Envoplakin, a Novel Precursor of the Comified Envelope That Has Homology to Desmoplaldn

The cornified envelope is a layer of transglutaminase cross-linked protein that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We present the sequence of one of the cornified envelope precursors, a protein with an apparent molecular mass of 210 kD. The 210-kD protein is translated from a 6.5-kb mRNA that is transcribed from a single copy gene. The mRNA was upregulated during suspension-induced terminal differentiation of cultured human keratinocytes. Like other envelope precursors, the 210-kD protein became insoluble in SDS and [3-mercaptoethanol on activation of transglutaminases in cultured keratinocytes. The protein was expressed in keratinizing and nonkeratinizing stratified squamous epithelia, but not in simple epithelia or nonepithelial cells. Immunofluorescence staining showed that in epidermal keratinocytes, both in vivo and in culture, the protein was upregulated during terminal differentiation and partially colocalized with desmosomal proteins. Immunogold EM confirmed the colocalization of the 210-kD protein and desmoplakin at desmosomes and on keratin filaments throughout the differentiated layers of the epidermis. Sequence analysis showed that the 210-kD protein is homologous to the keratin-binding proteins desmoplakin, bullous pemphigoid antigen 1, and plectin. These data suggest that the 210-kD protein may link the cornified envelope to desmosomes and keratin filaments. We propose that the 210-kD protein be named "envoplakin." T hE cornified envelope is a layer of insoluble protein, ~15 nm thick, that is deposited under the plasma membrane of keratinocytes in the outermost layers of the epidermis (reviewed by Reichert et al., 1993; Simon, 1994). The cornified envelope provides a protective barrier between the environment and the living layers of the skin, and is believed to play an important role in maintaining the structural integrity of the epidermis. The envelope is made of several precursor proteins that are cross-linked by e-(~/-glutamyl) lysine bonds in a calcium-dependent reaction that is catalyzed by epidermal transglutaminases. In lamellar ichthyosis, an autosomal recessive disorder of the skin, reduced activity of the membrane-bound, keratinocyte-specific transglutaminase (TGK) 1 results in severe perturbation of epidermal differentiation and function (Huber et al., 1995). Please address all correspondence to Dr. F. Watt, Keratinocyte Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Tel.: 44 171 269 3528; Fax: 44 171 269 3078. 1. Abbreviations used in this paper: BPAG1, bullous pemphigoid antigen 1; DPI, desmoplakin I; FSG/PBS, fish skin gelatin in PBS; TGK, keratinocyte transglutaminase. Current models propose that in the first step of cornified envelope assembly TGK catalyzes the cross-linking of involucrin at the plasma membrane, and that other, less abundant, envelope precursors such as cornifin, elafin, and the small proline-rich proteins are added subsequently (Eckert et al., 1993; Steinert and Marekov, 1995). The cytoplasmic surface of the envelope is believed to be composed of loricrin (Steinert and Marekov, 1995). All envelope precursors that have been characterized so far are soluble cytoplasmic proteins, with the exception of loricrin, which is a component of insoluble cytoplasmic aggregates (keratohyalin granules). It is not clear whether the cellular localization of TGK is sufficient to direct the assembly of the envelope to the inner face of the plasma membrane, or whether specific membrane-associated precursors are required for anchorage. A further unanswered question is how keratin filaments and desmosomes are linked to the cornified envelope (Haftek et al., 1991; Ming et al., 1994; Steinert and Marekov, 1995). In 1984, Simon and Green identified two membraneassociated proteins with apparent molecular masses of 195 and 210 kD that become incorporated into the cornified envelope on transglutaminase activation. Antibodies to the two proteins were absorbed by isolated cornified enve© The Rockefeller University Press, 0021-95251961081715115 $2.00 The Journal of Cell Biology, Volume 134, Number 3, August 1996 715-729 715 on M arch 5, 2007 w w w .jc.org D ow nladed fom lopes. Both proteins are expressed by epidermal keratinocytes, but not by dermal fibroblasts, and are upregulated during keratinocyte terminal differentiation. Simon and Green proposed that the two proteins might anchor other envelope proteins to the plasma membrane. We now report the sequencing of overlapping eDNA clones encoding the 210-kD cornified envelope precursor. The predicted structure of the 210-kD protein, its homology with other known proteins, and its expression