Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules.

Monoclonal antibodies to the constitutive desmosomal glycoprotein desmoglein were characterized whose epitopes are located intracellularly, i.e., in the cytoplasmic portion of this molecule, and contribute to the structure of the desmosomal plaque. Using one of these antibodies (DG3.10), a peptide was isolated from a proteolytic digest of desmoglein purified from isolated bovine muzzle demosomes, and its amino acid sequence was determined. In comparisons of this sequence with the amino acid sequence of desmoglein as deduced from the sequence of cDNA clones from the same tissue, encompassing most of approximately 7.6 kb mRNA and the complete coding region of 959 residues (calculated molecular weight approximately 102,400), the DG3.10 epitope was identified in a region starting 163 amino acids before the carboxy terminus in the first of four consecutive repeats of a homologous element of 29 +/- 1 amino acids. This topological information, together with the identification of a single hydrophobic region of sufficient length to provide a transmembrane segment and of several extended regions showing high sequence homology to various cadherins, has allowed the construction of a model of the molecular organization of desmoglein. We conclude that desmoglein is a member of the cadherin family of cell adhesion glycoproteins which is characterized by an unusually long cytoplasmic domain which exceeds those of the cadherins by more than 275 amino acids, contains special repetitive elements and spans the desmosomal plaque at least once.     

Loss of invasiveness in squamous cell carcinoma cells overexpressing desmosomal cadherins

The molecular and structural characteristics of intercellular adhesion were investigated in a squamous cell carcinoma (SCCA) cell line, originally derived from an oral tumor with an invasive growth pattern. The expression of adherens junction and desmosomal components were compared with that of cultured normal oral keratinocytes. Lack of membrane association in interdesmosomal areas, the disorganization of the actin cytoskeleton and the faster cell disassembly upon E-cadherin antibody binding in SCCA cells indicated decreased functional adherens junctions. These observations were supported by a significant reduction in E-, N-, and P-cadherin protein expression. In contrast, the level of desmosomal cadherin proteins, desmoglein 1/2 and desmocollin 2, were substantially upregulated and accompanied, ultrastructurally, by increased number and size of desmosomes. Since tumor invasion suppressor capacity has been proposed for desmosomal cadherins, we investigated the in vivo invasion potential of these SCCA cells by introducing them into SCID mice. Tumors developed, but with a benign phenotype. Based on these results, we hypothesize that the benign behavior of this SCCA cell line is a consequence of overexpressed desmosomal cadherins. This SCCA cell line, therefore, represents an excellent model system to further investigate the regulation and tumor invasion suppressor potential of desmosomal adhesion molecules.     

Desmoglein shows extensive homology to the cadherin family of cell adhesion molecules.

Desmoglein is a major adhesive component of the desmosome. It is also at least one of the antigenic targets of pathogenic antibodies circulating in the sera of patients with the blistering disease Pemphigus foliaceus. To examine the molecular basis of desmosomal adhesion and to further our understanding of its disruption in various bullous disorders we have cloned cDNAs encoding four of the extracellular domains of desmoglein. The predicted amino acid sequence of these clones shows extensive homology with the cadherin class of calcium-dependent cell adhesion molecules. Desmoglein represents a novel subtype of this family.     

Desmoglein 1–dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca²⁺-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor–Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.     

Defining domain-specific orientational order in the desmosomal cadherins

Desmosomes are large, macromolecular protein assemblies that mechanically couple the intermediate filament cytoskeleton to sites of cadherin-mediated cell adhesion, thereby providing structural integrity to tissues that routinely experience large forces. Proper desmosomal adhesion is necessary for the normal development and maintenance of vertebrate tissues, such as epithelia and cardiac muscle, while dysfunction can lead to severe disease of the heart and skin. Therefore, it is important to understand the relationship between desmosomal adhesion and the architecture of the molecules that form the adhesive interface, the desmosomal cadherins (DCs). However, desmosomes are embedded in two plasma membranes and are linked to the cytoskeletal networks of two cells, imposing extreme difficulty on traditional structural studies of DC architecture, which have yielded conflicting results. Consequently, the relationship between DC architecture and adhesive function remains unclear. To overcome these challenges, we utilized excitation-resolved fluorescence polarization microscopy to quantify the orientational order of the extracellular and intracellular domains of three DC isoforms: desmoglein 2, desmocollin 2, and desmoglein 3. We found that DC ectodomains were significantly more ordered than their cytoplasmic counterparts, indicating a drastic difference in DC architecture between opposing sides of the plasma membrane. This difference was conserved among all DCs tested, suggesting that it may be an important feature of desmosomal architecture. Moreover, our findings suggest that the organization of DC ectodomains is predominantly the result of extracellular adhesive interactions. We employed azimuthal orientation mapping to show that DC ectodomains are arranged with rotational symmetry about the membrane normal. Finally, we performed a series of mathematical simulations to test the feasibility of a recently proposed antiparallel arrangement of DC ectodomains, finding that it is supported by our experimental data. Importantly, the strategies employed here have the potential to elucidate molecular mechanisms for diseases that result from defective desmosome architecture.     

Desmosomal adhesion: structural basis,molecular mechanism and regulation (Review)

Desmosomes are adhesive intercellular junctions of epithelia and cardiac muscle. They have an essential function in maintaining the integrity of tissues, which is compromised in human genetic and autoimmune disease that targets desmosomal components. Recent evidence (1) suggests new roles for the function of desmosomal adhesion in tissue morphogenesis, (2) gives new insights into the molecular mechanism of adhesion, (3) indicates that the desmosomal adhesion molecules, desmocollin and desmoglein, may contribute to the regulation of epidermal diffentiation, and (4) shows that the affinity of desmosomal adhesion is regulated by protein kinase C.     

Desmosomal adhesion: structural basis,molecular mechanism and regulation (Review)

Desmosomes are adhesive intercellular junctions of epithelia and cardiac muscle. They have an essential function in maintaining the integrity of tissues, which is compromised in human genetic and autoimmune disease that targets desmosomal components. Recent evidence (1) suggests new roles for the function of desmosomal adhesion in tissue morphogenesis, (2) gives new insights into the molecular mechanism of adhesion, (3) indicates that the desmosomal adhesion molecules, desmocollin and desmoglein, may contribute to the regulation of epidermal diffentiation, and (4) shows that the affinity of desmosomal adhesion is regulated by protein kinase C.     

Desmosomal glycoproteins II and III. Cadherin-like junctional molecules generated by alternative splicing

We have cloned the human genes coding for desmosomal glycoproteins DGII and DGIII, found in desmosomal cell junctions, and sequencing shows that they are related to the cadherin family of cell adhesion molecules. Thus a new super family of cadherin-like molecules exists which also includes the other major desmosomal glycoprotein, DGI (Wheeler, G. N., Parker, A. E., Thomas, C. L., Ataliotis, P., Poynter, D., Arnemann, J., Rutman, A. J., Pidsley, S. C., Watt, F. M., Rees, D. A., Buxton, R. S., and Magee, A. I. (1991) Proc. Natl. Acad. Sci. U.S.A., in press). DGIII differs from DGII by the addition of a 46-base pair exon containing an in-frame stop codon resulting in mature protein molecular weights of 84,633 for DGII and 78,447 for DGIII. The unique carboxyl-terminal region of DGII contains a potential serine phosphorylation site explaining why only DGII is phosphorylated on serine. The cadherin cell adhesion recognition sequence (His-Ala-Val) is replaced by Phe-Ala-Thr, suggesting that DGII/III may be adhesive molecules using a different mechanism.     

Genomic organization of mouse desmocollin genes reveals evolutionary conservation.

