Trichohyalin Mechanically Strengthens the Hair Follicle
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m302037200
ISSN1083-351X
AutoresPeter M. Steinert, David Parry, Lyuben N. Marekov,
Tópico(s)Polysaccharides and Plant Cell Walls
ResumoTrichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks. Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks. Native trichohyalin (THH) 1The abbreviations used are: THH, trichohyalin; CE, cell envelope; IRS, inner root sheath; KIF, keratin intermediate filaments; PAD, peptidylarginine deiminase; SPR, small proline-rich protein; TGase, transglutaminase; Z, single-letter code for citrulline; HPLC, high performance liquid chromatography. is a large highly charged α-helix-rich and insoluble protein that is expressed in specialized mammalian epithelial cell types (1Rothnagel J.A. Rogers G.E. J. Cell Biol. 1986; 102: 1419-1429Crossref PubMed Scopus (106) Google Scholar, 2Lee S.-C. Kim I.-G. Marekov L.N. O'Keefe E.J. Parry D.A.D. Steinert P.M. J. Biol. Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar, 3Feitz M.J. McLaughlin C.J. Campbell M.T. Rogers G.E. J. Cell Biol. 1993; 121: 855-865Crossref PubMed Scopus (67) Google Scholar). The tissues in which it is most abundantly expressed include the inner root sheath (IRS) cells of the hair follicle (approximately one third of total protein) (4Vörner H. Dermatol. Z. (Berlin). 1903; 10: 357-376Crossref Scopus (9) Google Scholar, 5Auber L. Trans. R. Soc. Edinb. 1950; 62: 191-254Crossref Scopus (144) Google Scholar) and the medulla, a central column of cells within many coarse hairs (most of total protein) (4Vörner H. Dermatol. Z. (Berlin). 1903; 10: 357-376Crossref Scopus (9) Google Scholar, 5Auber L. Trans. R. Soc. Edinb. 1950; 62: 191-254Crossref Scopus (144) Google Scholar, 6Hamilton E.H. Payne J. Richard E. O'Keefe E.J. J. Invest. Dermatol. 1992; 96: 666-672Crossref Scopus (43) Google Scholar). THH is also expressed in trace amounts in other tissues such as newborn human foreskin epidermis, the hard palate, and rodent forestomach alone (6Hamilton E.H. Payne J. Richard E. O'Keefe E.J. J. Invest. 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Interestingly, each of these tissues is especially hardened or toughened to withstand mechanical abrasion and wear-and-tear during normal use. Thus, the question has arisen as to whether and how THH might contribute greater mechanical strength to these tissues (12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). THH can be recovered intact from these tissues only before their terminal differentiation (1Rothnagel J.A. Rogers G.E. J. Cell Biol. 1986; 102: 1419-1429Crossref PubMed Scopus (106) Google Scholar, 5Auber L. Trans. R. Soc. Edinb. 1950; 62: 191-254Crossref Scopus (144) Google Scholar). However, recovery from mature tissues requires proteolysis (13Rogers G.E. Nature. 1962; 194: 1149Crossref PubMed Scopus (59) Google Scholar, 14Steinert P.M. Dyer P.Y. Rogers G.E. J. Invest. Dermatol. 1969; 56: 49-54Abstract Full Text PDF Scopus (19) Google Scholar, 15Harding H.W.J. Rogers G.E. 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Steinert P.M. J. Biol. Chem. 1996; 271: 30709-30716Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). An important second modification is cross-linking by transglutaminase (TGase) enzymes (25Harding H.W.J. Rogers G.E. Biochemistry. 1971; 10: 624-630Crossref PubMed Scopus (104) Google Scholar, 26Harding H.W.J. Rogers G.E. Biochemistry. 1972; 11: 2858-2863Crossref PubMed Scopus (42) Google Scholar), which catalyze the formation of an isopeptide bond between peptide-bound glutamine and lysine residues; the net result is a stable insoluble protein polymer complex (27Lorand L. Conrad S.M. Mol. Cell Biochem. 1984; 58: 9-35Crossref PubMed Scopus (678) Google Scholar, 28Folk J.E Chung S.-I. Methods Enzymol. 1985; 113: 358-375Crossref PubMed Scopus (252) Google Scholar, 29Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (948) Google Scholar, 30Nemes Z. Steinert P.M. Exp. Mol. Med. 1999; 31: 5-19Crossref PubMed Scopus (465) Google Scholar, 31Kim S.-Y. Jeitner T.M. Steinert P.M. Neurosci. Lett. 2002; 40: 85-103Google Scholar). In the case of the medulla cells, the amorphous THH protein is extensively cross-linked to itself. In the nearby IRS cells of the hair follicle, there is also a very high content of cross-link (26Harding H.W.J. Rogers G.E. Biochemistry. 1972; 11: 2858-2863Crossref PubMed Scopus (42) Google Scholar, 32Steinert P.M. Biochemistry. 1978; 17: 5045-5052Crossref PubMed Scopus (21) Google Scholar). Earlier data suggested that these may link THH to the keratin intermediate filaments (KIF) characteristic of this tissue because KIF can be released from mature IRS cells only after a brief proteolytic digestion step (14Steinert P.M. Dyer P.Y. Rogers G.E. J. Invest. Dermatol. 