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hKAP1.6 and hKAP1.7, Two Novel Human High Sulfur Keratin-Associated Proteins are Expressed in the Hair Follicle Cortex

2002; Elsevier BV; Volume: 118; Issue: 2 Linguagem: Inglês

10.1046/j.0022-202x.2001.01653.x

1523-1747

Yutaka Shimomura, Noriaki Aoki, Masaaki Ito, Michael A. Rogers, Lutz Langbein, Jürgen Schweizer,

Silk-based biomaterials and applications

Hair fiber differentiation involves the expression of both hair keratin intermediate filament proteins and their associated proteins, termed keratin-associated proteins. In this study, cDNA clones encoding two novel keratin-associated proteins were isolated from human hair follicle mRNA. The predicted amino acid sequence derived from these clones revealed that these proteins represent members of the human keratin-associated protein 1 family. They show strong sequence homology to two previously described keratin-associated protein 1 family members hKAP1.1 A and hKAP1.1B. We have called these new proteins hKAP1.6 and hKAP1.7, respectively. RNA in situ hybridization studies of human anagen hair follicles using a conserved probe for these four keratin-associated protein 1 members demonstrated the expression of this group in the differentiated portions of the hair cortex. Hair fiber differentiation involves the expression of both hair keratin intermediate filament proteins and their associated proteins, termed keratin-associated proteins. In this study, cDNA clones encoding two novel keratin-associated proteins were isolated from human hair follicle mRNA. The predicted amino acid sequence derived from these clones revealed that these proteins represent members of the human keratin-associated protein 1 family. They show strong sequence homology to two previously described keratin-associated protein 1 family members hKAP1.1 A and hKAP1.1B. We have called these new proteins hKAP1.6 and hKAP1.7, respectively. RNA in situ hybridization studies of human anagen hair follicles using a conserved probe for these four keratin-associated protein 1 members demonstrated the expression of this group in the differentiated portions of the hair cortex. keratin-associated protein Hair is a strongly keratinized tissue formed within the hair follicle. The hair fiber possesses several distinctive morphologic features, among which are an external cuticular sheath, an inner cortical region, and, occasionally, a narrow, central lying medulla. The major structural proteins of hair are the keratin intermediate filament proteins, whose 8 nm filaments form the rigid hair shaft through crosslinking of these filaments with their associated proteins (KAPs) (Powell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser Verlag, Basel1997: 59-148Crossref Google Scholar). More than 60 hair KAPs have been found in various species and they have been classified into proteins exhibiting high sulfur (16–30 mol% cysteine, KAP1-KAP3, KAP10–KAP16 families) (Swart and Haylett, 1973Swart L.S. Haylett T. Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMKB-3A3.Biochem J. 1973; 133: 641-654Crossref PubMed Scopus (23) Google Scholar;Swart et al., 1976Swart L.S. Joubert F.J. Parris D. Homology in the amino acid sequences of the high-sulphur proteins from keratins,.in: Ziegler K. Proceedings of the 5th International Wool Textile Research Conference. German Wool Research Institute, Aachen1976: 254-264Google Scholar;Powell et al., 1983Powell B.C. Sleigh M.J. Ward K.A. Rogers G.E. Mammalian keratin gene families: organisation of genes coding for the B2 high-sulphur proteins of sheep wool.Nucl Acids Res. 1983; 11: 5327-5346Crossref PubMed Scopus (60) Google Scholar;Frenkel et al., 1989Frenkel M.J. Powell B.C. Ward K.A. Sleigh M.J. Rogers G.E. The keratin BIIIB gene family: isolation of cDNA clones and structure of a gene and a related pseudogene.Genomics. 1989; 4: 182-191Crossref PubMed Scopus (31) Google Scholar;Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar;Huh et al., 1994Huh N. Kashiwagi M. Konishi C. Hashimoto Y. Kohno Y. Nomura S. Kuroki T. Isolation and characterization of a novel hair follicle-specific gene, Hacl-1.J Invest Dermatol. 1994; 102: 716-720Abstract Full Text PDF PubMed Google Scholar;Powell et al., 1997Powell B.C. Rogers G.E. The role of keratin proteins and their genes in the growth, structure and properties of hair.in: Jolles P. Zahn H. Höcker H. Formation and Structure of Human Hair. Birkhäuser Verlag, Basel1997: 59-148Crossref Google Scholar;Aoki et al., 1998Aoki N. Ito K. Ito M. Hair follicle has a novel anagen-specific protein, mKAP13.J Invest Dermatol. 1998; 111: 804-809Crossref PubMed Scopus (11) Google Scholar;Cole and Reeves, 1998Cole S.E. Reeves R.H. A cluster of keratin-associated proteins on mouse chromosome 10 in the region of conserved linkage with human chromosome 21.Genomics. 1998; 54: 437-442Crossref PubMed Scopus (13) Google Scholar;Mitsui et al., 1998Mitsui S. Ohuchi A. Adachi-Yamada T. Hotta M. Tsuboi R. Ogawa H. Structure and hair follicle-specific expression of genes encoding the rat high sulfur protein B2 family.Gene. 1998; 208: 123-129Crossref PubMed Scopus (12) Google Scholar;Takaishi et al., 1998Takaishi M. Takata Y. Kuroki T. Huh N.-H. Isolation and characterization of a putative keratin-associated protein gene expressed in embryonic skin of mice.J Invest Dermatol. 1998; 111: 128-132Crossref PubMed Scopus (23) Google Scholar;Kuhn et al., 1999Kuhn F. Lassing C. Range A. Mueller M. Hunziker T. Ziemiecki A. Andres A.-C. Pmg-1 and Pmg-2 constitute a novel family of KAP genes differentially expressed during skin and mammary gland development.Mechanisms Dev. 1999; 86: 193-196Crossref PubMed Scopus (18) Google Scholar;Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar), ultra-high sulfur (>30% cysteine; KAP4, KAP5, KAP9, and KAP17 families) (McNab et al., 1989McNab A.R. Wood L. Theriault N. Gierman T. Vogeli G. An ultra-high sulfur keratin gene is expressed specifically during hair growth.J Invest Dermatol. 1989; 92: 263-266Abstract Full Text PDF PubMed Google Scholar;MacKinnon et al., 1990MacKinnon P.J. Powell B.C. Rogers G.E. Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles.J Cell Biol. 1990; 111: 2587-2600Crossref PubMed Scopus (70) Google Scholar;Fratini et al., 1994Fratini A. Powell B.C. Hynd P.I. Keough R.A. Rogers G.E. Dietary cysteine regulates the levels of mRNAs encoding a family of cysteine-rich proteins of wool.J Invest Dermatol. 1994; 102: 178-185Abstract Full Text PDF PubMed Google Scholar;Jenkins and Powell, 1994Jenkins B.J. Powell B.C. Differential expression of genes encoding a cysteine-rich keratin family in the hair cuticle.J Invest Dermatol. 1994; 103: 310-317Crossref PubMed Scopus (31) Google Scholar;Powell et al., 1995Powell B.C. Arthur J. Nesci A. Characterization of a gene encoding a cysteine-rich keratin associated protein synthesized late in rabbit hair follicle differentiation.Differentiation. 1995; 58: 227-232Crossref PubMed Scopus (20) Google Scholar;Perez et al., 1999Perez C. Auriol J. Gerst C. Bernard B.A. Egly J.M. Genomic organization and promoter characterization of two human UHS keratin genes.Gene. 1999; 227: 137-148Crossref PubMed Scopus (11) Google Scholar;Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar), and high glycine/tyrosine (KAP6–KAP8 families) amino acid content (Kuczek and Rogers, 1987Kuczek E.S. Rogers G.E. Sheep wool (glycine + tyrosine)-rich keratin genes: a family of low sequence homology.European J Biochem. 1987; 166: 79-85Crossref PubMed Scopus (51) Google Scholar;Fratini et al., 1993Fratini A. Powell B.C. Rogers G.E. Sequence expression, and evolutionary conservation of a gene encoding a glycine/tyrosine-rich keratin-associated protein of hair.J Biol Chem. 1993; 268: 4511-4518Abstract Full Text PDF PubMed Google Scholar;Aoki et al., 1997Aoki N. Ito K. Ito M. Isolation and characterization of mouse high-glycine/tyrosine proteins.J Biol Chem. 1997; 272: 30512-30518Crossref PubMed Scopus (23) Google Scholar). Furthermore, these KAPs have been subdivided into a total of 17 families (KAP1.n-KAP17.n) based on an initial proposal byRogers and Powell, 1993Rogers G.E. Powell B.C. Organization and expression of hair follicle genes.J Invest Dermatol. 1993; 101: 50s-55sAbstract Full Text PDF PubMed Google Scholar. This nomenclature is based on their cysteine/tyrosine-glycine content, as well as the degree of amino acid homology within each family and the nature of the repeat structures often found in these molecules (McNab et al., 1989McNab A.R. Wood L. Theriault N. Gierman T. Vogeli G. An ultra-high sulfur keratin gene is expressed specifically during hair growth.J Invest Dermatol. 1989; 92: 263-266Abstract Full Text PDF PubMed Google Scholar;Huh et al., 1994Huh N. Kashiwagi M. Konishi C. Hashimoto Y. Kohno Y. Nomura S. Kuroki T. Isolation and characterization of a novel hair follicle-specific gene, Hacl-1.J Invest Dermatol. 