TRICHOHYALIN - PURIFICATION FROM PORCINE TONGUE EPITHELIUM AND CHARACTERIZATION OF THE NATIVE PROTEIN

Trichohyalin, a protein of M(r) between 190 and 220 kDa in different species, was first demonstrated in large granules of the inner root sheath and medulla of hair follicles and may provide a matrix for keratin filaments. We have purified trichohyalin in milligram quantities from a citric acid - insoluble fraction derived from pig tongue epithelium. Trichohyalin was extracted under conditions of low ionic strength from the citric acid - insoluble fraction, separated by gel-filtration chromatography in buffer containing 1 M NaBr, and concentrated by ion-exchange chromatography in buffer containing 4 M urea. The purified material, which is soluble in buffers containing 1 M NaBr, was considered to be trichohyalin because of its characteristic molecular weight and amino acid composition and its localization to hair follicle inner root sheath and medulla by indirect immunofluorescence using antibodies against the purified protein. Immunofluorescence showed that trichohyalin is a major protein of filiform papillae of the tongue. Unlike trichohyalin from other animals examined, the porcine protein is a doublet on SDS polyacrylamide gels of 195 and 210 kDa; both bands are recognized by different antibodies, their two-dimensional peptide maps are nearly identical, and they have nearly identical isoelectric points of about 6.6. Trichohyalin has a Stokes radius of 124 A on gel filtration and a Svedberg constant of 6, consistent with an extended structure. The protein probably associates reversibly in solution, and the native protein we have isolated may be dimeric, because crosslinking of the iodinated purified protein with disuccinimidyl suberate demonstrated the presence of a dimer, which could be dissociated in the presence of high concentrations of urea. Rotary shadowing electron microscopy of the native protein showed a filamentous structure averaging 85 nm in length with a single globular-appearing end-domain. The purification of native trichohyalin provides a basis for future functional studies.