Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia.

Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75,000 basic protein does not serve general desmosomal functions but rather cell type-specific ones and that the composition of the desmosomal plaque can be different in different cell types. The possible diagnostic value of this protein as a marker in cell typing is discussed.

The complement of desmosomal plaque proteins in different cell types.

Desmosomal plaque proteins have been identified in immunoblotting and immunolocalization experiments on a wide range of cell types from several species, using a panel of monoclonal murine antibodies to desmoplakins I and II and a guinea pig antiserum to desmosomal band 5 protein. Specifically, we have taken advantage of the fact that certain antibodies react with both desmoplakins I and II, whereas others react only with desmoplakin I, indicating that desmoplakin I contains unique regions not present on the closely related desmoplakin II. While some of these antibodies recognize epitopes conserved between chick and man, others display a narrow species specificity. The results show that proteins whose size, charge, and biochemical behavior are very similar to those of desmoplakin I and band 5 protein of cow snout epidermis are present in all desmosomes examined. These include examples of simple and pseudostratified epithelia and myocardial tissue, in addition to those of stratified epithelia. In contrast, in immunoblotting experiments, we have detected desmoplakin II only among cells of stratified and pseudostratified epithelial tissues. This suggests that the desmosomal plaque structure varies in its complement of polypeptides in a cell-type specific manner. We conclude that the obligatory desmosomal plaque proteins, desmoplakin I and band 5 protein, are expressed in a coordinate fashion but independently from other differentiation programs of expression such as those specific for either epithelial or cardiac cells.

Plakoglobin is a component of the filamentous subplasmalemmal coat of lens cells.

The plasma membranes of the cells of the superficial layer of the eye lens and the lens fibres are in close intercellular contact, leaving an intermembrane space of approximately 20 nm or less throughout their entire length. This plasma membrane is underlaid by a filamentous, cytoplasmic web containing actin, proteins of the spectrin and band 4.1 families, alpha-actinin and vinculin. Using immunofluorescence microscopy and immunoblotting of gel electrophoretically separated proteins, we show that plakoglobin, the plaque protein common to desmosomal and nondesmosomal adhering junctions, is present in lens cells and is also a component of the subplasmalemmal coat of these cells. Plakoglobin also exists in the extended regions of intercellular contacts between cultured lenticular cells where it often colocalizes with vinculin but does not occur in other vinculin-rich plasma membrane regions such as the focal adhesions at the ventral cell surface. Plakoglobin associated with plasma membrane regions can also be identified in various other adhesive cultured cells, but it is not detected in cells and tissues that do not establish firm intercellular junctions such as erythrocytes, platelets, cultured myeloma cells and smooth muscle tissue. We conclude that plakoglobin occurs, at least in lens cells, throughout the entire subplasmalemmal coat, coexisting in this situation not only with vinculin but also with spectrin and 4.1 protein(s). This colocalization infers the presence of a distinct, complex type of membrane-skeleton assembly involving the actin filament-associated junctional plaque elements plakoglobin and vinculin together with actin-associated proteins of the spectrin and band 4.1 protein families.

Isolation and symmetrical splitting of desmosomal structures in 9 M urea.

A new way of isolating desmosomal structures from various epithelia is described which takes advantage of the unusual resistance of the desmosomal plaque and parts of the desmosomal membrane domain to denaturing agents such as 9 M urea and 5 M guanidinium hydrochloride (Gdn-HCl). The fractions obtained have been examined by electron microscopy and by gel electrophoresis. When cytoskeletal fractions from epithelial cells, notably those from multistratified epithelia such as bovine epidermis or tongue mucosa, are treated with urea or Gdn-HCl most of the cytoskeletal protein, including cytokeratin material, is removed. The desmosomal structures, however, are retained with well preserved plaque organization and desmoglea components and can be harvested by centrifugation. This simple and rapid procedure for the enrichment of desmosomal structures and proteins also express internal desmosomal domains as the result of "splitting" of the desmosome along the midline structure. These split desmosomal halves reveal regular arrays of desmogleal particles of 8 to 15 nm diameter projecting from the membrane surface. Gel electrophoresis of the polypeptides present in these residual structures has shown prominent amounts of desmoplakins I and II as well as components 3 and 5 whereas glycoproteins 4a and 4b are significantly reduced in relation to untreated or citric acid-treated fractions. Using immunoelectron microscopy on desmosomes split in urea we have also demonstrated the specific localization of desmoplakin on the cytoplasmic side. The observations suggest that the architectural components of the desmosome are among the cell structures most resistant to protein-denaturing treatments. The value of this procedure for preparations of desmosomal proteins and for the production of antibodies specifically reacting with internal domains of junctions, i.e., tools that may interfere with cell-to-cell coupling, is discussed.

Biochemical characterization of desmosomal proteins isolated from bovine muzzle epidermis: amino acid and carbohydrate composition.

The seven major desmosomal polypeptides from isolated bovine muzzle desmosomes ranging from Mr 75 000 to 250 000 were separated by gel electrophoresis, isolated and characterized with respect to their amino acid composition and sugar content. The two largest polypeptides (bands 1 and 2), i.e. desmoplakins I and II, are similar in their amino acid composition, confirming our previous immunological and biochemical data, and display a relatively high glycine content. In contrast, the other two cytoplasmic components also believed to be associated with the desmosomal plaque, i.e. polypeptides of bands 5 (Mr 83 000) and 6 (Mr 75 000), differ significantly in their amino acid composition from the desmoplakins and from each other. All four candidate polypeptides for plaque association, i.e. bands 1, 2, 5, and 6, show no significant glycosylation. The glycoproteins 4a and 4b (Mr 115 000 and 130 000) are similar in their amino acid composition, peptide analysis and immunological reactivity. Both are relatively rich in mannose and galactose but also contain sialic acid. Our determinations also indicate that the two polypeptides differ significantly in their N-acetylglucosamine and mannose content. Most, if not all, of the sugar residues are associated with a water-soluble fragment of Mr 15 500 obtained after limited digestion with V8 protease. The glycopolypeptides obtained in band 3 (Mr 164 000-175 000) are distinct from the glycopolypeptides 4a and 4b in amino acid composition, sugar content, isoelectric pH values, certain antigenic determinants and in their pattern of cleavage products obtained by treatment with proteases or cyanogen bromide. The results identify polypeptides of bands 3, 4a and 4b as glycosylated with characteristic sugar compositions. It is suggested that the major glycoproteins (bands 3, 4a, 4b) of the desmosome are integral membrane components arranged in a special way conferring resistance to detergent treatment. The possible roles of these glycoproteins in cell recognition and in adhesive functions of the desmosome are discussed.

Demonstration of specific detection of anti-HCV IgM core antibodies.

The specificity of IgM isotype response directed against the putative core of hepatitis C virus (HCV core IgM) was demonstrated in HCV IgM EIA reactive samples. No interference was noted when samples with increasing levels of rheumatoid factor (RF) alone, or in combination with graded concentrations of anti-HCV IgG (HCV IgG), were tested. No deterioration in assay specificity was seen in 30 sera from patients wih monoclonal gammapathies (all isotypes). With Protein G affinity chromatography, RF/HCV IgG immune complexes and HCV core IgM antibodies displayed different binding characteristics, HCV core IgM appeared in the buffer eluate while HCV IgG/RF remained bound. With sucrose density gradient centrifugation HCV core IgM sedimented at 17-19S.

Performance characteristics of an improved version of the ABBOTT IMx HBsAg (V2) MEIA.

The ABBOTT IMx HBsAg (V2) microparticle enzyme immunoassay (MEIA) is a fully automated two-step assay for the qualitative determination of hepatitis B surface antigen (HBsAg). HBsAg is the most important serological marker of acute and chronic hepatitis B infection. Therefore, sensitivity of the currently used detection systems for HBsAg is critical to blood screening, diagnosis of HBV infection and therapy monitoring of HBV infected individuals. The design of the assay has been modified and performance characteristics of the modified test were compared to its predecessor. Precision and specificity of the modified assay are comparable to its predecessor. PEI standard subtype ad is detected at 0.026 U/ml versus 0.053 U/ml with the previous assay version. HBsAg subtypes ad and ay are detected at 0.22 ng/ml and 0.17 ng/ml, respectively. The seroconversion window is reduced by 2-28 days versus the predecessor test. The modified assay has the capability to detect HBsAg mutants not detected by some other commercial assays. The modified version of the ABBOTT IMx HBsAg (V2) MEIA provides significantly improved sensitivity combined with the capability to detect prevalent and less prevalent HBsAg mutants.