1994; 102: 716-720Abstract Full Text PDF PubMed Google Scholar;Aoki et al., 1998Aoki N. Ito K. Ito M. Hair follicle has a novel anagen-specific protein, mKAP13.J Invest Dermatol. 1998; 111: 804-809Crossref PubMed Scopus (11) Google Scholar;Cole and Reeves, 1998Cole S.E. Reeves R.H. A cluster of keratin-associated proteins on mouse chromosome 10 in the region of conserved linkage with human chromosome 21.Genomics. 1998; 54: 437-442Crossref PubMed Scopus (13) Google Scholar;Takaishi et al., 1998Takaishi M. Takata Y. Kuroki T. Huh N.-H. Isolation and characterization of a putative keratin-associated protein gene expressed in embryonic skin of mice.J Invest Dermatol. 1998; 111: 128-132Crossref PubMed Scopus (23) Google Scholar;Kuhn et al., 1999Kuhn F. Lassing C. Range A. Mueller M. Hunziker T. Ziemiecki A. Andres A.-C. Pmg-1 and Pmg-2 constitute a novel family of KAP genes differentially expressed during skin and mammary gland development.Mechanisms Dev. 1999; 86: 193-196Crossref PubMed Scopus (18) Google Scholar). So far genes encoding KAPs have been isolated from humans, sheep, rabbit, rat, and mouse (Wood et al., 1990Wood L. Mills M. Hatzenbuhler N. Vogeli G. Serine-rich ultra high sulfur protein gene expression in murine hair and skin during the hair cycle.J Biol Chem. 1990; 265: 21375-21380Abstract Full Text PDF PubMed Google Scholar;Fratini et al., 1993Fratini A. Powell B.C. Rogers G.E. Sequence expression, and evolutionary conservation of a gene encoding a glycine/tyrosine-rich keratin-associated protein of hair.J Biol Chem. 1993; 268: 4511-4518Abstract Full Text PDF PubMed Google Scholar;Powell et al., 1995Powell B.C. Arthur J. Nesci A. Characterization of a gene encoding a cysteine-rich keratin associated protein synthesized late in rabbit hair follicle differentiation.Differentiation. 1995; 58: 227-232Crossref PubMed Scopus (20) Google Scholar;Aoki et al., 1997Aoki N. Ito K. Ito M. Isolation and characterization of mouse high-glycine/tyrosine proteins.J Biol Chem. 1997; 272: 30512-30518Crossref PubMed Scopus (23) Google Scholar). In humans, six KAP1 genes and five KAP5 genes have been published (MacKinnon et al., 1990MacKinnon P.J. Powell B.C. Rogers G.E. Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles.J Cell Biol. 1990; 111: 2587-2600Crossref PubMed Scopus (70) Google Scholar;Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar;Emonet et al., 1997Emonet N. Michaille J.J. Dhouailly D. Isolation and characterization of genomic clones of human sequences presumably coding for hair cysteine-rich proteins.J Dermatol Sci. 1997; 14: 1-11Abstract Full Text PDF PubMed Scopus (12) Google Scholar;Perez et al., 1999Perez C. Auriol J. Gerst C. Bernard B.A. Egly J.M. Genomic organization and promoter characterization of two human UHS keratin genes.Gene. 1999; 227: 137-148Crossref PubMed Scopus (11) Google Scholar;Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). We have isolated two cDNAs encoding new members of the human KAP1 family, hKAP1.6 and hKAP1.7, found during the analysis of the hKAP1.1 A/B genes in patients possessing a congenital fragile hair disorder (for a description of the hKAP1.1 A/B genes and their nomenclature, see Discussion). In this study, we have characterized these novel KAP1 members and examined their expression in the hair follicle. Peripheral leukocyte DNAs were prepared from healthy individuals using standard protocols. The human hKAP1 A/B and hKAP1.6 genes were amplified by polymerase chain reaction (PCR) using human genomic DNAs as templates and hKAP1.1 A/B-specific oligonucleotide primers (KAP1.5′-1, 5′-ACT TATAAAAAGCCGGCAGTGG-3′; and KAP1.3′-1, 5′- GGTACT GGAGTTCAGAAGATTG-3′) (Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar). PCR was performed using KOD-Plus-DNA polymerase (Toyobo, Tokyo, Japan). Amplification conditions were 94°C for 2 min, followed by 35 cycles of 94°C for 15 s, 53°C for 30 s, and 68°C for 1 min, with a final extension at 68°C for 7 min. The amplified fragments were analyzed on a 6% polyacrylamide gel. After extraction of the fragments from the gel, direct fluorescent chain termination DNA cycle sequencing of the PCR products was performed (Big Dye DNA sequencing kit, Applied Biosystems, Foster City, CA). The DNA sequences were analyzed on an ABI373 DNA sequencer (Applied Biosystems). The resulting sequences were