The most widespread desmosomal cadherin, desmoglein 2, is a novel target of caspase 3-mediated apoptotic machinery

Apoptotic cells are known to regulate the ordered dismantling of intercellular contacts through caspase activity. Despite the important role of desmoglein (Dsg) 2 in epithelial cell-cell adhesion, the fate of this widespread desmosomal cadherin during apoptosis is yet poorly understood. Here, by means of pharmacological approaches, we investigated whether Dsg2 was targeted by caspases in HaCaT and HT-29 cell lines undergoing staurosporine (STS)-induced apoptosis. Results showed that STS induced a caspase-dependent form of cell-death in both keratinocytes (HaCaT) and enterocytes (HT-29), that associated with progressive depletion of Dsg2 from cell lysates. The proteolytic processing of full-length Dsg2 resulted in the appearance of a 70-kDa fragment which was released into the cytosol. Consistently, immunofluorescence studies revealed that Dsg2 staining was abolished from cell surface whereas the cytoplasmic region of Dsg2 did localize intracellularly. Plakoglobin (Pg) also underwent cleavage and detached from Dsg2. Apoptotic changes paralleled with progressive loss of intercellular adhesion strength. All these biochemical, morphological, and functional changes were regulated by caspase 3. Indeed, in the presence of the caspase 3-inhibitor z-DEVD-fmk, full-length Dsg2 protein levels were preserved, whereas the amount of the 70-kDa fragment was maintained on control levels. Furthermore, cells pretreated with z-DEVD-fmk retained the membrane labeling of Dsg2. Taken together, our data demonstrate that the apoptotic processing of Dsg2 is mediated by caspase 3 in epithelial cells.     

Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation

Desmogleins are members of the cadherin superfamily which form the core of desmosomes. In vitro studies indicate that the cytoplasmic domain of desmogleins associates with plakoglobin; however, little is known about the role of this domain in desmosome recognition or assembly in vivo, or about the possible relation of desmoglein mutations to epidermal differentiation and disease. To address these questions we used transgenic mouse technology to produce an NH2-terminally truncated desmoglein (Pemphigus Vulgaris Antigen or Dsg3) in cells known to express its wild-type counterpart. Within 2 d, newborn transgenic animals displayed swelling of their paws, flakiness on their back, and blackening of the tail tip. When analyzed histologically and ultrastructurally, widening of intercellular spaces and disruption of desmosomes were especially striking in the paws and tail. Desmosomes were reduced dramatically in number and were smaller and often peculiar in structure. Immunofluorescence and immunoelectron microscopy revealed no major abnormalities in localization of hemidesmosomal components, but desmosomal components organized aberrantly, resulting in a loss of ultrastructure within the plaque. In regions where desmosome loss was prevalent but where some adhesive structures persisted, the epidermis was thickened, with a marked increase in spinous and stratum corneum layers, variability in granular layer thickness, and parakeratosis in some regions. Intriguingly, a dramatic increase in cell proliferation was also observed concomitant with biochemical changes, including alterations in integrin expression, known to be associated with hyperproliferation. An inflammatory response was also detected in some skin regions. Collectively, these findings demonstrate that a mutation in a desmoglein can perturb epidermal cell-cell adhesion, triggering a cascade of changes in the skin.     

The differentiation-dependent desmosomal cadherin desmoglein 1 is a novel caspase-3 target that regulates apoptosis in keratinocytes

Although a number of cell adhesion proteins have been identified as caspase substrates, the potential role of differentiation-specific desmosomal cadherins during apoptosis has not been examined. Here, we demonstrate that UV-induced caspase cleavage of the human desmoglein 1 cytoplasmic tail results in distinct 17- and 140- kDa products, whereas metalloproteinase-dependent shedding of the extracellular adhesion domain generates a 75-kDa product. In vitro studies identify caspase-3 as the preferred enzyme that cleaves desmoglein 1 within its unique repeating unit domain at aspartic acid 888, part of a consensus sequence not conserved among the other desmosomal cadherins. Apoptotic processing leads to decreased cell surface expression of desmoglein 1 and re-localization of its C terminus diffusely throughout the cytoplasm over a time course comparable with the processing of other desmosomal proteins and cytoplasmic keratins. Importantly, whereas classic cadherins have been reported to promote cell survival, short hairpin RNA-mediated suppression of desmoglein 1 in differentiated keratinocytes protected cells from UV-induced apoptosis. Collectively, our results identify desmoglein 1 as a novel caspase and metalloproteinase substrate whose cleavage likely contributes to the dismantling of desmosomes during keratinocyte apoptosis and also reveal desmoglein 1 as a previously unrecognized regulator of apoptosis in keratinocytes.     