1969; 56: 49-54Abstract Full Text PDF Scopus (19) Google Scholar) that clips off keratin head and/or tail domain sequences (32Steinert P.M. Biochemistry. 1978; 17: 5045-5052Crossref PubMed Scopus (21) Google Scholar). In addition, we have identified a few cross-linked peptides involving THH linked to several barrier protein components of the cell envelope (CE) of mouse forestomach tissue (12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The purpose of this study is to fully characterize the utilization of THH in the IRS tissue in which it is abundantly expressed. Our new protein sequence data provide robust support for the biomechanical role of THH in stabilization of cell structure by coordination of the CE with the KIF·THH complex within the IRS cells. IRS tissue was harvested from the hair follicles of newborn albino mice (32Steinert P.M. Biochemistry. 1978; 17: 5045-5052Crossref PubMed Scopus (21) Google Scholar). Hair follicles isolated from trunk skin dermis of newborn (<1 day old) albino mice (33Weinberg W.C. Goodman L.V. George C. Morgan D.L. Ledbetter S. Yuspa S.H. Lichti U. J. Invest. Dermatol. 1993; 100: 229-236Abstract Full Text PDF PubMed Google Scholar) were extracted in a buffer of 25 mm Tris-HCl (pH 8.0) containing 8 m urea. This buffer dissolves the lower portions of the hair follicles, thereby releasing the mature hardened IRS tissues as discrete conical, cylindrical multicellular structures. It does not appreciably dissolve the detached hair fiber material. The urea suspension was filtered through nylon gauze (pore size approximately 0.2 mm) through which the IRS tissues pass but hair fibers do not. In our experience, approximately 60% of the mass of these hair follicles is IRS tissue; the hair fiber cortical tissue is still poorly developed, and there are no hair fibers. The highly enriched IRS tissue was pelleted by centrifugation at 100 × g and gently washed in phosphate-buffered saline to remove urea but not to dissociate the IRS. We found in preliminary sequencing experiments that mouse IRS tissue was contaminated by solubilized outer root sheath proteins, particularly K6a and K16. To obtain a fraction enriched in KIF proteins (34Steinert P.M. Aynardi-Whitman M. Zackroff R.V. Goldman R.D. Wilson L. Methods and Perspectives in Cell Biology; The Cytoskeleton, Part A: Cytoskeletal Proteins, Isolation and Characterization. Vol. 24. Academic Press Inc., New York1982: 399-419Google Scholar), newborn mouse hair follicles were homogenized by sonication in phosphate-buffered saline containing 0.5 m KCl, and a protease mixture kit (Roche). This suspension was made to 5 mm MgCl2 and 100 mg/ml DNase I and incubated at 30% acetonitrile, covalently attached them to a solid support, and performed sequencing for up to 15 Edman degradation cycles as described previously (7Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.-I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 12Steinert P.M. Kartasova T. Marekov L.N. J. Biol. Chem. 1998; 273: 11758-11769Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Empirically we found that such peptide species contained >15 residues and collectively accounted for >90% of the total cross-link content of the samples. Additionally, on sequencing, these typically contained two or more peptide "branches" adjoined by one or more cross-links. Sequences could be assigned from data base searches. The phenylthiohydantoin derivative of citrulline eluted at the same time as threonine in our system (Porton LF-3000 gas phase), and assignment was rarely in doubt because of the paucity of threonine in the proteins. Peptides corresponding the carboxyl terminus of mouse c29 protein (36Bawden C.S. McLaughlin C. Nesci A. Rogers G.E. J. Invest. Dermatol. 2001; 116: 157-166Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) of sequence STKVNKTEQRIPS (IRSwt) and STKVNKTEQZIPS (IRScit) were synthesized, coupled to an 8-branched inert matrix, and used to generate polyclonal antibodies in guinea pigs, which were subsequently affinity-purified using the peptide antigen. Frozen serial sections of 5-day-old mouse skin (7 mm) were used for indirect immunofluorescence methods exactly as described previously (7Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.-I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Primary antibodies used were: polyclonal rabbit anti-THH-8 (dilution 1:30) (7Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.-I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), polyclonal goat anti-human TGase 1 (dilution 1:50) (37Kim S.-Y. Chung S.-I. Steinert P.M. J. Invest. Dermatol. 