Human Dsg3 Research Articles

Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B.

We identified a novel pathogenicity island in Staphylococcus aureus which contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, and edin-B gene in tandem and the phage resistance gene, flanked by hsdM, hsdS (restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), and Staphylococcus hyicus ETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced by S. aureus and is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive for edin-B were suggested to be clonally associated, and all edin-B-positive strains tested were positive for etd. Among 18 etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.

Suprabasal desmoglein 3 expression in the epidermis of transgenic mice results in hyperproliferation and abnormal differentiation.

The desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) isoforms of the desmosomal cadherins are expressed in the suprabasal layers of epidermis, whereas Dsg3 and Dsc3 are more strongly expressed basally. This differential expression may have a function in epidermal morphogenesis and/or may regulate the proliferation and differentiation of keratinocytes. To test this hypothesis, we changed the expression pattern by overexpressing human Dsg3 under the control of the keratin 1 (K1) promoter in the suprabasal epidermis of transgenic mice. From around 12 weeks of age, the mice exhibited flaking of the skin accompanied by epidermal pustules and thinning of the hair. Histological analysis of affected areas revealed acanthosis, hypergranulosis, hyperkeratosis, localized parakeratosis, and abnormal hair follicles. This phenotype has some features in common with human ichthyosiform diseases. Electron microscopy revealed a mild epidermal spongiosis. Suprabasally, desmosomes showed incorporation of the exogenous protein by immunogold labeling but were normal in structure. The epidermis was hyperproliferative, and differentiation was abnormal, demonstrated by expression of K14 in the suprabasal layer, restriction of K1, and strong induction of K6 and K16. The changes resembled those found in previous studies in which growth factors, cytokines, and integrins had been overexpressed in epidermis. Thus our data strongly support the view that Dsg3 contributes to the regulation of epidermal differentiation. Our results contrast markedly with those recently obtained by expressing Dsg3 in epidermis under the involucrin promoter. Possible reasons for this difference are considered in this paper.

The humanized SCID mouse model to study HLA class II-linked autoimmunity to desmoglein 3 in pemphigus vulgaris.

Pemphigus vulgaris (PV) is a severe autoimmmune skin disorder induced by antibodies against desmoglein (Dsg) 3 on epidermal keratinocytes. To establish an active animal model of PV to analyse the T-cell-regulated production of pathogenic antibodies in vivo. Immunodeficient SCID mice were injected with peripheral blood lymphocytes (PBL) from an HLA-DRB1*0402/DQ8+ patient with PV or a DRB1*0402/DQ8+ healthy donor, with or without subsequent injections of human Dsg3 or preincubation of PBL with Dsg3. Human immunoglobulins (2.7-18.5 mg mL-1) were detected in all the mice after 8 weeks. Only one of 30 PBL-treated mice developed IgM against Dsg3 and showed intercellular IgM deposits in skin, nostrils and tongue. In contrast, in a previous study, 41% of SCID mice injected with PBL from patients with PV developed anti-Dsg3 antibodies in vivo. Our inability to reproduce these findings may be due to the transfer of slightly lower numbers of PBL (20 x 10(6) vs. 25-30 x 10(6)).

Isoform-Specific Differences in the Size of Desmosomal Cadherin/Catenin Complexes

Isoform-Specific Differences in the Size of Desmosomal Cadherin/Catenin Complexes

Immunoreactivity against intracellular domains of desmogleins in pemphigus

In pemphigus vulgaris (PV) and pemphigus foliaceus (PF), most of the autoantibodies direct against the extracellular domains of desmoglein 1 (Dsg1) or Dsg3, and those antibodies are proved to play a pathogenic role in blister formation in the skin and mucous membranes. However, some pemphigus sera have been reported to react with the intracellular domains of these antigens. In the present study, we examined the reactivity of the sera from various types of pemphigus with recombinant proteins of extracellular and intracellular domains of human Dsg1 and Dsg3 by immunoblot analysis. We produced the entire extracellular domain of Dsg1 or Dsg3 fused with mouse IgG2a by baculovirus expression. We prepared the intracellular domain of Dsg1 or Dsg3 fused with glutathione- S-transferase by bacterial expression. All of the 31 PV sera reacted with the extracellular domain of Dsg3 and four reacted with the intracellular domain. Six out of 19 PF sera reacted with the extracellular domain of Dsg1 and five reacted with the intracellular domain. In addition, some sera of Brazilian PF patients or cases with mixed features of PV and PF also reacted with the intracellular domains of Dsg1 or Dsg3. Although the frequency was low, some sera did react with the intracellular domain of Dsgs.

Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.