used to search for DNA homologies with genes registered in the GenBank/EMBO/DDBJ databases using the BLASTN program. Total RNA was isolated using an Isogen kit (Nippongene, Tokyo, Japan) from 15 freshly plucked anagen hair follicles according to the manufacturer's recommendations. The resulting RNA was reverse transcribed using an oligo(dT) primer and was preceded by 10 min digestion with RNase-free DNase (Roche, Mannheim, Germany) in order to ensure the removal of contaminating genomic DNA. The cDNAs were amplified by PCR using the same primers and with the same PCR conditions as described above. The PCR products were directly sequenced after gel purification. To identify the sequence of the 3′-noncoding region in hKAP1.6 mRNA, 3′-RACE was performed using a standard 3′-RACE kit according to the manufacturer's instructions (Takara, Tokyo, Japan). After the synthesis of the first strand cDNAs using an oligo(dT)-adaptor primer derived from total RNA of anagen hair follicles, the 3′-cDNA end of hKAP1.6 was amplified by PCR using the adaptor primer (5′-GTTTCCCAGTCACGAC-3′) and internal gene-specific primer (KAP1.5′-4, 5′-TTCAGATTCAACTCCTGACACC-3′) derived from the 5′-noncoding region of the partial hKAP1.6 cDNA. The amplified cDNA fragments were directly sequenced after extraction from the gel. The preparation and arraying of a cDNA library from human scalp tissue has been described previously (Rogers et al., 1995Rogers M.A. Nischt R. Korge B. et al.Sequence data and chromosomal localization of human type I and type II hair keratin genes.Exp Cell Res. 1995; 220: 357-362https://doi.org/10.1006/excr.1995.1326Crossref PubMed Scopus (51) Google Scholar;Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). A PCR product specific for the 3′-noncoding region of the hKAP1.1 A/B gene (Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar) was prepared by amplification of genomic DNA (upper primer, 5′-CCATGG CCTGCTGTCAGAC-3′; lower primer, 5′-GTGGCTGTGTGA TGAGAGGTTG-3′). The hKAP1.1 A/B PCR product was randomly labeled with 32P-dCTP (Amersham-Pharmacia-Biotech, Bucks, U.K.) and used as a probe in order to screen the human scalp cDNA library according to methods described previously (Rogers et al., 1995Rogers M.A. Nischt R. Korge B. et al.Sequence data and chromosomal localization of human type I and type II hair keratin genes.Exp Cell Res. 1995; 220: 357-362https://doi.org/10.1006/excr.1995.1326Crossref PubMed Scopus (51) Google Scholar). Briefly, the hybridization was performed overnight at 65°C using a hybridization solution containing 6 × sodium citrate/chloride buffer (SSC), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, 1% sodium dodecyl sulfate (SDS), and 1 × 106 cpm per ml of the radiolabeled probe. Thereafter, multiple washings were performed with 0.5 × SSC/1% SDS at 65°C, and the filters were autoradiographed overnight using Kodak AR5 film. The screening resulted in many positive clones. Twenty of these clones were analyzed. The in situ hybridization protocol with human scalp cryostat sections using hybridization probes derived from cloned 3′-noncoding region probes of keratin and KAP genes has been described previously (Rogers et al., 1997Rogers M.A. Langbein L. Praetzel S. Moll I. Krieg T. Winter H. Schweizer J. Sequences and differential expression of three novel human type-II hair keratins.Differentiation. 1997; 61: 187-194https://doi.org/10.1007/s002580050184Crossref PubMed Scopus (0) Google Scholar;Langbein et al., 1999Langbein L. Rogers M.A. Winter H. Praetzel S. Beckhaus U. Rackwitz H.-R. Schweizer J. The catalog of human hair keratins: I Expression of the nine type I members in the hair follicle.J Biol Chem. 1999; 274: 19874-19884Crossref PubMed Scopus (203) Google Scholar). In these experiments, the 130 bp PCR product used for the human scalp cDNA library screening was cloned into the plasmid pCR2.1 (TA cloning kit, Invitrogen, Gröningen, Netherlands). This construct was used to generate a 35S cRNA transcript of hKAP1.1 A/B. The bright field microscopy and In situ hybridization signals were visualized using a confocal laser scanning microscope (LSM510, Carl Zeiss, Jena/Oberkochen, Germany). The instrument allows the simultaneous visualization of the reflected light from epi-illumination for the detection of hybridization signals (red channel) and transmitted light in bright field (green channel). The two channels were combined by an overlay in pseudocolors (transmission image in green, electronically changed into black/white; reflection image, i.e., hybridization signals, in red). The hKAP1.6, and hKAP1.7 sequences are available under the accession numbers AB052868, AB055057 in the EMBL/GenBank/DDBJ databases. In an attempt to analyze the sequences of human KAP1 genes, we amplified the hKAP1.1 A/B genes from human genomic DNA by PCR using primers derived from the original DNA sequence (Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar). The result of polyacrylamide gel electrophoresis showed the presence of two DNA fragments (Figure 1a). The direct DNA sequencing of these products indicated that the 700 bp PCR product corresponded to the hKAP1.1 A/B genes and that the 550 bp product might be a putative novel KAP1 gene. Therefore, RT-PCR was performed using total RNA extracted from plucked human hair follicles in order to confirm the expression of these putative genes at the mRNA level. Care was needed in the analysis of the PCR products because KAP genes, in general, consist of only one exon, thus making the discrimination between PCR products from true cDNA and contaminating genomic DNA difficult. Therefore, predigestion of human scalp RNA with DNase I was performed, as well as the amplification of a multiple exon containing housekeeping gene, in order to ensure the specificity of the RT-PCR reaction. This experiment also detected two cDNA products, 700 bp and 550 bp in size (Figure 1b). The sequence of each cDNA was completely consistent with that of the hKAP1.1 A/B genes or the novel gene, indicating that these genes are not pseudogenes and are actually expressed in the human hair follicle. In order to isolate the 3′-end of these cDNAs, we developed a 2-fold strategy. First, we amplified human hair follicle cDNAs using a 3′-RACE strategy, and obtained two cDNA fragments corresponding to hKAP1.1 A/B and the novel gene via PCR amplification (data not shown). DNA sequencing of the shorter fragment allowed the construction of a full-length cDNA of the novel gene (Figure 2a), and we have termed this gene hKAP1.6 for reasons explained below. In addition, we also attempted to isolate the hKAP1.6 cDNA by screening of a human scalp cDNA library with a hKAP1.1 A/B 3′-noncoding region probe, on the assumption that the high sequence identity of the originally identified hKAP1.1 A/B and hKAP1.6 PCR products might extend into their 3′-noncoding region. Although the analysis of 20 cDNA clones failed to lead to the discovery of the hKAP1.6 cDNA, it resulted in the identification of a further, weakly expressed, new member of the hKAP1 family, termed hKAP1.7 (Figure 2b).Figure 2Nucleotide sequence of hKAP1.6 and hKAP1.7, and the deduced amino acid sequence of the protein products. (A) hKAP1.6; (B) hKAP1.7. Nucleotides are numbered consecutively at the left-hand side. The derived amino acid sequence appears below the nucleotide sequence and the pentapeptide repeats are boxed. Polyadenylation signal is underlined. The accession numbers for the respective nucleotide sequences are: hKAP1.6, AB052868; hKAP1.7, AB055057.View Large Image Figure ViewerDownload (PPT) Comparisons between hKAP1.1 A/B, hKAP1.6, and hKAP1.7 not only showed high nucleotide sequence identity in their coding regions, but also complete sequence identity in their 3′-noncoding regions. This high degree of identity was also seen in the 5′-noncoding region of hKAP1.1 A/B and hKAP1.6. Translation of the open reading frame of hKAP1.6 yielded a protein of 131 residues with a calculated molecular weight of 13.6 kDa (Figure 2a). The cysteine and serine content of this protein was high, i.e., 24% and 15%, respectively. In the deduced amino acid sequence, the pentapeptide repeat C(C/Y)Q(p/T)S appeared four times, and a similar sequence, CCETS, appeared once (Figure 2a). In contrast, the deduced hKAP1.7 protein consisted of 85 amino acid residues with a molecular mass of 9.1 kDa, and also had a high cysteine (26%) and serine (13%) content (Figure 2b). In this amino acid sequence the double cysteine containing pentapeptide CCQTS could be found only once. In addition, hKAP1.6 and hKAP1.7 had further variants of these double cysteine containing pentapeptides in their C-terminal region. Database searching for homologous protein sequences revealed that hKAP1.6 and hKAP1.7 shared a high identity with members of the sheep, rat, and human KAP1 family (Figure 