1995; 104: 211-217Abstract Full Text PDF PubMed Scopus (93) Google Scholar), polyclonal guinea pig anti-IRSwt (dilution 1:100), polyclonal guinea pig anti-IRScit (dilution 1:200), and mouse monoclonal anti-K6 (Roche Molecular Biochemicals). Primary antibodies were then visualized using the appropriate affinity-purified secondary antibodies (Cappel Organon Technika Corp., Durham, NC) (7Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.-I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Western blotting, dot blotting, and immunoprecipitation methods were used exactly as described usbpreviously (7Tarcsa E. Marekov L.N. Andreoli J. Idler W.W. Candi E. Chung S.-I. Steinert P.M. J. Biol. Chem. 1997; 272: 27893-27901Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The purpose of this study is to more fully characterize the utilization and function of THH in a tissue in which it is abundantly expressed: the IRS of the mouse hair follicle. We generated three different fractions of peptides (Fig. 2) designed to separately explore the possible multiple functions of THH. The amounts of cross-link and citrulline were measured. Numerous peptides containing cross-links from each fraction were sequenced. Table I lists and sorts the protein partners. Each will be discussed in detail below.Table ISummary of numbers of protein partners in sequenced peptidesPartnersFraction AFraction BFraction CTHH-THH773THH-keratin45aListed in Table III.30aListed in Table III.6bListed in Table IV.THH-CE proteinsbListed in Table IV.1034CE-CEbListed in Table IV.41CE-keratinbListed in Table IV.13a Listed in Table III.b Listed in Table IV. Open table in a new tab Comparison of THH Sequences—The sequences of human (1897 residues; Ref. 2Lee S.-C. Kim I.-G. Marekov L.N. O'Keefe E.J. Parry D.A.D. Steinert P.M. J. Biol. Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar), rabbit (1407 residues; accession no. P37709), mouse (1439 residues, accession no. XP_177952), and sheep (1549 residues; Ref. 3Feitz M.J. McLaughlin C.J. Campbell M.T. Rogers G.E. J. Cell Biol. 1993; 121: 855-865Crossref PubMed Scopus (67) Google Scholar) THH proteins are available. All display marked similarities, yet notable differences; they consist of varying numbers of predominantly α-helical, highly charged quasi-repeating peptide motifs that are poorly conserved although recognizably similar between species. Human THH has been divided into nine domains, based on regions of either high or low sequence regularity among adjacent repeats (2Lee S.-C. Kim I.-G. Marekov L.N. O'Keefe E.J. Parry D.A.D. Steinert P.M. J. Biol. Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar). By use of the University of Wisconsin Genomics Computer Group software, domain 1 of mouse THH consists of two well defined EF-hand repeats (residues 1–95), domain 2 (96–243), and domains 3 and 4 (244–373), each similar to human THH. Next, its domain 5 (residues 374–595) consists of irregular sequence repeats of lower overall predicted α-helical content, roughly corresponding to human domains 5 and 7. Residues 596–697 define a region here termed domain 5* of weakly defined repeats unique to mouse THH. Residues 698–880 and 881–1406 of mouse THH define sets of well ordered sequences of different repeat motifs that are equivalent to human domains 6 and 8, respectively. Finally, domain 9 (residues 1407–1439) is homologous to human. Direct Evidence That THH Functions as an Interfilamentous KIF Cross-bridging Protein in the IRS—KIF were harvested from mature IRS by use of a preliminary limited trypsin digestion (14Steinert P.M. Dyer P.Y. Rogers G.E. J. Invest. Dermatol. 1969; 56: 49-54Abstract Full Text PDF Scopus (19) Google Scholar, 32Steinert P.M. Biochemistry. 1978; 17: 5045-5052Crossref PubMed Scopus (21) Google Scholar), which generated a KIF yield of approximately 40% of total IRS protein mass. Some of the cellular debris after brief trypsinization consisted of clumps of KIF apparently still cross-linked together. Unlike all other types of IF we have investigated, these IRS KIF are rigid straight rods that have a central densely staining core (Fig. 3A). This is reminiscent of the core observed in the center of the distinctly different trichocyte KIF of the adjacent hair fiber cortical cells (38Watts N.R. Jones L.N. Cheng N. Wall J.S. Parry D.A. Steven A.C. J. Struct. Biol. 2002; 137: 109-119Crossref PubMed Scopus (41) Google Scholar). The KIF were pelleted and dissolved in SDS buffer. Upon fractionation by column chromatography, three peaks were recovered (Fig. 3B). The first and second peaks contained only traces of cross-link (<1 residue/1000 residues) and citrulline (ratio < 0.01), and presumably contained somewhat pruned IF chains, as judged by their high α-helix contents (data not shown). However, the peak eluted at Vt contained >90% of the citrulline and isopeptide cross-link of the isolated KIF. This Vt material, termed fraction A, was further digested to completion with trypsin and the peptides resolved by HPLC (Fig. 4A). From the resultant reproducible profile, we recovered 38 well resolved peaks that reflect quantitatively major species. Sequencing revealed that 35 possessed one or more cross-links; 28 peptides had one cross-link, 4 had two cross-links, and 3 had three cross-links, so that a total of 44 peptide partners was found (Tables I and III). Together, these are likely to be highly representative, as they accounted for 69% of the total cross-link and 73% of the total citrulline content of the Vt fraction. It remains likely, however, that many other minor peptide/protein species have been overlooked. Another 20 peptides involving KIF chains, having a total of 30 partners, were recovered from fraction B (Fig. 4C and Table III).Table IIIKIF chains are cross-linked by way of their head and tail domains exclusively to domains 6 and 8 of mouse THHTable IIIKIF chains are cross-linked by way of their head and tail domains exclusively to domains 6 and 8 of mouse THH Analyses of the combined data revealed two types of sequences: those from THH, and those from type I and type II keratin chains. For THH, all cross-links involved sequences of the highly regular domain 6 and 8 regions and the consensus THH peptide sequences involved were DZK(F/I)(Z/R)(Z/R) for the lysine residues and (E/Z)EQE(Z/L)Z for the glutamine residues. Based on ongoing studies of murine, ovine, and human data, the IRS uniquely expresses a set of least three (ovine) type I keratin chains (39Sato H. Koide T. Sagai T. Ishiguro S. Tamai M. Saitou N. Shiroshi T. Genomics. 1999; 56: 303-309Crossref PubMed Scopus (14) Google Scholar, 40Carninci P. Shinata Y. Hayatsu N. Sugahara Y. Shinata K. Itoh M. Konro H. Okazaki Y. Muramatsu M. Hayashizaki Y. Genome Res. 2000; 10: 1617-1630Crossref PubMed Scopus (255) Google Scholar, 41Hesse M. Magin T.M. Weber K. J. Cell Sci. 2001; 114: 2569-2575Crossref PubMed Google Scholar, 42Aoki N. Sawada S. Rogers M.A. Schweizer J. Shimomura Y. Tsujimoto T. Ito K. Ito M. J. Invest. Dermatol. 2001; 116: 359-365Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). One mouse sequence (KRT1-c29) has been reported (39Sato H. Koide T. Sagai T. Ishiguro S. Tamai M. Saitou N. Shiroshi T. Genomics. 1999; 56: 303-309Crossref PubMed Scopus (14) Google Scholar), which has highest homology to the sheep type I IRSa3 proteins (36Bawden C.S. McLaughlin C. Nesci A. Rogers G.E. J. Invest. Dermatol. 2001; 116: 157-166Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Our new sequencing data reveal that tail domain sequences of c29 were commonly found to participate in cross-linking, and that its arginines were usually modified to citrullines (Tables II and III). Further, three other homologous sequences were found (Table II) that may represent polymorphisms of this protein, or other mouse type I IRS keratins that perhaps correspond to the additional known sheep proteins for which only limited data have been reported. Several other sequences matched a known type II keratin chain expressed in IRS tissue (41Hesse M. Magin T.M. Weber K. J. Cell Sci. 2001; 114: 2569-2575Crossref PubMed Google Scholar, 42Aoki N. Sawada S. Rogers M.A. Schweizer J. Shimomura Y. Tsujimoto T. Ito K. Ito M. J. Invest. Dermatol. 2001; 116: 359-365Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), but we cannot exclude the possibility that other K6-like type II IRS chains might be present in minor amounts, or might possess identical sequences around cross-linking sites. All of the keratin sequences were from end domains, with most from the tail. This may be because there are few Gln and Lys residues in the head domain sequences and because the tails may be more readily accessible for cross-linking. No links were obtained from central rod domain/linker Gln and Lys residues, but we cannot exclude that such linkages occur in minor amounts. This may be because the rod domain Gln and Lys residues are relatively inaccessible because of intra- and/or interchain molecular interactions.Table IIIdentities and similarities of mouse IRS type I keratin tail sequences recovered in this work The data suggest that cross-linking of the IRS KIF is managed differently from the epidermis and in other epithelia studied heretofore. In the case of normal epidermis for example, essentially only the "KSISIS" Lys residue in the head domain of the type II keratin chains is used for KIF cross-linking and at only approximate
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