Exfoliative toxin A, produced by Staphylococcus aureus, causes blisters in bullous impetigo and its more generalized form, staphylococcal scalded-skin syndrome. The toxin shows exquisite specificity in causing loss of cell adhesion only in the superficial epidermis. Although exfoliative toxin A has the structure of a serine protease, a target protein has not been identified. Desmoglein (Dsg) 1, a desmosomal cadherin that mediates cell-cell adhesion, may be the target of exfoliative toxin A, because it is the target of autoantibodies in pemphigus foliaceus, in which blisters form with identical tissue specificity and histology. We show here that exfoliative toxin A cleaved mouse and human Dsg1, but not closely related cadherins such as Dsg3. We demonstrate this specific cleavage in cell culture, in neonatal mouse skin and with recombinant Dsg1, and conclude that Dsg1 is the specific receptor for exfoliative toxin A cleavage. This unique proteolytic attack on the desmosome causes a blister just below the stratum corneum, which forms the epidermal barrier, presumably allowing the bacteria in bullous impetigo to proliferate and spread beneath this barrier.

Characterization of the regulatory regions in the human desmoglein genes encoding the pemphigus foliaceous and pemphigus vulgaris antigens.

The adhesive proteins in the desmosome type of cell junction consist of two members of the cadherin superfamily, the desmogleins and desmocollins. Both desmogleins and desmocollins occur as at least three different isoforms with various patterns of expression. The molecular mechanisms controlling the differential expression of the desmosomal cadherin isoforms are not yet known. We have begun an investigation of desmoglein gene expression by cloning and analysing the promoters of the human genes coding for the type 1 and type 3 desmogleins (DSG1 and DSG3). The type 1 isoform is restricted to the suprabasal layers of the epidermis and is the autoantigen in the autoimmune blistering skin disease pemphigus foliaceous. The type 3 desmoglein isoform is also expressed in the epidermis, but in lower layers than the type 1 isoform, and is the autoantigen in pemphigus vulgaris. Phage lambda genomic clones were obtained containing 4.2 kb upstream of the translation start site of DSG1 and 517 bp upstream of the DSG3 start site. Sequencing of 660 bp upstream of DSG1 and 517 bp upstream of DSG3 revealed that there was no obvious TATA box, but a possible CAAT box was present at -238 in DSG1 and at -193 in DSG3 relative to the translation start site. Primer extension analysis and RNase protection experiments revealed four putative transcription initiation sites for DSG1 at positions -163, -151, -148 and -141, and seven closely linked sites for DSG3, the longest being at -140 relative to the translation start site. The sequences at these possible sites at -166 to -159 in DSG1 (TTCAGTCC) and at -124 to -117 in DSG3 (CTTAGACT) have some similarity to the initiator sequence (CTCANTCT) described for a TATA-less promoter often from -3 to +5, and the true transcription initiator site might therefore be the A residue in these sequences. There were two regions of similarity between the DSG1 and DSG3 promoters just upstream of the transcription initiation sites, of 20 and 13 bp, separated by 41 bp in DSG1 and 36 bp in DSG3. The significance of these regions of similarity remains to be elucidated, but the results suggest that they represent a point at which these two desmoglein genes are co-ordinately regulated. Analysis of the upstream sequences revealed GC-rich regions and consensus binding sites for transcription factors including AP-1 and AP-2. Exon boundaries were conserved compared with the classical cadherin E-cadherin, but the equivalent of the second cadherin intron was lacking. A 4.2 kb region of the human DSG1 promoter sequence was linked to the lacZ gene reporter gene in such a way that there was only one translation start site, and this construct was used to generate transgenic mice. We present the first transgenic analysis of a promoter region taken from a desmosomal cadherin gene. Our results suggest that the 4.2 kb upstream region of DSG1 does not contain all the regulatory elements necessary for correct expression of this gene but might have elements that regulate activity during hair growth.

Cloning of the Gene for Human Pemphigus Vulgaris Antigen (Desmoglein 3), a Desmosomal Cadherin: CHARACTERIZATION OF THE PROMOTER REGION AND IDENTIFICATION OF A KERATINOCYTE-SPECIFIC cis-ELEMENT

Pemphigus vulgaris antigen is a cadherin-like desmosomal cell adhesion molecule expressed primarily in suprabasal keratinocytes within the epidermis. Previously characterized structural features have defined this molecule as a desmoglein, DSG3. In this study, we have cloned the human DSG3 gene and examined the transcriptional regulation of its expression. The total gene consisted of 15 exons and was estimated to span >23 kilobases. Comparison of exon-intron organization of DSG3 with bovine DSG1 and several classical cadherin genes revealed striking conservation of the structure. Up to 2.8 kilobases of the upstream genomic sequences were sequenced and found to contain several putative cis-regulatory elements. The promoter region was GC-rich and TATA-less, similar to previously characterized mammalian cadherin promoters. The putative promoter region was subcloned into a vector containing chloramphenicol acetyl transferase reporter gene. Transient transfections with a series of deletion clones indicated that the DSG3 promoter demonstrated keratinocyte-specific expression, as compared with dermal fibroblasts examined in parallel, and fine mapping identified a 30-base pair segment at -200 to -170 capable of conferring epidermal specific expression. The results provide evidence for the transcriptional regulation of the pemphigus vulgaris antigen gene, potentially critical for development of the epidermis and physiologic terminal differentiation of keratinocytes.

Antigen-Specific Immunoadsorption of Pathogenic Autoantibodies in Pemphigus Foliaceus

Patients with the autoimmune blistering disease pemphigus foliaceus (PF) have circulating autoantibodies directed against the desmosomal cadherin desmoglein 1 (Dsg1). Based on the fact that purified IgG fractions from PF patients induce loss of cell adhesion in organ culture and in a neonatal mouse model, it has been proposed that these anti-Dsg1 antibodies play a pathogenic role in blister formation. To directly address whether antibodies in PF sera specific for the Dsg1 extracellular domain are indeed pathogenic in the disease, PFIg, a chimeric protein containing the entire extracellular domain of human Dsg1 and the constant region of human IgG1, was produced by baculovirus expression. Incubation of PF patients' sera with the PFIg baculoprotein removed the immunoreactivity of autoantibodies against keratinocyte cell surfaces in all 20 PF and eight Brazilian PF patients' sera tested. This adsorption was conformation dependent, because PFIg protein denatured by low pH or heat was no longer able to adsorb the immunoreactivity of PF sera. Furthermore, the incubation with the PFIg baculoprotein eliminated the pathogenic activity of PF patients' sera and prevented gross blister formation in a neonatal mouse model of pemphigus. Anti-Dsg1 antibodies eluted from the PFIg protein column were pathogenic as they resulted in the appearance of gross blisters in neonatal mice with typical histologic findings of PF. These observations indicate that the extracellular domain of Dsg1 expressed by baculovirus is capable of specifically immunoadsorbing pathogenic autoantibodies from PF patients' sera and provide direct evidence that the anti-Dsg1 autoantibodies in PF sera are indeed pathogenic. The availability of this Dsg1 recombinant protein may facilitate the development of antigen-specific plasmapheresis as a novel therapeutic strategy in pemphigus.

Plakoglobin Binding by Human Dsg3 (Pemphigus Vulgaris Antigen) in Keratinocytes Requires the Cadherin-Like Intracytoplasmic Segment

Desmogleins are transmembrane desmosomal cadherins. Two desmogleins, Dsg3 and Dsg1, have been shown to bind plakoglobin, an intracytoplasmic (IC) desmosomal plaque protein. This binding may be critical for desmosome assembly or stability. The IC domain of desmogleins consists of subdomains that are either desmoglein specific or homologous with the IC region of classical cadherins. Here we identify the domains of human Dsg3 that are critical for plakoglobin binding in human keratinocytes. We constructed eukaryotic expression vectors containing chimeric cDNAs that encode the extracellular domain of mouse E-cadherin (Ecad) with the transmembrane and IC domains of Dsg3, with increasing truncations eliminating various IC subdomains from the carboxy-terminus. These constructs were used for transient transfection of HaCaT cells. Extracts were subjected to immunoprecipition with an anti-mouse Ecad antibody (that does not precipitate human Ecad), thus precipitating the chimeric protein and any tightly associated plakoglobin. Co-precipitation of plakoglobin was confirmed by immunoblotting. These data show that the desmoglein-specific IC subdomains are not necessary for plakoglobin binding, but the carboxy-terminal 87 amino acids of the IC-cadherin-like segment subdomain are critical. Finally, we confirmed these results outside cells with in vitro transcription and translation, which also demonstrates that the Dsg3-plakoglobin interaction is direct and does not depend on other cellular factors. These results underscore the importance of a region, highly conserved in all desmogleins, in the carboxy terminus of the IC-cadherin-like subdomain for the localization of plakoglobin to desmosomes.

A simple epithelial cell line (MDCK) shows heterogeneity of desmoglein isoforms, one resembling pemphigus vulgaris antigen

The epidermal blistering disease, pemphigus vulgaris (PV), is caused by circulating autoantibodies that react with a desmosomal glycoprotein desmoglein (Dsg3). This antigen is expressed only in stratified epithelial tissues. Here we show that the simple epithelial canine kidney cell line, MDCK, expresses at least two desmoglein isoforms recognised by different monoclonal antibodies. One of these isoforms is a 130 x 10(3) M(r) polypeptide that is recognised by both PV autoantisera and a monoclonal antibody reactive with a cytoplasmic domain of human Dsg3. Antibodies in PV sera bind to the surface of MDCK cells but not cause loss of intercellular adhesion. This is the first demonstration of the expression of a polypeptide related to human PV antigen by a simple epithelial cell type.