Desmosomal cadherins are a family of calcium regulated proteins involved in the formation of desmosomes, a type of cell junction important in maintaining cell adhesion and tissue stability. The desmosomal plaque consists of members of the desmosomal cadherin, plakin and armadillo family of proteins. Desmosomal cadherins are transmembrane glycoproteins that interact with desmosomal cadherins of the adjacent cells via their extracellular repeat domains and are divided in two subfamilies, the desmogleins (Dsg) and the desmocollins (Dsc). On the cytoplasmic side, the cadherins connect to the intermediate filament (IF) network indirectly by interacting with plakin and armadillo proteins. Here, we report the elucidation of the genomic structure of two mouse desmocollin genes, Dsc2 and Dsc3. Interestingly, at the genomic level, desmocollins show a higher degree of similarity to the classical cadherins, such as E-cadherin, than to the desmogleins.     

Membrane-impermeable cross-linking provides evidence for homophilic, isoform-specific binding of desmosomal cadherins in epithelial cells

Desmosomes and adherens junctions are cadherin-based protein complexes responsible for cell-cell adhesion of epithelial cells. Type 1 cadherins of adherens junctions show specific homophilic adhesion that plays a major role in developmental tissue segregation. The desmosomal cadherins, desmocollin and desmoglein, occur as several different isoforms with overlapping expression in some tissues where different isoforms are located in the same desmosomes. Although adhesive binding of desmosomal cadherins has been investigated in a variety of ways, their interaction in desmosome-forming epithelial cells has not been studied. Here, using extracellular homobifunctional cross-linking, we provide evidence for homophilic and isoform-specific binding between the Dsc2, Dsc3, Dsg2, and Dsg3 isoforms in HaCaT keratinocytes and show that it represents trans interaction. Furthermore, the cross-linked adducts are present in the detergent-insoluble fraction, and electron microscopy shows that extracellular cross-linking probably occurs in desmosomes. We found no evidence for either heterophilic or cis interaction, but neither can be completely excluded by our data. Mutation of amino acid residues Trp-2 and Ala-80 that are important for trans interaction in classical cadherin adhesive binding abolished Dsc2 binding, indicating that these residues are also involved in desmosomal adhesion. These interactions of desmosomal cadherins may be of key importance for their ordered arrangement within desmosomes that we believe is essential for desmosomal adhesive strength and the maintenance of tissue integrity.     

Coordinated expression of desmoglein 1 and desmocollin 1 regulates intercellular adhesion

Desmoglein 1 (Dsg1) is a component of desmosomes present in the upper epidermis and can be targeted by autoimmune antibodies or bacterial toxins, resulting in skin blistering diseases. These defects in tissue integrity are believed to result from compromised desmosomal adhesion; yet, previous attempts to directly test the adhesive roles of desmosomal cadherins using normally non-adherent L cells have yielded mixed results. Here, two complementary approaches were used to better resolve the molecular determinants for Dsg1-mediated adhesion: (1) a tetracycline-inducible system was used to modulate the levels of Dsg1 expressed in L cell lines containing desmocollin 1 (Dsc1) and plakoglobin (PG) and (2) a retroviral gene delivery system was used to introduce Dsg1 into normal human epidermal keratinocytes (NHEK). By increasing Dsg1 expression relative to Dsc1 and PG, we were able to demonstrate that the ratio of Dsg1:Dsc1 is a critical determinant of desmosomal adhesion in fibroblasts. The distribution of Dsg1 was organized at areas of cell-cell contact in the multicellular aggregates that formed in these suspension cultures. Similarly, the introduction of Dsg1 into NHEKs was capable of increasing the aggregation of single cell suspensions and further enhanced the adhesive strength of intact epithelial sheets. Endogenous Dsc1 levels were also increased in NHEKs containing Dsg1, providing further support for the coordination of these two desmosomal cadherins in regulating adhesive structures. These Dsg1-mediated effects on intercellular adhesion were directly related to the presence of an intact extracellular domain as ETA, a toxin that specifically cleaves this desmosomal cadherin, inhibited adhesion in both fibroblasts and keratinocytes. Collectively, these observations demonstrate that Dsg1 promotes the formation of intercellular adhesion complexes and suggest that the relative level of Dsg and Dsc expressed at the cell surface regulates this adhesive process.     

Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains

Desmosomal glycoproteins 2 and 3 (dg2 and 3) or desmocollins have been implicated in desmosome adhesion. We have obtained a 5.0-kb-long clone for dg3 from a bovine nasal epidermal lambda gt11 cDNA library. Sequence analysis of this clone reveals an open reading frame of 2,517 bases encoding a polypeptide of 839 amino acids. The sequence consists of a signal peptide of 28 amino acids, a precursor sequence of 104 amino acids, and a mature protein of 707 amino acids. The latter has the characteristics of a transmembrane glycoprotein with an extracellular domain of 550 amino acids and a cytoplasmic domain of 122 amino acids. The sequence of a partial clone from the same library shows that dg2 has an alternative COOH terminus that is extended by 54 amino acids. Genomic DNA sequence data show that this arises by splicing out of a 46-bp exon that encodes the COOH-terminal 11 amino acids of dg3 and contains an in-frame stop codon. The extracellular domain of dg3 shows 39.4% protein sequence identity with bovine N-cadherin and 28.4% identity with the other major desmosomal glycoprotein, dg1, or desmoglein. The cytoplasmic domain of dg3 and the partial cytoplasmic domain of dg2 show 23 and 24% identity with bovine N-cadherin, respectively. The results support our previous model for the transmembrane organization of dg2 and 3 (Parrish, E.P., J.E. Marston, D.L. Mattey, H.R. Measures, R. Venning, and D.R. Garrod. 1990. J. Cell Sci. 96:239-248; Holton, J.L., T.P. Kenny, P.K. Legan, J.E. Collins, J.N. Keen, R. Sharma, and D.R. Garrod. 1990. J. Cell Sci. 97:239-246). They suggest that these glycoproteins are specialized for calcium-dependent adhesion in their extracellular domains and, cytoplasmically, for the molecular interactions involved in desmosome plaque formation. Moreover this represents the first example of alternative splicing within the cadherin family of cell adhesion molecules.     

Pemphigus vulgaris antigen, a desmoglein type of cadherin, is localized within keratinocyte desmosomes

Pemphigus vulgaris antigen (PVA) is a member of the desmoglein subfamily of cadherin cell adhesion molecules. Because autoantibodies in this disease cause blisters due to loss of epidermal cell adhesion, and because desmoglein is found in the desmosome cell adhesion junction, we wanted to determine if PVA is also found in desmosomes. By immunofluorescence, PV IgG bound, in a dotted pattern, to the cell surface of cultured human keratinocytes induced to differentiate with calcium, suggesting junctional staining. However, by preembedding, immunogold electron microscopic studies only slight labeling could be detected in desmosomes, presumably because of difficulty in gold penetration of intact desmosomes. We therefore treated the keratinocytes with 0.01% trypsin in 1 mM calcium, conditions known to preserve cadherin antigenicity but that caused slight separation of desmosomes, before immunogold staining. In this case there was extensive labeling of the extracellular part of desmosomes but not of the interdesmosomal cell membrane which was stained with anti-beta 2- microglobulin antibodies. To confirm the specificity of this binding we showed that antibodies raised in rabbits against the extracellular portions of PVA also bound desmosomes in these cultures. In intact mouse epidermis we could also show slight, but specific, immunogold desmosomal labeling with PV IgG. Furthermore, neonatal mice injected with PV IgG affinity purified on PVA showed desmosomal separation with the IgG localized to desmosomal cores. These results indicate that PVA is organized and concentrated within the desmosome where it presumably functions to maintain the integrity of stratifying epithelia.     

Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.

Pemphigus vulgaris (PV) is a life-threatening skin disease in which autoantibodies against a keratinocyte cell surface 130 kd glycoprotein, PV antigen (PVA), cause loss of cell-cell adhesion, with resultant epidermal blisters. We used affinity-purified PV IgG to isolate cDNA, containing the entire coding sequence for PVA, from human keratinocyte expression libraries. Northern blot analysis indicated PV mRNA expression only in stratified squamous epithelia. The deduced amino acid sequence of PVA was unique but showed significant homology with members of the cadherin family of Ca(2+)-dependent cell adhesion molecules, most markedly to desmoglein I. These findings demonstrate that a novel epithelial cadherin is the target of autoantibodies in PV.     