3). In particular, the degree of identity between hKAP1.1 A/B, hKAP1.6, and hKAP1.7 was striking. Comparison of hKAP1.1 A/B and hKAP1.6 revealed one amino acid change between the proteins as well as a 46 residue deletion in the amino terminal region of hKAP1.6. Similarly, comparison between hKAP1.1 A/B and hKAP1.7 revealed complete identity between the proteins, but with a 92 residue amino acid deletion in hKAP1.7. In order to determine the region of mRNA expression of hKAP1.1 A/B, hKAP1.6, and hKAP1.7, we analyzed hair follicles from human scalp biopsies by means of in situ hybridization. Due to the high sequence identity between hKAP1.1 A/B, hKAP1.6, and hKAP1.7 it was not possible to create in situ hybridization probes specific for any single family member. The probe used in these studies therefore recognized all four members presented here. In longitudinal sections of anagen hair follicles, mRNA transcripts were exclusively detected predominantly in the highly differentiated portions of the uppermost cortex region (Figure 4a). At higher magnifications, no signal could be detected in the hair shaft cuticle cells (Figure 4b). Moreover, no mRNA expression for these genes was found in the hair fiber medulla (Figure 4c). In addition, no expression was observed in the hair follicle inner root sheath or outer root sheath (Figure 4a–c) or the interfollicular epidermis, sebaceous glands or sweat glands (data not shown). In this study, we identified two cDNAs encoding the novel human KAPs, hKAP1.6 and hKAP1.7. In 1993, Rogers and Powell proposed a modification of the nomenclature used to classify KAPs and termed a group of sheep and human high sulfur KAP proteins found to be expressed in the cortical layer of the hair follicle the KAP1 family (Rogers and Powell, 1993Rogers G.E. Powell B.C. Organization and expression of hair follicle genes.J Invest Dermatol. 1993; 101: 50s-55sAbstract Full Text PDF PubMed Google Scholar). To date, eight members of the KAP1 family have been reported from sheep, rabbit, and humans (Elleman and Dopheide, 1972Elleman T.C. Dopheide T.A. The sequence of SCMK-B2B, a high-sulfur protein from wool keratin.J Biol Chem. 1972; 247: 3900-3909Abstract Full Text PDF PubMed Google Scholar;Powell et al., 1983Powell B.C. Sleigh M.J. Ward K.A. Rogers G.E. Mammalian keratin gene families: organisation of genes coding for the B2 high-sulphur proteins of sheep wool.Nucl Acids Res. 1983; 11: 5327-5346Crossref PubMed Scopus (60) Google Scholar;Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar;Mitsui et al., 1998Mitsui S. Ohuchi A. Adachi-Yamada T. Hotta M. Tsuboi R. Ogawa H. Structure and hair follicle-specific expression of genes encoding the rat high sulfur protein B2 family.Gene. 1998; 208: 123-129Crossref PubMed Scopus (12) Google Scholar), and four additional human members have been described in a recent report (Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). In humans, two KAP1 family genes were originally identified and termed hB2A and hB2B (Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar). In keeping with the newer nomenclature, these genes were renamed hKAP1.1 A and hKAP1.2 (Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). In addition, recent evidence arose showing another identical copy of the hKAP1.1 A gene present on a separate human gene locus. This gene has been designated hKAP1.1B (Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). Like the KAP1 members described previously, the new hKAP1.6 and hKAP1.7 proteins had a high cysteine residue content and showed significant homology with all of the known KAP1 family members, especially the two initially described human members. The multialignment of the members of the human, sheep, and rat KAP1 family revealed that most of these proteins can be divided into five individual domains: an N-terminal domain, a repetitive I domain, a central nonrepetitive domain, a repetitive II domain, and a C-terminal domain (Figure 3). The N-terminal, central nonrepetitive, and repetitive II domains appear to be highly conserved in all three species, which could indicate that they play an essential role in the function of the KAP1 family. This is strikingly seen in the hKAP1.7 protein, which contains these three domains but does not have the multiple double cysteine containing amino terminal repeat structure seen in the other proteins isolated thus far. In contrast, the C-terminal domain is the smallest region found, consisting of 3–18 residues, and is conserved to a lesser degree in the three species analyzed. This region, however, shows high similarity within each respective species. One further region that is striking among the KAP1 family members is the repetitive I domain, with its large degree of repetitiveness and size variability. Decapeptide repeat structures have been previously reported in the KAP1 family members of sheep and rat (Powell et al., 1983Powell B.C. Sleigh M.J. Ward K.A. Rogers G.E. Mammalian keratin gene families: organisation of genes coding for the B2 high-sulphur proteins of sheep wool.Nucl Acids Res. 1983; 11: 5327-5346Crossref PubMed Scopus (60) Google Scholar;Mitsui et al., 1998Mitsui S. Ohuchi A. Adachi-Yamada T. Hotta M. Tsuboi R. Ogawa H. Structure and hair follicle-specific expression of genes encoding the rat high sulfur protein B2 family.Gene. 1998; 208: 123-129Crossref PubMed Scopus (12) Google Scholar), and fundamentally appeared to be composed of two pentapeptide subelements. These elements are CCQPT and SIQTS in sheep, and CCQP(S/T) and C(S/T)QSS in rat. In human, they consist essentially of amino acid variations of a double cysteine-containing pentapeptide, CCQ(p/T)S and CCETS. Therefore, like the C-terminal domain, a certain degree of sequence repeat specificity is present in individual species. In addition, the comparisons of KAP1 members in the respective species showed exclusively high homologies at the N-terminal, central nonrepetitive, repetitive II, and C-terminal domains in all species, indicating that the distinctions among individual KAP1 members in the same species might be dependent upon the number of pentapeptide repeats in the repetitive I domain. Therefore, the repetitive I domain may decide the individual functional characteristics of KAP1 members in a specific species, and may participate in species-specific interactions with the C-terminal domain of other KAP1 family members. Interestingly, hKAP1.1 A/B and hKAP1.2 have another unique amino acid sequence (consensus sequence, FCGFPS(F/C)ST(G/S)GTC(D/G)SS) inserted into the repetitive I region of these proteins. This sequence cannot be detected in the repetitive I domains of rat and sheep KAP1 members already reported. The analysis of amino acid multialignment of KAP1 members could not provide convincing evidence for the presence of orthologous proteins among the three species. Recently four new members of the KAP1 family (hKAP1.1B, hKAP1.3, hKAP1.4, hKAP1.5), possessing DNA sequences unique to hKAP1.6 and hKAP1.7 as well as to the other previously described human KAP1 family members (Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar), were identified on the human genome and cDNA sequences for three of these genes could be found (Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). An orthologous relationship of these sequences to that of the KAP1 family members in sheep and rat could also not be found in these studies. Therefore, at present it is uncertain whether there are identifiable orthologs of sheep and rat KAP1 members in the human genome. In addition, it is difficult to decide whether one human KAP1 member corresponds to a member in another species because not all of the KAP1 family members in any one species have yet been found. Due to this lack of orthologous relationships between species, the decision taken in the naming of the two new KAP1 family members was based exclusively on what was, as yet, known in humans (Zhumbaeva et al., 1992Zhumbaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar;Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). Therefore, we have termed the novel KAP1 members identified in this study hKAP1.6 and hKAP1.7. The in situ hybridization performed here showed the cumulative expression of the transcripts of hKAP1.1 A/B, hKAP1.6, and hKAP1.7 exclusively in the differentiated portion of the hair follicle cortex, which was consistent with the previous reports on sheep and rat KAP1 family members (Powell et al., 1991Powell B.C. Nesci A. Rogers G.E. Regulation of keratin gene expression in hair follicle differentiation.Ann NY Acad Sci. 1991; 642: 1-20Crossref PubMed Scopus (73) Google Scholar;Mitsui et al., 1998Mitsui S. Ohuchi A. Adachi-Yamada T. Hotta M. Tsuboi R. Ogawa H. Structure and hair follicle-specific expression of genes encoding the rat high sulfur protein B2 family.Gene. 