Isolation and Characterization of Human Repetin, a Member of the Fused Gene Family of the Epidermal Differentiation Complex Marcel Huber, Georges Siegenthaler,

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Isolation and Characterization of Human Repetin, a Member of the Fused Gene Family of the Epidermal Differentiation Complex Marcel Huber, Georges Siegenthaler, Nicolae Mirancea, Ingo Marenholz, Dean Nizetic, Dirk Breitkreutz, Dietmar Mischke, Daniel Hohl Journal of Investigative Dermatology Volume 124, Issue 5, Pages 998-1007 (May 2005) DOI: 10.1111/j.0022-202X.2005.23675.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Genomic organization of the human repetin gene. (A) The genomic structure of cosmid 31H23 containing the repetin gene with exons I–III and the different cDNA clones are shown. Probes A and B designate DNA fragments used for cDNA cloning and hybridization experiments. (B) Depicts the repetin domain structure with the N-terminal part (amino acids 1–248) containing EF hands, the central repetitive domain (amino acids 249–584), and the C-terminal part (amino acids 585–784). P1, P3, and P4 indicate the positions of three peptides that were used for production of affinity-purified polyclonal antibodies. Journal of Investigative Dermatology 2005 124, 998-1007DOI: (10.1111/j.0022-202X.2005.23675.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Nucleotide and deduced amino acid sequence of human repetin. These data have been compiled from cosmid clone 31H23 and cDNA clones λhrep and pTcR3.8 (Figure 1a). The single-letter code amino acid sequence is numbered with the initiation methionine as amino acid 1. Exons I–III are marked in bold, the putative TATA box is underlined, and the polyadenylation signal sequence is in italics. This sequence has been deposited in Genbank under accession number AY219924. Journal of Investigative Dermatology 2005 124, 998-1007DOI: (10.1111/j.0022-202X.2005.23675.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Protein expression of human repetin. Immunoblot analysis of repetin expression in cytosolic (lane 1), membrane (lane 2) and cytoskeletal (lane 3) extracts from suspension-induced human keratinocytes (A) and human foreskin biopsy (B) detected with antibody AF646. (C) Depicts cytoskeletal fractions of suspension-induced human keratinocytes immunoblotted with antibodies anti-peptide P1 (lane 1), anti-peptide P3 (lane 2), and reaction omitting the first antibody (lane 3). (D) Demonstrates immunodetection using AF646 with whole cells extracts of p28HR transformed BL21 CodonPlus(DE3)-RIL bacteria 180 min after IPTG induction (lane 1) and the cytoskeletal fraction of suspension-induced human keratinocytes (lane 2). The positions of molecular weight markers in kDa are indicated on the left. Journal of Investigative Dermatology 2005 124, 998-1007DOI: (10.1111/j.0022-202X.2005.23675.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Amino acid comparison of EF hand proteins. Comparison of the N-terminal amino acid sequences of human repetin with mouse repetin (Genbank T30251), human profilaggrin (A48118), human trichohyalin (A45973), human S100A1 (NP_006262), human S100A2 (NP_005969), human calpactin I light chain (JC1139), human calcyclin (P06703), human tumor-related protein (AAD55747), and mouse hornerin (AAK15791). The letters h and m stand for human and mouse, respectively. Journal of Investigative Dermatology 2005 124, 998-1007DOI: (10.1111/j.0022-202X.2005.23675.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 45Ca2+ overlay assay. (A) Autoradiograph after 45Ca2+ overlay assay with protein extracts from psoriatic scales (lane 1), pET28HREF transformed BL21 (DE3) pLysS bacteria before IPTG induction (lane 2), and 180 min after IPTG induction (lane 3). (B) Autoradiograph after removing radioactive Ca2+ by incubation with cold Ca2+. The positions of molecular weight markers in kDa are indicated on the left. Journal of Investigative Dermatology 2005 124, 998-1007DOI: (10.1111/j.0022-202X.2005.23675.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Repetin expression analysis in cultured human keratinocytes and epidermal biopsies. (A) RNA isolated from normal human keratinocytes cultured in high calcium medium on irradiated 3T3 feeder layer at confluency (lanes 1 and 2), 5 d (lanes 3 and 4) and 7 d (lanes 5 and 6) postconfluency. (B) Normal human keratinocytes were suspended in 1.75% methylcellulose and RNA isolated before suspension (lanes 1 and 2), and 17 h (lane 3), 24 h (lanes 4 and 5), or 48 h (lanes 6 and 7) after suspension. (C) Total RNA from keratinocytes after suspension culture of 48 h (lanes 1–4) and from biopsies from human skin (lane 5) or human foreskin (lane 6) was hybridized with probes for human repetin (Rep; probe B; Figure 1a), loricrin (Lor;Hohl et al, 1991), and glyceraldehyde-phosphate dehydrogenase (GAPDH). The positions of 28S and 18S rRNA are indicated on the left. Journal of Investigative Dermatology 2005 124, 998-1007DOI: (10.1111/j.0022-202X.2005.23675.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Repetin is a late epidermal differentiation marker highly expressed in tongue. Immunofluorescence analysis of repetin was carried out using antibody AF 646 and FITC-labeled anti-rabbit IgG antibody on frozen section of human skin (A), human foreskin (B), human tongue (C and D), and human hair follicles (E and F). Journal of Investigative Dermatology 2005 124, 998-1007DOI: (10.1111/j.0022-202X.2005.23675.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Repetin is associated with keratohyalin granules and is expressed in the transition zone in human skin. Immunelectron microscopic analysis was carried out with anti-repetin antibody AF646 and gold-labeled (15 nm) secondary antibody. (A) Association of repetin with keratohyalin granules in the upper granular layers. (B) Repetin expression in the zone between upper granular layer and the stratum corneum. (C) Higher magnification of the same region as in (B). (D) Demonstration that repetin immunoreactivity is drastically reduced in the upper stratum corneum. Scale bar: 440 nm (A), 1100 nm (B), 360 nm (C), 250 nm (D). Journal of Investigative Dermatology 2005 124, 998-1007DOI: (10.1111/j.0022-202X.2005.23675.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions