Abstract
Based on cDNA cloning and sequencing, human epiplakin has been classified as a member of the plakin protein family of cytolinkers. We report here the characterization of the mouse epiplakin gene locus and the isolation of full-length mouse epiplakin cDNA using BAC vectors. We found that the protein is encoded by a single remarkably large exon (>20 kb) that consists of a series of 0.8-1.5-kb-long DNA repeats, eight of which are virtually identical. Consequently, mouse epiplakin contains 16 plakin repeat domains, three more than reported for the human protein and eight more than predicted for the mouse protein based on the contig characterized by the Mouse Genome Sequencing Consortium. Using antibodies raised to a highly conserved repeating epiplakin sequence domain, we show that the protein in cells is expressed in its full length (725 kDa), and we provide evidence that the size of human epiplakin previously may have been underestimated. In addition we show on transcript and protein levels that epiplakin is restricted to epithelial tissues and that its gene maps to mouse chromosome 15 (human chromosome 8). This study lays the groundwork for future genetic approaches aimed at defining the biological role of this unique protein.
Highlights
Epiplakin was originally identified as a 450-kDa epidermal autoantigen showing immunoreactivity with the serum of a patient suffering from a subepidermal blistering disease [1, 2]
Too, it could be shown that residues downstream of its third plakin repeat domains (PRDs) module were important for keratin binding, whereas the linker between the second and third module as well as the second PRD module itself have been reported to be required for vimentin interaction [12,13,14,15]
Isolation of cDNA and DNA Sequencing—cDNA clones were isolated from a mouse skin cDNA library using a 830-bp-long rat epiplakin cDNA fragment as probe. 5Ј- and 3Ј-rapid amplification of cDNA ends as well as PCR analyses of MarathonReadyTM cDNA derived from mouse kidney and 11-day-old mouse embryo (Clontech, Palo Alto, CA) were performed using Advantage cDNA polymerase (Clontech) in a Perkin-Elmer GeneAmp 9700 thermal cycler, following the protocols supplied by the manufacturers
Summary
Isolation of cDNA and DNA Sequencing—cDNA clones were isolated from a mouse skin cDNA library (strain C57BL/6; Stratagene) using a 830-bp-long rat epiplakin cDNA fragment as probe. 5Ј- and 3Ј-rapid amplification of cDNA ends as well as PCR analyses of MarathonReadyTM cDNA derived from mouse kidney and 11-day-old mouse embryo (Clontech, Palo Alto, CA) were performed using Advantage cDNA polymerase (Clontech) in a Perkin-Elmer GeneAmp 9700 thermal cycler, following the protocols supplied by the manufacturers. Isolation of cDNA and DNA Sequencing—cDNA clones were isolated from a mouse skin cDNA library (strain C57BL/6; Stratagene) using a 830-bp-long rat epiplakin cDNA fragment as probe. RNase Protection Assays—cDNA sequences used as probes were subcloned into pSP64 (Promega, Madison, WI) by PCR cloning using primers flanked with suitable restriction sites. Maltose binding protein fusion proteins were brought into solution by sonication in 20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EGTA and purified using amylose affinity chromatography. The blots were developed using antibodies to epiplakin (diluted 1:75,000), and a rabbit antiserum was raised against a recombinant amino-terminal protein fragment corresponding to exons 9 –12 (Glu419–Val451) of rat plectin (diluted 1:6000), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories; dilution, 1:12,500) and visualization of proteins with SuperSignal substrate (Pierce). All of the sections (2 m) were incubated with antibodies to epiplakin at a dilution of 1:10000, followed by incubation with Texas Red-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories)
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