pattern strongly suggest that it is associated with the plasma membrane and may link keratin filaments and desmosomes to the cornified envelope. Materials and Methods Screening of cDNA Libraries and cDNA Sequencing A mouse polyclonal antiserum (M) raised against the 210-kD protein (Simon and Green, 1984) was used to screen a random-primed keratinocyte hgtll expression library, as described previously (Hudson et al., 1992), and a eDNA clone (p210-1) containing a 1-kbp insert was isolated. A probe (P1) derived from this clone was used to screen an oligo-dT-primed plasmid library (kindly provided by P. Jones, Imperial Cancer Research Fund, London) and a second random-primed kgtll library (a gift from R. Buxton, National Institute for Medical Research, London), and two eDNA clones were isolated, p210-21 from the plasmid library and p210141 from the kgtll library. A further eDNA clone, p210-23, was isolated from the second hgtll library using a probe (P141) containing the 5' end of clone p210-141. For screening of libraries with DNA probes, eDNA fragments were radiolabeled by random priming (Sambrook et al., 1989). Hybridizations were performed at 65°C for 16 h in a hybridization buffer containing 0.5 M sodium phosphate buffer, pH 7.2, 1 mM EDTA, pH 8.0, 1% BSA, 7% SDS, and 100 i~g/ml denatured and fragmented herring sperm DNA (Boehringer Mannheim, Lewes, UK). Washing was performed at 65°C in 0.1 × SSC (1 × SSC = 150 mM NaCI, 15 mM sodium citrate) and 0.1% SDS. The inserts of the hgtll clones were subcloned into pBluescript II KS(+/-) (Stratagene Ltd., Cambridge, UK) for sequencing. The eDNA clones were sequenced by the dideoxy chain termination method using the Sequenase II kit (Amersham International pie., Bucks, UK) and oligonucleotides synthesized by Oligonucleotide Synthesis Services, ICRF. Southern and Northern Blot Analyses Genomic DNA was isolated from cultured human foreskin keratinocytes, digested to completion with restriction enzymes (New England Biolabs, Hitchin, UK), electrophoresed in a 1% agarose gel, and transferred to HybondN membrane (Amersham International plc.), according to the manufacturer's instructions. A DNA molecular weight standard (1-kbp ladder; GIBCO BRL, Paisley, UK) was electrophorased in the same gel. Hybridization was performed at 65°C for 16 h with a DNA probe (radiolabeled as described above) in a hybridization buffer containing 0.9 M NaCI, 50 mM sodium phosphate buffer, pH 7.2, 5 mM EDTA, pH 8.0, 0.1% BSA, 0.1% polyvinylpyrrolidine, 0.1% Ficoll (Pharmacia, St. Albans, UK), and 100 p.g/ml denatured and fragmented herring sperm DNA (Boehringer Mannheim). Washing was performed as described above. Total RNA was isolated from cultured human keratinocytes by extraction with guanidine thiocyanate (Sambrook et al., 1989). Poly(A) ÷ RNA was purified from total RNA using oligo(dT)-cellulose spin columns (Pharmacia). 2 Ixg of poly(A) + RNA per lane and RNA molecular weight standards (RNA ladder; GIBCO BRL) were separated on 1% formaldehyde gels, as described (Sambrook et al., 1989). To facilitate the transfer of large RNA species, RNA was partially hydrolyzed by soaking the gel for 20 rain in 50 mM NaOH, then neutralizing the gel for 30 min in 20 × SSC. The RNA was transferred to an HybondN membrane, as recommended by the manufacturer. Hybridizations were performed at 42°C for 16 h with probes, labeled as described above, in a hybridization buffer containing 50% formamide, 0.9 M NaCI, 50 mM sodium phosphate buffer, pH 7.2, 5 mM EDTA, pH 8.0, 0.1% BSA, 0.1% polyvinylpyrrolidine, 0.1% Fieoll (Pharmaeia), and 100 p.g/ml denatured and fragmented herring sperm DNA (Boehringer Mannheim). Washing was performed as described above. The involucrin probe was derived from plasmid pI-2 (Eckert and Green, 1986). Computational Analysis of the Predicted Amino Acid Sequence Secondary structure predictions were performed using the algorithms of Gamier et al. (1978) or Chou and Fasman (1978), as implemented by MacVector 3.5 (International Biotechnologles Inc., Cambridge, UK). Coiled coil analyses were performed with the program MacStripe (Knight and Kendriek-Jones, 1993), based on the algorithm described by Lupas et al. (1991). The predicted protein sequence was examined for potential transmembrane domains using MacVector 3.5, based on the method of Kyte and Doolittle (1982), and Top Pred II (Claros and yon Heijne, 1994), based on the method of Argos and Rao (1986). The SwissProt and PIR protein databases were searched with the program BLAST at the National Center for Biotechnology Information (Bethesda, MD). Dotplot homology comparisons were performed using the programs COMPARE and DOTPLOT in the UWGCG (University of Wisconsin Genetics Computer Group) software suite.