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.     

Human heparanase is localized within lysosomes in a stable form

Heparanase is an endo-beta-D-glucuronidase involved in degradation of heparan sulfate (HS) and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mechanism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflammation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previously suggested that heparanase subcellular localization is a major determinant in regulating the enzyme's biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron microscopy. Our results indicate that heparanase is localized primarily within lysosomes and the Golgi apparatus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that following synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angiogenesis.     

Pv12, a 6-Cys antigen of Plasmodium vivax, is localized to the merozoite rhoptry

Pf12 in Plasmodium falciparum has been characterized as a merozoite surface protein and the Pf12 gene is actively transcribed during schizont stage. An orthologous gene, Pv12, has been identified in genome of P. vivax, but the protein product has not been characterized. The Pv12 is a 362 amino acid long polypeptide encoded by a single exon gene PVX_113775, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Pv12 contains two predicted six-cysteine (6-Cys) domains, which may be constrained by predicted disulfide bonds, and a transmembrane domain and a predicted GPI anchor attachment site in C-terminal region. The recombinant Pv12 protein is recognized by serum antibodies of patients naturally exposed to P. vivax and the native Pv12 protein from parasite extract is also recognized by immune mouse serum. The Pv12 is localized in rhoptry; an apical organelle of the merozoite, and the localization pattern of Pv12 is distinct from that of Pf12 in P. falciparum. The present study suggests that Pv12 is immunogenic in humans during parasite infection and it could play an important role in erythrocyte invasion.     

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.     

Expression of cadherin10, a type II classic cadherin gene, in the nervous system of the embryonic zebrafish

Cadherins are cell surface adhesion molecules that play important roles in development of tissues and organs. In this study, we analyzed expression pattern of cadherin10, a member of the type II classic cadherin subfamily, in the embryonic zebrafish using in situ hybridization methods. cadherin10 message (cdh10) is first and transiently expressed by the notochord. In the developing nervous system, cdh10 was first detected in a subset of the cranial ganglia, then in restricted brain regions and neural retina. As development proceeds, cdh10 expression domain and/or expression levels increased in the embryonic nervous system. Our results show that cdh10 expression in the zebrafish developing nervous system is both spatially and temporally regulated.     

Characterization of cadherin-24, a novel alternatively spliced type II cadherin

Cadherins comprise a superfamily of calcium-dependent cell-cell adhesion molecules. Within the superfamily are six subfamilies including type I and type II cadherins. Both type I and type II cadherins are composed of five extracellular repeat domains with conserved calcium-binding motifs, a single pass transmembrane domain, and a highly conserved cytoplasmic domain that interacts with beta-catenin and p120 catenin. In this study, we describe a novel cadherin, cadherin-24. It is a type II cadherin with a 781-codon open reading frame, which encodes a type II cadherin protein complete with five extracellular repeats containing calcium-binding motifs, a transmembrane domain, and a conserved cytoplasmic domain. Cadherin-24 has the unusual feature of being alternatively spliced in extracellular repeat 4. This alternative exon encodes 38 in-frame amino acids, resulting in an 819-amino-acid protein. Sequence analysis suggests the presence of beta-catenin and p120 catenin-binding sequences, and immunoprecipitation experiments confirm the ability of both forms of the novel cadherin to associate with alpha-catenin, beta-catenin, and p120 catenin. In addition, aggregation assays show that both forms of cadherin-24 mediate strong cell-cell adhesion.     

The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody- based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose treatment, and to evaluate the possible biological involvement of the antigen in carcinogenesis, we set out to molecularly characterize the antigen. A cDNA clone encoding the E48 antigen was isolated by expression cloning in COS cells. Sequence analysis revealed that the clone contained an open reading frame of 128 amino acids, encoding a core protein of 13,286 kD. Database searching showed that the E48 antigen has a high level of sequence similarity with the mouse ThB antigen, a member of the Ly-6 antigen family. Phosphatidylinositol-specific (PI-specific) phospholipase-C treatment indicated that the E48 antigen is glycosylphosphatidylinositol-anchored (GPI-anchored) to the plasma membrane. The gene encoding the E48 antigen is a single copy gene, located on human chromosome 8 in the 8q24-qter region. The expression of the gene is confined to keratinocytes and squamous tumor cells. The putative mouse homologue, the ThB antigen, originally identified as an antigen on cells of the lymphocyte lineage, was shown to be highly expressed in squamous mouse epithelia. Moreover, the ThB expression level is in keratinocytes, in contrast to that in lymphocytes, not mouse strain related. Transfection of mouse SV40-polyoma transformed mouse NIH/3T3 cells with the E48 cDNA confirmed that the antigen is likely to be involved in cell-cell adhesion.     

Intracellular domain of desmoglein 3 (pemphigus vulgaris antigen) confers adhesive function on the extracellular domain of E-cadherin without binding catenins

For the extracellular (EC) domain of E-cadherin to function in homophilic adhesion it is thought that its intracytoplasmic (IC) domain must bind alpha- and beta-catenins, which link it to the actin cytoskeleton. However, the IC domain of pemphigus vulgaris antigen (PVA or Dsg3), which is in the desmoglein subfamily of the cadherin gene superfamily, does not bind alpha- or beta-catenins. Because desmogleins have also been predicted to function in the cell adhesion of desmosomes, we speculated that the PVA IC domain might be able to act in a novel way in conferring adhesive function on the EC domain of cadherins. To test this hypothesis we studied aggregation of mouse fibroblast L cell clones that expressed chimeric cDNAs encoding the EC domain of E-cadherin with various IC domains. We show here that the full IC domain of PVA as well as an IC subdomain containing only 40 amino acids of the PVA intracellular anchor (IA) region confer adhesive function on the E-cadherin EC domain without catenin-like associations with cytoplasmic molecules or fractionation with the cell cytoskeleton. This IA region subdomain is evolutionarily conserved in desmogleins, but not classical cadherins. These findings suggest an important cell biologic function for the IA region of desmogleins and demonstrate that strong cytoplasmic interactions are not absolutely necessary for E- cadherin-mediated adhesion.     

Reduction of disulfide bonds within lysosomes is a key step in antigen processing.

Reduction of disulfide bonds is a key step in antigen processing both to allow the unfolding of protein antigens, increasing the access of proteolytic processing enzymes, and to expose free Cys residues within linear peptide epitopes recognized by T cells. We show here that reduction and alkylation of Ag (hen egg lysozyme and ribonuclease A) vastly increased their proteolysis (by specific enzymes or lysosomal fractions) and the production of specific immunogenic peptides that bound to class II MHC molecules recognized by T hybridoma cells. We also show that the lysosome is the vesicular compartment that mediates protein disulfide reduction. We coupled [125I]tyrosine to 131I-alpha 2-macroglobulin or [131I] transferrin via a reducible disulfide linker. Removal of [125I]tyrosine from the alpha 2-macroglobulin conjugate was initiated only after 15 to 20 min of uptake by macrophages, suggesting that reduction occurred late in the endocytic pathway. No reduction of transferrin conjugates was seen, indicating that early, recycling endosomes did not contain reducing activity. Subcellular fractionation showed that the disulfide bonds were reduced only in heavy density (lysosome) fractions and remained intact in fractions of light density (endosomes and plasma membrane). These results indicate the importance of lysosomes in the biochemical processing of protein Ag presented to T cells.     

P120-catenin is a novel desmoglein 3 interacting partner: identification of the p120-catenin association site of desmoglein 3

P120-catenin (p120ctn) is an armadillo-repeat protein that directly binds to the intracytoplasmic domains of classical cadherins. p120ctn binding promotes the stabilization of cadherin complexes on the plasma membrane and thus positively regulates the adhesive activity of cadherins. Using co-immunoprecipitation, we show here that p120ctn associates to desmogleins (Dsg) 1 and 3. To determine which region is involved in the association between Dsg3 and p120ctn, we constructed mutant Dsg3 proteins, in which various cytoplasmic subdomains were removed. The tailless Dsg3 constructs Delta IA:AA1-641Dsg3 and Delta 641-714Dsg3, which do not contain the intracellular anchor (IA) region, did not coprecipitate with p120cn, nor did they colocalize at the plasma membrane. Immunocytochemical analysis revealed that p120ctn does not localize to desmosomes, but colocalizes with Dsg3 at the cell surface. A biotinylation assay for Dsg3 showed that biotinylated Delta 641-714Dsg3 was turned over more rapidly than wild-type Dsg3. These results indicate that the membrane proximal region (corresponding to residues 641-714) in the IA region of Dsg3 is necessary for complex formation with p120ctn, and to maintain free Dsg3 at the cell surface before it is integrated into desmosomes. In summary, we show that p120ctn is a novel interactor of the Dsg proteins, and may play a role in desmosome remodeling.     

Movement protein of a closterovirus is a type III integral transmembrane protein localized to the endoplasmic reticulum

Cell-to-cell movement of beet yellows closterovirus requires four structural proteins and a 6-kDa protein (p6) that is a conventional, nonstructural movement protein. Here we demonstrate that either virus infection or p6 overexpression results in association of p6 with the rough endoplasmic reticulum. The p6 protein possesses a single-span, transmembrane, N-terminal domain and a hydrophilic, C-terminal domain that is localized on the cytoplasmic face of the endoplasmic reticulum. In the infected cells, p6 forms a disulfide bridge via a cysteine residue located near the protein's N terminus. Mutagenic analyses indicated that each of the p6 domains, as well as protein dimerization, is essential for p6 function in virus movement.     

Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum merozoites

Immunoelectron microscopy demonstrated the presence of Pf155/RESA in dense granules of Plasmodium falciparum merozoites rather than in micronemes as previously suggested. Since the dense granules are released after the merozoite enters the parasitophorous vacuole, the role of Pf155/RESA in invasion and subsequent steps of parasite development may differ from that of a molecule located in the micronemes.     

Immunoreactive GABA transaminase within the pancreatic islet is localized in mitochondria of the B-cell

Subcellular localization of gamma aminobutyrate-alpha-ketoglutarate transaminase (GABA-T) in the pancreatic islets of Langerhans was determined by use of an electron microscopic, immunogold post-embedding protocol. The objective of this study was to define the islet cell distribution and subcellular localization of GABA-T. Within the islet, GABA-T was found only in the B-cells and was localized in mitochondria; 78 mitochondria contained 336 gold particles, whereas 245 secretory granules contained only 18 gold particles. Although studies utilizing either the isolated perfused pancreas or cultured islets have shown that exogenous GABA modulates D-cell secretion, in this study immunoreactive GABA-T, the catabolic enzyme for GABA, was not detectable in A- and D-cells of the islet. Control studies substituting normal rabbit serum for the GABA-T antiserum resulted in absence of labeling. These results indicate that the high concentration of GABA present in islet B-cells is catabolized by GABA-T in the mitochondrial compartment, consistent with the possibility that GABA functions as a mediator of B-cell activity.     

TEX101, a germ cell-marker glycoprotein, is associated with lymphocyte antigen 6 complex locus k within the mouse testis

In adult male mice, the glycosylphosphatidyl inositol-anchored glycoprotein TEX101 is expressed only in germ cells and is thought to be involved in spermatogenesis. However, the details regarding the function of TEX101 remain to be clarified. We previously identified Ly6k as a candidate TEX101-associated protein, but as molecular probes are not currently available to detect Ly6k, we do not have conclusive evidence of the association between TEX101 and Ly6k. In this study, we confirmed the biological interaction between TEX101 and Ly6k using an established anti-mouse Ly6k polyclonal antibody (pAb). A combination of immunoprecipitation, Western blot, and immunohistochemical analyses using the pAb revealed that TEX101 is physically associated with Ly6k within the testis. In addition, these proteins simultaneously co-migrate into the detergent-resistant membrane fractions, suggesting that TEX101 collaborates with Ly6k on the cell membrane and may play a role in spermatogenesis.