Antigenic interrelationship between the 40-kilodalton cytokeratin polypeptide and desmoplakins

We describe here antigenic cross-reactivity between the human 40-kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF-7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40-kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes. In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40-kD cytokeratin.     

150th Anniversary Series: Desmosomes and Autoimmune Disease,Perspective of Dynamic Desmosome Remodeling and Its Impairments in Pemphigus

Abstract

Desmosomes are the most important intercellular adhering junctions that adhere two adjacent keratinocytes directly with desmosomal cadherins, that is, desmogleins (Dsgs) and desmocollins, forming an epidermal sheet. Recently, two cell–cell adhesion states of desmosomes, that is, “stable hyper-adhesion” and “dynamic weak-adhesion” conditions have been recognized. They are mutually reversible through cell signaling events involving protein kinase C (PKC), Src and epidermal growth factor receptor (EGFR) during Ca²⁺-switching and wound healing. This remodeling is impaired in pemphigus vulgaris (PV, an autoimmune blistering disease), caused by anti-Dsg3 antibodies. The antibody binding to Dsg3 activates PKC, Src and EGFR, linked to generation of dynamic weak-adhesion desmosomes, followed by p38MAPK-mediated endocytosis of Dsg3, resulting in the specific depletion of Dsg3 from desmosomes and acantholysis. A variety of pemphigus outside-in signaling may explain different clinical (non-inflammatory, inflammatory, and necrolytic) types of pemphigus. Pemphigus could be referred to a “desmosome-remodeling disease involving pemphigus IgG-activated outside-in signaling events”.     

非洲爪蟾孵化酶分子对卵黄膜作用方式的研究

非洲爪蟾的孵化液对卵黄膜和二甲基酷蛋白具有降妥活性。用非洲爪蟾孵化酶的特异性抗ＧＳＴ－ＵＶ．２抗体进行Ｗｅｓｔｅｒｎ杂交的结果表明,孵化液中出现一种分子量为６０ｋＤ的大组分,有时也会出现一种分子量为４０ｋＤ的小组分。     

Desmosomal cadherins utilize distinct kinesins for assembly into desmosomes

The desmosomal cadherins, desmogleins (Dsgs) and desmocollins (Dscs), comprise the adhesive core of intercellular junctions known as desmosomes. Although these adhesion molecules are known to be critical for tissue integrity, mechanisms that coordinate their trafficking into intercellular junctions to regulate their proper ratio and distribution are unknown. We demonstrate that Dsg2 and Dsc2 both exhibit microtubule-dependent transport in epithelial cells but use distinct motors to traffic to the plasma membrane. Functional interference with kinesin-1 blocked Dsg2 transport, resulting in the assembly of Dsg2-deficient junctions with minimal impact on distribution of Dsc2 or desmosomal plaque components. In contrast, inhibiting kinesin-2 prevented Dsc2 movement and decreased its plasma membrane accumulation without affecting Dsg2 trafficking. Either kinesin-1 or -2 deficiency weakened intercellular adhesion, despite the maintenance of adherens junctions and other desmosome components at the plasma membrane. Differential regulation of desmosomal cadherin transport could provide a mechanism to tailor adhesion strength during tissue morphogenesis and remodeling.     

Expression of cell adhesion molecule E-cadherin in Xenopus embryos begins at gastrulation and predominates in the ectoderm