1998; 208: 123-129Crossref PubMed Scopus (12) Google Scholar). This is also consistent with recent data showing expression of hKAP1.5 in the human hair follicle cortex (Rogers et al., 2001Rogers M.A. Langbein L. Winter H. Ehmann C. Praetzel S. Schweizer J. Characterization of a cluster of human high/ultrahigh sulfur keratin associated protein (KAP) genes imbedded in the type I keratin gene domain on chromosome 17q12–21.J Biol Chem. 2001; 276: 19440-19451Crossref PubMed Scopus (84) Google Scholar). It must also be kept in mind, however, that due to the high homology occurring between these four gene sequences, the probe used for the in situ hybridization cannot differentiate between members. Therefore probable quantitative differences among the individual members cannot be distinguished. Moreover, if there were individual differences in the initiation and termination of mRNA synthesis, this would also not be detected. In any case, the isolation of both a specific RT-PCR product of hKAP1.6 from human scalp mRNA and a clone from a human scalp cDNA library for hKAP1.7 point to the expression of these genes in human scalp. The presence of only one hKAP1.7 transcript in the 20 cDNA clones isolated and the absence of an hKAP1.6 transcript indicate low expression levels of these two genes compared to hKAP1.1 A/B. Mutations in the hair keratin genes hHb1 and hHb6 have previously been shown to be present in patients suffering from the congenital hair disease monilethrix, which is characterized by beaded and fragile hairs (Winter et al., 1997aWinter H. Rogers M.A. Langbein L. et al.Mutations in the hair cortex keratin hHb6 cause the inherited hair disease monilethrix.Nature Genet. 1997; 16: 372-374Crossref PubMed Scopus (156) Google Scholar, Winter et al., 1997bWinter H. Rogers M.A. Gebhardt M. et al.A new mutation in the type II hair cortex keratin hHb1 involved in the inherited hair disorder monilethrix.Human Genet. 1997; 101: 165-169Crossref PubMed Scopus (89) Google Scholar;Korge et al., 1999Korge B.P. Hamm H. Jury C.S. et al.Identification of novel mutations in basic hair keratins hHb1 and hHb6 in monilethrix: implications for protein structure and clinical phenotype.J Invest Dermatol. 1999; 113: 607-612Crossref PubMed Scopus (52) Google Scholar). In an attempt to find a causal mutation in a family of patients with fragile, but not moniliform, hairs, we analyzed all type II hair keratin genes, including hHb1 and hHb6, of these patients, and detected no pathogenic mutations. As KAPs have also been postulated as playing a role in hereditary hair genodermatoses, we decided to analyze these putative candidate genes, which eventually led to the discovery of the new KAP1 family members presented here. We could not detect mutations in either the hKAP1.1 A/B or hKAP1.6 genes of the patients analyzed, however. More recently, it has been reported that transgenic mice overexpressing Hoxc 13 in differentiating keratinocytes of hair follicles developed alopecia and several KAP genes were downregulated in postnatal skin of these mice (Tkatchenko et al., 2001Tkatchenko A.V. Visconti R.P. Shang L. et al.Overexpression of Hoxc 13 in differentiating keratinocytes results in downregulation of a novel hair keratin gene cluster and alopecia.Development. 2001; 128: 1547-1558PubMed Google Scholar). Thus, the production of transgenic mice expressing a mutated form of the KAPs is probably more likely to provide insight into its function in the hair follicles, and may represent a putative model for a human hair disorder in the future. Furthermore, the discovery of the complete set of human KAP gene, of which hKAP1.6 and hKAP1.7 are a part, should advance our knowledge concerning the characteristics of hair between individuals as well as the role of KAPs in hereditary hair diseases. We wish to thank Claudia Ehmann and Silka Praetzel for their technical expertise. We also wish to thank Dr. Herbert Spring for his help with confocal laser microscopy. The arrayed human scalp cDNA library used in this report is available from the German Human Genome Resource Center (see http://www.rzpd.de). The library number is p636. Likewise the clone hKAP1.7, which was isolated from this library, is available there under the number DKFZp636L2317Q4. We wish to thank the members of the RZPD for their continuing support. Part of this work was supported by the German Research Council under the number SCHW539/4-1.