Abstract
Trichohyalin is an intermediate filament-associated protein that associates in regular arrays with keratin intermediate filaments (KIF) of the inner root sheath cells of the hair follicle and the granular layer of the epidermis and is a known substrate of transglutaminases. We have determined the full-length sequence of human trichohyalin by use of RNA-mediated anchored polymerase chain reaction methods and from a genomic clone and analyzed its potential secondary structure. We show here that trichohyalin may have at least three important functions in these cells. The protein of 248 kDa is unusual in that it contains one of the highest contents of charged residues of any protein. Of several defined domains, domains 2-4, 6, and 8 are almost entirely alpha-helical, configured as a series of peptide repeats of varying regularity, and are thought to form a single-stranded alpha-helical rod stabilized by ionic interactions between successive turns of the alpha-helix. Domain 6 is the most regular and may bind KIF directly by ionic interactions. Domains 5 and 7 are less well organized and may introduce folds in the molecule. Thus, human trichohyalin is predicted to be an elongated flexible rod at least 215 nm long and to function as a KIF-associated protein by cross-linking the filaments in loose networks. In addition, trichohyalin is similar to, but several times longer than, involucrin, a known cell envelope constituent, so that together, involucrin and trichohyalin may serve as scaffold proteins in the organization of the cell envelope of these cells or even anchor the cell envelope to the KIF network. Finally, trichohyalin possesses a pair of functional calcium-binding domains of the EF-hand type at its amino terminus that may be involved in its calcium-dependent postsynthetic processing during terminal differentiation.
Highlights
From the Skin Biology Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, the $Department of Dermatology, University of North Carolina, Chapel Hill, North Carolina27514, and the $Department of Physics and Biophysics, Massey University, Palmerston North, NewZealand
In the inner root sheath, the TRHYprotein becomes enmeshed with the keratin intermediate filaments (KIF)of the cells (4, 6,7) with an apparent periodicity of -200 nm (8) or 400 nm(range of200-500 nm) (9); in the medulla, the protein forms amorphous deposits that are not organized in any specific way(2-4, 8)
Trichohyalin is like TRHY molecules emerging from the granules into filasimilar to, but several times longer than, involucrian, ments (8).This concept would require that the TRHYprotein known cell envelope constituent, so that together, in- contain the characteristic canonical a-helical central rod dovolucrin and trichohyalin may serve as scaffold pro- main sequences of IF (17), but insufficient sequence inforteins in the organization of the cell envelope of these cells or even anchor the cell envelope to the KIF network
Summary
We have previously described the isolation and characterization of a 504-bp cDNA clone (24) obtained by PCR ampli-. The level of expression of TRHY is and a second intron of 864-bp splices sequences 223 bp from elevated by a factorof -25 in mouse hair follicles, as expected the 5'-end of our cDNA sequence information. These introns from its abundant expression in the developing inner root define an exon I of at least 54 bp and an exon I1of 169 bp. For quantitation purposes (see Table I), the x-ray films were exposed for several different times (4 h to 46 days); this figure shows an exposure of 6 days
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