Trichohyalin is a major differentiation product of the inner root sheath cells of hair follicle, the medulla of the hair fiber, and is expressed in minor amounts in the epidermis. It is thought to function as a keratin intermediate filament associated protein in these tissues. In addition, trichohyalin is a substrate for transglutaminases (TGases), and for the enzyme peptidylarginine deiminase (PAD) which can convert arginines into citrullines. We have expressed domain 8 of human trichohyalin in bacteria, purified it to homogeneity, and used it as a substrate for TGases and PAD. PAD can convert up to 60% or the arginines of trichohyalin into citrullines. In another PAD substrate, filaggrin, the conversion efficiency is near 100%. By use of a series of model proteins and peptide substrates, the reaction by PAD is clearly related to structure. By circular dichroism and fluorescence spectroscopy, the trichohyalin is a highly ordered approximately 90% alpha- helical protein in solution. Following reaction with PAD, all organized secondary structure is lost, presumably because the substituted urea derivative (citrulline) interfers with protein structure. The trichohyalin is used as a substrate by the three TGases epxressed in the skin, but by calculation of kinetic parameters, TGase 3 uses it most efficiently. Multiple glutamines and lysines are utilized, indicating that trichohyalin is a complete TGase substrate. The kcat for the TGase 3 enzyme increases by a factor of 10 after PAD modification. These data suggest that the highly insoluble trichohaylin is first modified to a soluble form in cells by PAD before it is preferentially crosslinked by TGase 3. An antibody elicited against the expressed trichohyalin is being used to explore tissue expression. An analysis of the regulatory sequences which control expression of the trichohyalin gene have identified several sequences within the first 150 bp above the transcription start site which define the proximal promoter and epithelial-specific expression. However, 1.5 kb of upstream sequences coupled to a beta-galactosidase reporter gene are required to connote expression in transgenic mice in the hair follicle cells, hard palate, tongue epithelium and nail beds.