The expression of the Ca2+-dependent epithelial cell adhesion molecule E-cadherin (also known as uvomorulin and L-CAM) in the early stages of embryonic development of Xenopus laevis was examined. E-Cadherin was identified in the Xenopus A6 epithelial cell line by antibody cross-reactivity and several biochemical characteristics. Four independent mAbs were generated against purified Xenopus E-cadherin. All four mAbs recognized the same polypeptides in A6 cells, adult epithelial tissues, and embryos. These mAbs inhibited the formation of cell contacts between A6 cells and stained the basolateral plasma membranes of A6 cells, hepatocytes, and alveolar epithelial cells. The time of E-cadherin expression in early Xenopus embryos was determined by immunoblotting. Unlike its expression in early mouse embryos, E-cadherin was not present in the eggs or early blastula of Xenopus laevis. These findings indicate that a different Ca2+-dependent cell adhesion molecule, perhaps another member of the cadherin gene family, is responsible for the Ca2+-dependent adhesion between cleavage stage Xenopus blastomeres. Detectable accumulation of E-cadherin started just before gastrulation at stage 9 1/2 and increased rapidly up to the end of gastrulation at stage 15. In stage 15 embryos, specific immunofluorescence staining of E-cadherin was discernible only in ectoderm, but not in mesoderm and endoderm. The ectoderm at this stage consists of two cell layers. The outer cell layer of ectoderm was stained intensely, and staining was localized to the basolateral plasma membrane of these cells. Lower levels of staining were observed in the inner cell layer of ectoderm. The coincidence of E-cadherin expression with the process of gastrulation and its restriction to the ectoderm indicate that it may play a role in the morphogenetic movements of gastrulation and resulting segregation of embryonic germ layers.     

Xenopus MHC class II molecules. II. Polymorphism as determined by two-dimensional gel electrophoresis

The class II antigens from four inbred strains of Xenopus laevis (r, f, g, and j haplotypes) and six gynogenetic LG clones (two Xenopus laevis, two Xenopus gilli haplotypes) with functionally well-defined MHC types have been immunoprecipitated with the rabbit anti-human class II beta-chain serum anti-p29boost and analyzed by two-dimensional gel electrophoresis. The glycosylated material from 15-hr biosynthetically labeled cells runs as a broad fuzzy band around 33kD that, upon removal of N-linked glycans by Endo F, resolves into upper beta-chain bands and lower alpha-chain bands. Both the glycosylated and deglycosylated class II antigens give rise to multiple IEF spots in evenly spaced arrays (alpha-chain: two to eight spots in one to three arrays, beta-chain: two to 12 spots in one to five arrays). Both chains are polymorphic and both map to the functionally defined MHC. The large number of spots argues for multiple class II antigens; by radioactive N-terminal sequencing, two homologous alpha-chains and five beta-chains are present in the f haplotype. By comparison with MHC-linked alloantisera, anti-p29boost recognizes all major polymorphic class II molecules in Xenopus laevis. A selection of outbred animals were typed by using an IEF procedure requiring only a million PHA-stimulated blood cells.     

Xenopus MHC class II molecules. I. Identification and structural characterization

Class II antigens from the Xenopus laevis MHC (f haplotype) were identified by using a rabbit antihuman class II beta-chain serum (anti-p29boost). This xenoantiserum inhibits bidirectional Xenopus MLR (but not PHA-stimulation), recognizes the same molecules as certain MHC-linked Xenopus alloantisera, and immunoprecipitates class II molecules from Xenopus cells consistent with the tissue distribution of mammalian class II molecules. The Xenopus class II molecules are composed of two different chains, both of which are 30 to 35kD transmembrane glycoproteins. The alpha-chains have some N-terminal sequence homology with mammalian class II alpha-chains (both I-E/DR and I-A/DC); the beta-chains are directly recognized by anti-p29boost and have a markedly increased SDS gel mobility under nonreducing conditions. During biosynthesis, they are noncovalently associated with a number of other chains, including ones at 25kD, 33kD, and 40 to 45kD. The alpha-chains bear three N-linked glycans (two Endo H insensitive in mature material) and the beta-chains bear two (one Endo H insensitive). Unlike most mammalian class II molecules, the deglycosylated beta-chains are significantly larger and more acidic than the alpha-chains.     

A new high molecular mass protein showing unique localization in desmosomal plaque

A high molecular mass protein of 680 kD was identified and purified from the isolated desmosomes in bovine muzzle epidermal cells. This protein, called "desmoyokin" (from the English, yoke) here, showed no binding ability with keratin filaments in vitro, and its molecule had a characteristic dumbell shape approximately 170 nm in length. We have succeeded in obtaining one monoclonal antibody specific to desmoyokin. By the use of this monoclonal antibody and antidesmoplakin monoclonal antibody, desmoyokin was shown to be colocalized with desmoplakin at the immunofluorescence microscopic level; desmoyokin occurred only in the stratified epithelium, not in the simple epithelium nor in the other tissues. At the electron microscopic level, these two proteins were clearly seen to be sorted out in the plaque of desmosomes with desmoyokin at the periphery and desmoplakin at the center of the disk- shaped desmosomal plaque, suggesting that these two plaque proteins play distinct roles in forming and maintaining the desmosomes in stratified epithelium.     

The C-terminal unique region of desmoglein 2 inhibits its internalization via tail–tail interactions

Desmosomal cadherins, desmogleins (Dsgs) and desmocollins, make up the adhesive core of intercellular junctions called desmosomes. A critical determinant of epithelial adhesive strength is the level and organization of desmosomal cadherins on the cell surface. The Dsg subclass of desmosomal cadherins contains a C-terminal unique region (Dsg unique region [DUR]) with unknown function. In this paper, we show that the DUR of Dsg2 stabilized Dsg2 at the cell surface by inhibiting its internalization and promoted strong intercellular adhesion. DUR also facilitated Dsg tail–tail interactions. Forced dimerization of a Dsg2 tail lacking the DUR led to decreased internalization, supporting the conclusion that these two functions of the DUR are mechanistically linked. We also show that a Dsg2 mutant, V977fsX1006, identified in arrhythmogenic right ventricular cardiomyopathy patients, led to a loss of Dsg2 tail self-association and underwent rapid endocytosis in cardiac muscle cells. Our observations illustrate a new mechanism desmosomal cadherins use to control their surface levels, a key factor in determining their adhesion and signaling roles.     

Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae).

Seven peptides (XT-1-XT-7) with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions of the diploid clawed frog, Xenopus tropicalis. Structural characterization of the peptides demonstrated that amino acid sequence similarity to antimicrobial peptides previously isolated from Xenopus laevis was low, suggesting that the species are not closely related phylogenetically. Peptides XT-5 and XT-3 are probably the orthologs of X. laevis peptide glycine-leucine amide (PGL(a)) and the N-terminal spacer region of prolevitide, respectively. XT-1, XT-6 and XT-7 show limited structural similarity to the spacer region of X. laevis procaeruleins and the paralogs XT-2 and XT-4 are similar to corresponding regions of proxenopsin. Orthologs of the magainins were not identified. The C-terminally alpha-amidated peptide XT-7 (GLLGPLLKIAAKVGSNLL.NH2) showed the lowest minimum inhibitory concentrations against reference microorganisms (Staphylococcus aureus 5 microM, Escherichia coli 5 microM, and Candida albicans 40 microM) and was also active against clinical isolates of methicillin-resistant S. aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus group C, Shigella sonnei, Pseudomonas aeruginosa and Enterobacter cloacae. The peptide was, however, hemolytic against human erythrocytes (50% lysis at 70 microM). Circular dichroism studies showed that XT-7 has a random structure in aqueous solution, pH 7.0 but adopts an alpha-helical conformation in the presence of 50% trifluoroethanol. Decreasing the cationicity of XT-7 either by replacement of the C-terminal CONH2 group by COOH or by deletion of the Lys(8) residue produced analogs with greatly (>10-fold) decreased antimicrobial potencies.     

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

Cell to cell adhesion systems in Xenopus laevis, the South African clawed toad. II: Monoclonal antibody against a novel Ca2+-dependent cell-cell adhesion glycoprotein on amphibian cells

We isolated a mouse monoclonal antibody that disrupts Ca2+-dependent cell-cell adhesion of amphibian (Xenopus laevis) cells. When added to culture medium, the monoclonal antibody completely disrupted cell-cell adhesion of amphibian cells in monolayer culture and specifically inhibited Ca2+-dependent cell-cell adhesion of dissociated cells in reaggregation experiments. The monoclonal antibody recognized a 140 kDa cell surface glycoprotein antigenically different from the previously reported Ca2+-dependent cell-cell adhesion molecules (cadherins).     

Low-density caveolae-like membrane from Xenopus laevis oocytes is enriched in Ras

Detergent-free discontinuous sucrose density gradient centrifugation was used to resolve low- and high-density membrane fractions from Xenopus laevis oocytes. Compared to high-density membrane, low-density oocyte membrane is enriched two-fold in cholesterol and highly enriched in ganglioside GM1. Protein immunoblotting of membrane fractions from whole cells with polyclonal anti-human caveolin antibody detected multiple bands, including a distinctive triad with apparent molecular weights of 21, 33, and 48 kDa. To more clearly determine which of these caveolin-like protein(s) is associated with the oocyte plasma membrane, microdissection was used to separate external membrane (cortical preparations containing plasma membrane) from intracellular membrane. Cortical membrane preparations displayed a single 21-kDa caveolin-like protein in low-density membrane. Internal oocyte membrane displayed the higher molecular weight bands of 33 and 48 kDa and a lesser amount of the 21-kDa protein in low-density membrane fractions. Monoclonal anti-human Ras antibody detected a single 23-kDa immunoblot band that is enriched an average of eight-fold in low-density membrane fractions prepared from whole cells. This is the first report of caveolin-associated, low-density membrane in amphibian oocytes, and is consistent with a role for caveolin and caveolae-like microdomains in oocyte signal transduction.     

The origin of yolk-DNA in Xenopus laevis.

Xenopus laevis serum and plasma was found to contain an average of 25 microgram DNA/ml. Isolated X. laevis oocytes incubated in medium containing 25 microgram DNA/ml labeled with either 125I, 32P or 14C and from three different sources (bovine, E. coli and X. laevis), incorporated the label at an average rate of 0.11 ng.mm-2.hr-1. Sucrose gradient fractionation of oocytes revealed that 40-75% of the acid-precipitable label incorporated was associated with the yolk platelets. Additional incubations of oocytes in unlabeled medium demonstrated that the DNA incorporated into the yolk platelets was undergoing turnover; only 20% of the yolk-associated DNA was still present after a one-week incubation. Our data suggest that yolk-DNA arises by the adventitious uptake of DNA present in the maternal serum by vitellogenic oocytes.     

Isolation of the intercellular glycoproteins of desmosomes

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.     

An immunoelectron microscopic comparison of desmosomal constituents and hemidesmosomal ones originating from the same tissue of the same animal

The molecular constituents of desmosomes and hemidesmosomes were compared by examining bovine muzzle epidermis under immunoelectron microscopy using a postembedding method, first with antibodies prepared to four desmosomal antigens (DP1/2, DP3, DG1, DG2/3), followed by protein A-gold (PAG) complexes. The four antibodies showed almost negative labeling at hemidesmosomes as compared with the labeling observed at the desmosomes in the same tissue. By counting the number of PAG particles/200 millimicrons at hemidesmosomes and desmosomes, the above qualitative observation was confirmed quantitatively. These results support a new concept which has recently been proposed by several researchers that hemidesmosomes and desmosomes are immunochemically distinct.     

Regulation of Xenopus embryonic cell adhesion by the small GTPase,rac

TGF-beta family signalling pathways are important for germ layer formation and gastrulation in vertebrate embryos and have been studied extensively using embryos of Xenopus laevis. Activin causes changes in cell movements and cell adhesion in Xenopus animal caps and dispersed animal cap cells. Rho family GTPases, including rac, mediate growth factor-induced changes in the actin cytoskeleton, and consequently, in cell adhesion and motility, in a number of different cell types. Ectopic expression of mutant rac isoforms in Xenopus embryos was combined with animal cap adhesion assays and a biochemical assay for rac activity to investigate the role of rac in activin-induced changes in cell adhesion. The results indicate that (1) the perturbation of rac signalling disrupts embryonic cell-cell adhesion, (2) that rac activity is required for activin-induced changes in cell adhesive behavior on fibronectin, and (3) that activin increases endogenous rac activity in animal cap explants.