Isolation of cDNAs encoding desmosomal plaque proteins: evidence that bovine desmoplakins I and II are derived from two mRNAs and a single gene.

Abstract

Desmoplakins (DPs) I and II (%240 and -210 kDa) are major components of the internal portion of the desmosomal cytoplasmic plaque. Desmosomes play a crucial role in cell-cell adhesion and serve as specific attachment sites for cytoplasmic intermediate riaments. Although DP-I and -II are closely related molecules, their structure (i.e., amino acid or DNA sequence) has not been determined. In addition, it is not known whether these proteins are derived from one or more genes or whether they result from posttranscriptional or posttranslational events. This paper describes the isolation and characterization of eight DP cDNA clones from a bovine Agtll expression library. Fusion proteins from six of these clones selected antibodies that reacted with DP-I and -II and two selected antibodies that reacted with DP-I alone. Antibodies made against fusion protein produced by the DP1A clone reacted specifically with DP-I and -II on immunoblots. When used for indirect immunofluorescence on bovine tongue cryostat sections and cultured mouse keratinocytes, these antibodies produced a typical desmosomal staining pattern. RNA blot analysis demonstrated hybridization of three DP-I/iI cDNA probes with two messages of %7.5 and %9.5 kilobases in bovine tongue RNA. In contrast, a cDNA clone that affinitypurified antibodies reacting with DP-I only hybridized exclusively with the 9.5-kilobase band. Southern blots of genomic DNA digested with a panel of restriction enzymes were hybridized with one probe derived from a DP-I/ll clone and with one from a DP-I clone. Both probes hybridized with single bands of the same size in each digested sample of DNA. Together, these data suggest that DP-I and DP-ll are translated from two separate messages in bovine tongue and that these messages may be derived from a single gene.Desmoplakins (DPs) I and II (-=240 and "210 kDa) are closely related proteins that have been localized to the internal portion of the cytoplasmic plaque of spot desmosomes (1-3). Desmosomes (macula adhaerens) are intercellular junctions regarded as playing a crucial role in cell-cell adhesion as well as providing specific attachment sites for cytoplasmic intermediate filaments (4-6). Although the DPs are major constituents of the desmosome, little is known regarding their molecular structure (including amino acid and DNA sequence as well as their higher-order structure), their synthesis and expression in cells and tissues, or their possible interactions with other desmosomal and cytoplasmic proteins such as intermediate filaments.The conclusion that the two DP polypeptides are closely related is based on similarities in their solubility characteristics, isoelectric points, tryptic peptide maps, amino acid composition, and immunological criteria (7,8). Monospecific polyclonal and monoclonal antibodies react with both DP-I and -II (9). Although there are several monoclonal antibodies that react only with DP-I, none specific for DP-II has been reported (9). The distribution of the DPs in cells and tissues of a variety of o...

Human autoantibodies against desmoplakins in paraneoplastic pemphigus.

“…Desmoplakin I has been found in all desmosomes studied (13,18), whereas DP II has been found predominantly, but not exclusively, in stratified squamous epithelia (19). Overlapping cDNA clones encoding two major domains of the human desmoplakins have been identified (4), and suggest that DP I and II are encoded by separate mRNAs derived from a single gene (5 Although we have previously reported that passive transfer of whole immunoglobulin fractions from patients with paraneoplastic pemphigus into neonatal mice can induce cutaneous and esophageal acantholysis (cell-cell detachment, 1, 2), the role of the autoantibodies that are directed specifically against the desmoplakins in the induction of lesions is not known. Any speculation that they might be causative is tempered by the fact that the desmoplakins are intracellular proteins with no transmembrane or extracellular domains, and it is not clear how serum autoantibodies could bind to and presumably compromise the function of such intracellular antigens.…”

“…Their sequence and structure are known (3,4) and desmoplakin II is apparently a product of alternate splicing of the desmoplakin I transcript (5). Desmoplakin I has a M, = 250,000 in SDS-PAGE and a calculated mol wt of 201,000.…”

Discovering the Molecular Components of Intercellular Junctions--A Historical View

“…1D). Later, K. Green and her group determined that desmoplakins I and II are two splice mRNA forms derived from the same gene and that this large protein presents a very special organization with a near-aminoterminal plakin domain, a central coiled-coil region, and a so-called plakin-repeat domain in the carboxy-terminal portion (Green et al 1988(Green et al , 1990Virata et al 1992;Godsel et al 2004), so to say the prototype of what later was termed the "plakin family" of proteins (Ruhrberg and Watt 1997). The identification of the desmoplakins was soon followed by the localization of epidermaltype desmosomal proteins and glycoproteins in various cell types and species, although not in all cells that were known to contain true desmosomes (cf.…”

Structural and Functional Diversity of Desmosomes

“…Initial cloning studies used antibodies to desmosomal components or degenerate oligonucleotide probes derived from peptide sequencing data to screen cDNA libraries (Green et al, 1988;Cowin et al, 1986;Goodwin et al, 1990). In 1989, Franke et al (1989) published the fi rst human sequence encoding a desmosomal protein, plakoglobin (abbreviated here as PG, offi cial gene symbol-JUP ).…”