Desmoglein 2 can undergo Ca2+-dependent interactions with both desmosomal and classical cadherins including E-cadherin and N-cadherin

Desmoglein (Dsg) 2 is a ubiquitously expressed desmosomal cadherin. Particularly, it is present in all cell types forming desmosomes, including epithelial cells and cardiac myocytes and is upregulated in the autoimmune skin disease pemphigus. Thus, we here characterized the binding properties of Dsg2 in more detail using atomic force microscopy (AFM). Dsg2 exhibits homophilic interactions and also heterophilic interactions with the desmosomal cadherin desmocollin (Dsc) 2, and further with the classical cadherins E-cadherin (E-Cad) and N-cadherin (N-Cad), which may be relevant for cross talk between desmosomes and adherens junctions in epithelia and cardiac myocytes. We found that all homo- and heterophilic interactions were Ca²⁺-dependent. All binding forces observed are in the same force range, i.e., 30 to 40 pN, except for the Dsg2/E-Cad unbinding force, which with 45 pN is significantly higher. To further characterize the nature of the interactions, we used tryptophan, a critical amino acid required for trans-interaction, and a tandem peptide (TP) designed to cross-link Dsg isoforms. TP was sufficient to prevent the tryptophan-induced loss of Dsg2 interaction with the desmosomal cadherins Dsg2 and Dsc2; however, not with the classical cadherins E-Cad and N-Cad, indicating that the interaction modes of Dsg2 with desmosomal and classical cadherins differ. TP rescued the tryptophan-induced loss of Dsg2 binding on living enterocytes, suggesting that interaction with desmosomal cadherins may be more relevant. In summary, the data suggest that the ubiquitous desmosomal cadherin Dsg2 enables the cross talk with adherens junctions by interacting with multiple binding partners with implications for proper adhesive function in healthy and diseased states.     

Localization of HIV RNA in mitochondria of infected cells: potential role in cytopathogenicity

A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive S2 cells. cDNA clones were isolated using a polyclonal antibody inhibiting Ca(2+)-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca(2+)-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected S2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca(2+)-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI- cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine.     

Identification of a cadherin cell adhesion recognition sequence

The molecular mechanisms by which the cadherins interact with one another to promote cell adhesion have not been elucidated. In particular, the amino acid sequences of the cadherin cell adhesion recognition sites have not been determined. Here we demonstrate that synthetic peptides containing the sequence HAV, which is common to all of the cadherins, inhibit two processes (compaction of eight-cell-stage mouse embryos and rat neurite outgrowth on astrocytes) that are known to be mediated by cadherins. The data suggest that the tripeptide HAV is a component of a cadherin cell adhesion recognition sequence.     

Sequence and structural determinants of strand swapping in cadherin domains: do all cadherins bind through the same adhesive interface?

Cadherins are cell surface adhesion proteins important for tissue development and integrity. Type I and type II, or classical, cadherins form adhesive dimers via an interface formed through the exchange, or “swapping”, of the N-terminal β-strands from their membrane-distal EC1 domains. Here, we ask which sequence and structural features in EC1 domains are responsible for β-strand swapping and whether members of other cadherin families form similar strand-swapped binding interfaces. We created a comprehensive database of multiple alignments of each type of cadherin domain. We used the known three-dimensional structures of classical cadherins to identify conserved positions in multiple sequence alignments that appear to be crucial determinants of the cadherin domain structure. We identified features that are unique to EC1 domains. On the basis of our analysis, we conclude that all cadherin domains have very similar overall folds but, with the exception of classical and desmosomal cadherin EC1 domains, most of them do not appear to bind through a strand-swapping mechanism. Thus, non-classical cadherins that function in adhesion are likely to use different protein-protein interaction interfaces. Our results have implications for the evolution of molecular mechanisms of cadherin-mediated adhesion in vertebrates.