Michael F. Naso, Bailin Liang, C

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Dermokine: An Extensively Differentially Spliced Gene Expressed in Epithelial Cells Michael F. Naso, Bailin Liang, C. Chris Huang, Xiao-Yu Song, Lillian Shahied-Arruda, Stanley M. Belkowski, Michael R. D'Andrea, Debbie A. Polkovitch, Danielle R. Lawrence, Don E. Griswold, Ray W. Sweet, Bernard Y. Amegadzie Journal of Investigative Dermatology Volume 127, Issue 7, Pages 1622-1631 (July 2007) DOI: 10.1038/sj.jid.5700779 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Agarose gel analysis of RT-PCR-amplified transcripts expressed in adult human skin. Molecular weight is a 1-kb DNA ladder. Oligonucleotide pairs specific for each isoform were used in the analysis and are listed above the gel image. Arrows and brackets point to bands that were cloned and sequenced. Journal of Investigative Dermatology 2007 127, 1622-1631DOI: (10.1038/sj.jid.5700779) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Dermokine gene structure. Schematic representation of (a) the human and (b) mouse dermokine loci. Exons are indicated by filled lines and arrows. Exons that have been found to be differentially spliced in either the in silico or RT-PCR analysis are indicated by asterisks above it. Representative transcripts isolated by RT-PCR are shown below the genomic structure. Position of the 5′ and 3′ in situ hybridization probes are also shown. Journal of Investigative Dermatology 2007 127, 1622-1631DOI: (10.1038/sj.jid.5700779) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Determination of the mouse γ isoform. (a) Genomic sequence of the mouse locus showing the potential new termination exon and translation. (b) Nucleotide and amino-acid translation of the 3′ end of clones isolated by 3′RACE using an oligonucleotide specific for this new exon. (c) RT-PCR amplification of the mouse dermokine γ. Brackets indicate amplification products cloned and sequenced. Only clones of around 1.2kb were found to be specific for mouse dermokine. (d) Nucleotide and translated amino-acid sequence of mouse dermokine γ. Journal of Investigative Dermatology 2007 127, 1622-1631DOI: (10.1038/sj.jid.5700779) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Differential splice donor usage in dermokine δ and ε transcripts. (a) Genomic organization of this region including the initiation site in exon 7, exon 8, and the intron between them. Exons are boxed and are in uppercase, whereas introns are in lowercase. The two different splice donors are underlined and numbered. (b–d) Three different splice variants isolated from dermokine δ and ε transcripts. Journal of Investigative Dermatology 2007 127, 1622-1631DOI: (10.1038/sj.jid.5700779) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 In situ hybridization analysis of dermokine expression in various tissues. A digoxigenin-labeled anti-sense riboprobe specific for the 5′ and 3′ end of the dermokine β gene was used in the analysis. Dermokine-specific hybridization is indicated by arrows. (a) skin (arrows to epidermis); (b) skin with sense-strand as a negative control; (c) gut (arrows to epithelial cells); (d) lung (arrows on macrophages, arrow-head on endothelial cells) (e) breast (arrows on epithelial cells); (f) prostate (arrows on epithelial cells); (g) liver (arrows on hepatocytes); and (h) pancreas (arrows on acinar cells). All are at original magnification × 40 or final magnification × 600. Bar=25μm. Journal of Investigative Dermatology 2007 127, 1622-1631DOI: (10.1038/sj.jid.5700779) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Biochemical characterization of recombinant dermokine gamma. (a, Left panel) SDS-PAGE and Western blot analysis of recombinantly expressed and purified dermokine γ. Three micrograms of purified recombinant dermokine γ with a C-terminal Hist tag was run under non-reduced and reduced conditions on a 4–12% gradient SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane for Western blotting. The Western blot was probed with the rabbit polyclonal anti-dermokine antibody. Notice the existence of multimers in the non-reduced sample in lane 1, which are confirmed to be dermokine by the Western blot. Molecular weight standards are indicated to the left of the gel. (a, Right panel) Total human skin proteins were similarly run under non-reduced conditions and probed with the polyclonal antibody used in the left panel and showing the existence of multimers as well. (b) Matrix-assisted laser desorption ionization–mass spectrometry analysis of recombinant dermokine γ. Matrix-assisted laser desorption ionization–mass spectrometry was performed under reduced (top) and non-reduced (bottom) conditions. The existence of the multimers seen in the SDS-PAGE analysis is also seen in this analysis. Molecular weight of individual species is indicated. (c) Carbohydrate analysis of recombinant dermokine γ. Expressed and purified dermokine γ was treated with O-glycosidase with and without sialidase. Released glycans were labeled with anthranilic acid and analyzed by HPLC. The peak around 29minutes corresponds to the O-linked glycans released by either O-glycosidase or sialidase. Journal of Investigative Dermatology 2007 127, 1622-1631DOI: (10.1038/sj.jid.5700779) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Immunohistochemical analysis of dermokine expression in normal human tissues. A rabbit polyclonal antibody raised against the dermokine gamma protein was used to detect dermokine expression. (a) Skin (arrows to epidermis, note intense labeling in the squamous layer of the epidermis); (b) skin without primary antibody as a negative control; (c) kidney (arrows to ductal epithelial cells); (d) liver (arrows on punctate, granular labeling in the hepatocytes); (e) lung (arrows on macrophages, arrowhead on endothelial cells): (f) pancreas (arrows on islet cells); (g) vessel (arrows on the endothelial cells; arrowhead on the smooth muscle cell); and (h) spleen, showing lack of labeling. All are at original magnification × 40 or final magnification × 600. Bar=25μm. Journal of Investigative Dermatology 2007 127, 1622-1631DOI: (10.1038/sj.jid.5700779) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Molecular features of dermokine protein isoforms. (a) Proposed domain architecture for dermokine isoforms. (b) Primary amino-acid sequence of the collagen-like domain. Gly-X-Y repeats are indicated, with proline residues highlighted in bold. Journal of Investigative Dermatology 2007 127, 1622-1631DOI: (10.1038/sj.jid.5700779) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions