Journal:

Article Title: A Homozygous Missense Mutation in TGM5 Abolishes Epidermal Transglutaminase 5 Activity and Causes Acral Peeling Skin Syndrome

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Figure Lengend Snippet: Biochemical studies of TG5 mutant forms. NHEK (a) and HEK-293 (b) cells were transfected for 48 h with the use of wild-type TG5 and the three mutant forms indicated. Total TG5 activities were measured for insoluble and fractions and standardized for transfection efficiency (GFP). The control vector indicates the endogenous TG5 activity (cells transfected only with the GFP control vector). The data obtained are reported as percentages of TG activity, with 100% corresponding to the activity for cells transfected with the wild-type construct. The data presented are the averages of two independent experiments. c, View of the entire TG5 model with the four main structural domains. The mutations described here are located in the N-terminal domain at the interface between the N-terminal domain and the catalytic domain. d, Effects of the mutations in a local environment. The G113C mutation, close to the active site, would probably cause a local change in the main protein structure. The poorly conserved residue T109 is present on the surface and is, therefore, unlikely to be functionally critical. Green indicates the mutated residues, and red represents wild-type TG5 residues.

Article Snippet: Cell Culture and Transfection Cryopreserved NHEK cells obtained from BioWhittaker were grown in calf skin collagen–coated dishes (Sigma Chemical) in serum-free keratinocyte medium (BioWhittaker) at 0.05 mM Ca 2+ and supplemented with Single-Quots (BioWhittaker) containing 7.5 μg/ml bovine pituitary extract, 0.5 mg/ml insulin, 0.5 mg/ml hydrocortisone, and 0.1 μg/ml hEGF.

Techniques: Mutagenesis, Transfection, Control, Plasmid Preparation, Activity Assay, Construct, Residue