31K): [in a new window]   Fig. 1. Detection of K8 and K18 sequence variations in inflammatory bowel disease patients. (A) Patient DNA sequence from an ulcerative colitis patient showing heterozygous KRT8 g.1296G>T causing K8 Gly-62Cys. (B) Patient DNA sequence showing heterozygous KRT8 g.1299A>G causing K8 Ile63Val. (C) Patient DNA sequence showing KRT8 g.8510G>T causing K8 Lys464Asn. (D) Patient DNA sequence showing KRT18 g.4918G>C causing K18 Ser230Thr. (E) Positions of the amino acid substitutions in the K8 and K18 proteins. The helical domains are represented by boxes (shaded regions are hotspots for mutations causing severe disease in epidermal keratins) and the non-helical domains are represented by the solid line. Solid red triangles represent familial ulcerative colitis, solid green triangles represent familial Crohn disease and open green triangles represent sporadic Crohn disease.

View larger version (138K): [in a new window]   Fig. 2. In vitro filament assembly properties of variant keratins. (A) SDS polyacrylamide gel electrophoresis plus Coomassie Blue staining to show (left to right) molecular weight markers (Mr in kDa as indicated), recombinant K8(wt) and K18(wt), respectively. (B) Coomassie Blue-stained SDS-PAGE of samples from a sedimentation assay of K8(wt), K8(G62C), K8(I63V) or K8(K464N) each with K18(wt), polymerized in vitro under reducing conditions. `P' denotes pelleted (i.e. filamentous) material; `S' denotes material recovered from the supernatant. K8(wt) and K18(wt) readily assemble into pelletable polymers, whereas the mutant K8 forms smaller amounts of pelleted material in the sedimentation assay. (C) Coomassie Blue-stained SDS-PAGE of a sedimentation assay of K8(wt) with K18(wt) or K18(S230T), polymerized in vitro under reducing conditions: K18(S230T) mutant behaves as wild type. (D) Electron micrograph showing long, uniform filaments formed by in vitro polymerization of K8(wt) with K18(wt). Polymerization of wild-type K18 with (E): K8(G62C), (F): K8(I63V) or (G): K8(K464N) produces shorter, less uniform filaments and more small particulate aggregates. (H) K18(S230T) forms filaments with K8 (wild type) that are similar to those formed by wild-type K18. Bars, 200 nm.

View larger version (23K): [in a new window]   Fig. 3. Plasmon resonance analysis of K8/K18 binding. (A) Binding of 20 µg/ml K8 (wild type), K8(G62C), K8(I63V), K8(K464N) or BSA to immobilized wild-type recombinant K18 protein for 5 minutes, followed by washing for 5 minutes, under reducing conditions. K8(G62C) and K8(K464N) binding was less efficient than K8 (wild type), and K8(I63V) was similar to wild type. (B) Binding of K18(S230T) to wild-type immobilized K8 was impaired compared with K18 (wild type), and BSA binding was negligible. Once formed, all K8-K18 interactions were stable as shown by the persistent plateau through the wash period.

View larger version (111K): [in a new window]   Fig. 4. De novo filament assembly in transfected keratin-free SW13 epithelial cells. At 24 hours after transfection, most cells coexpressing K8 (wild type) and K18 (wild type) contained extended filament networks (A), whereas most cells coexpressing K8(G62C) and K18 (wild type) contained short lengths of disorganized filaments and aggregates (B). By 120 hours after tranfection with wild-type K8 and K18, most transfected cells contained extended arrays of ordered keratin filaments (C). However, cells expressing the K8(G62C) mutant contained predominantly disordered filament fragments (D). Bar, 20 µm.

View larger version (26K): [in a new window]   Fig. 5. Oxidative stress of nontransfected SW480 intestinal epithelial cells and SW480 cells stably expressing FLAG-tagged K8(G62C). Extracts were prepared from control cells incubated under normal growing conditions `C' or from cells subjected to oxidative stress `S' by treatment with 20 mM H2O2 for 1 hour. (A) Anti-FLAG immunoblot of proteins extracted from cells separated under nonreducing conditions. FLAG-tagged K8(G62C) was only detected in the transfected cultures and a putative K8 homodimer with an apparent molecular weight of just over 100 kDa was also detected in cells expressing K8(G62C) after oxidative stress. (B) Same samples as in (A) but run under reducing conditions: the high Mr band in K8(G62C) cells is no longer apparent as the disulphide bond in the homodimer has been reduced and all the K8 runs as monomers. (C) Immunoblotting extracts run under nonreducing conditions with monoclonal antibody M20 (detects K8 and K18) showed that endogenous K8 did not form putative homodimers in nontransfected cells. The monomeric FLAG-tagged K8(G62C) is detected by M20 as a weaker signal than endogenous K8 with an apparent molecular weight of 54 kDa in the unstressed transfected cells. The homodimeric FLAG-tagged K8(G62C) is also detected by M20 in the stressed cells.

View larger version (23K): [in a new window]   Fig. 6. Protein sequence alignment of type II keratins in the head domain V1/H1 region. Gly62 and Ile63 (bold type) are conserved in all type II keratins, as are the other shaded amino acids, suggesting that substitutions at these locations are likely to have functional consequences.