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Disruption of desmosome assembly by monovalent human pemphigus vulgaris monoclonal antibodies.

ABSTRACT: The intercellular interactions of the desmosomal cadherins, desmoglein and desmocollin, are required for epidermal cell adhesion. Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterized by autoantibodies against desmoglein (Dsg) 3. During calcium-induced desmosome assembly, treatment of primary human keratinocytes with pathogenic monovalent anti-Dsg3 mAbs produced from a PV patient causes a decrease of Dsg3 and desmoplakin but not desmocollin (Dsc) 3 in the Triton-insoluble fraction of cell lysates within 2 hours. Immunofluorescence and antibody ELISA studies suggest that pathogenic mAbs cause internalization of cell-surface Dsg3 but not Dsc3 through early endosomes. Electron microscopy demonstrated a lack of well-formed desmosomes in keratinocytes treated with pathogenic compared to nonpathogenic mAbs. In contrast, pathogenic mAbs caused late depletion of Dsg3 from preformed desmosomes at 24 hours, with effects on multiple desmosomal proteins including Dsc3 and plakoglobin. Together, these studies indicate that pathogenic PV mAbs specifically cause internalization of newly synthesized Dsg3 during desmosome assembly, correlating with their pathogenic activity. Monovalent human PV anti-Dsg mAbs reproduce the effects of polyclonal PV IgG on Dsg3 and will facilitate future studies to further dissect the cellular mechanisms for the loss of cell adhesion in pemphigus.

SUBMITTER: Mao X

PROVIDER: S-EPMC2743719 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Disruption of desmosome assembly by monovalent human pemphigus vulgaris monoclonal antibodies.

Mao Xuming X Choi Eun Jung EJ Payne Aimee S AS

The Journal of investigative dermatology 20081127 4

The intercellular interactions of the desmosomal cadherins, desmoglein and desmocollin, are required for epidermal cell adhesion. Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterized by autoantibodies against desmoglein (Dsg) 3. During calcium-induced desmosome assembly, treatment of primary human keratinocytes with pathogenic monovalent anti-Dsg3 mAbs produced from a PV patient causes a decrease of Dsg3 and desmoplakin but not desmocollin (Dsc) 3 in the Tri ...[more]

PMID: 19037235

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Project description:BackgroundA number of autoimmune diseases have been clinically and pathologically characterized. In contrast, target antigens have been identified only in a few cases and, in these few cases, the knowledge of the exact epitopic antigenic sequence is still lacking. Thus the major objective of current work in the autoimmunity field is the identification of the epitopic sequences that are related to autoimmune reactions. Our labs propose that autoantigen peptide epitopes able to evoke humoral (auto)immune response are defined by the sequence similarity to the host proteome. The underlying scientific rationale is that antigen peptides acquire immunoreactivity in the context of their proteomic similarity level. Sequences uniquely owned by a protein will have high potential to evoke an immune reaction, whereas motifs with high proteomic redundancy should be immunogenically silenced by the tolerance phenomenon. The relationship between sequence redundancy and peptide immunoreactivity has been successfully validated in a number of experimental models. Here the hypothesis has been applied to pemphigus diseases and the corresponding desmoglein autoantigens.MethodsDesmoglein 3 sequence similarity analysis to the human proteome followed by dot-blot/NMR immunoassays were carried out to identify and validate possible epitopic sequences.ResultsComputational analysis led to identifying a linear immunodominant desmoglein-3 epitope highly reactive with the sera from Pemphigus vulgaris as well as Pemphigus foliaceous. The epitopic peptide corresponded to the amino acid REWVKFAKPCRE sequence, was located in the extreme N-terminal region (residues 49 to 60), and had low redundancy to the human proteome. Sequence alignment showed that human desmoglein 1 and 3 share the REW-KFAK-RE sequence as a common motif with 75% residue identity.ConclusionThis study 1) validates sequence redundancy to autoproteome as a main factor in shaping desmoglein peptide immunogenicity; 2) offers a molecular mechanicistic basis in analyzing the commonality of autoimmune responses exhibited by the two forms of pemphigus; 3) indicates possible peptide-immunotherapeutical approaches for pemphigus diseases.

Project description: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.

Dataset Information

Disruption of desmosome assembly by monovalent human pemphigus vulgaris monoclonal antibodies.

Publications

Disruption of desmosome assembly by monovalent human pemphigus vulgaris monoclonal antibodies.

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