Early detection of hepatitis B surface antigen--prototype of a new fully automated HBsAg microparticle enzyme immunoassay (MEIA).

HBsAg is the most important serological marker of acute and chronic hepatitis B infection. Therefore, sensitivity of the currently used detection systems for HBsAg is of major importance for blood screening, diagnosis of HBV infection and therapy monitoring of HBV infected individuals. A Prototype Microparticle Enzyme Immunoassay (MEIA) for qualitative determination of hepatitis B surface antigen (HBsAg) has recently been developed for the fully automated AxSYM analyzer with the intention to provide a test displaying improved sensitivity when compared to the currently marketed HBsAg assays. The present study was conducted to demonstrate sensitivity of the new Prototype AxSYM HBsAg MEIA and the corresponding, fully automated Prototype AxSYM HBsAg Confirmatory MEIA. Analytical sensitivity of the Prototype AxSYM HBsAg MEIA was assessed by testing quantitative HBsAg panels. Sensitivity for HBsAg subtypes ad and ay was found to be 0.15 ng/ml and 0.09 ng/ml, respectively. The detection level for HBsAg subtype ad Standard provided by the Paul Ehrlich Institute was estimated to be 0.03 PEI U/ml. Analysis of hepatitis B seroconversion panels suggests that the new Prototype AxSYM HBsAg MEIA significantly reduces the seroconversion window when being compared to other highly sensitive commercial HBsAg assays. The corresponding Prototype AxSYM HBsAg Confirmatory MEIA demonstrated the ability to confirm low titer seroconverters found reactive with the Prototype AxSYM HBsAg Screening MEIA.

Effect of Ca2+ on binding of the calpains to calpastatin.

Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.

Immunolocalization of plakoglobin in endothelial junctions: identification as a special type of Zonulae adhaerentes.

We have characterized the junctions between endothelial cells of diverse blood vessels at the light and electron microscopic level using various antibodies to plakoglobin (polypeptide Mr 83,000) and vinculin. Endothelial cells from fenestrated and non-fenestrated capillaries to large arteries are connected to each other by extended junctions that are coated on their cytoplasmic face by plaques of loosely matted filamentous material that form a continuous belt system along the cell circumference. These plaques are devoid of desmosome-specific proteins such as desmoplakin(s) and desmoglein, but contain plakoglobin. Immunofluorescence microscopic reactions of these regions with vinculin antibodies have also been observed, although they are much weaker and less consistent. This composition, together with their association with actin microfilaments, classifies this extended plaque system as Zonulae adhaerentes. Our results also show that such endothelia may be distinguished from truly epithelial cells by the absence of desmosomes and intermediate filaments of the cytokeratin type. The relationship of the various kinds of adhering junctions and the physiological importance of these junctions are discussed.

Biochemical characterization of the soluble form of the junctional plaque protein, plakoglobin, from different cell types.

A polypeptide of identical molecular mass (Mr 83,000) and charge to desmosomal plakoglobin from bovine snout epidermis was identified in soluble and pelletable fractions from diverse tissues and cells of different mammalian species, including cells and tissues devoid of desmosomes (e.g. endothelial, retinal, lenticular cells, fibroblasts). The protein, however, was not detected in erythrocytes and platelets and in myeloma cells, nor in smooth muscle tissue. In all cells examined, the plakoglobin soluble upon cell lysis in buffers of near-physiological pH and ionic strength (21-31% of the total plakoglobin in the different cell types) was found to exist in a distinct molecular form. On sucrose gradient centrifugation it appeared at about 7 S and gel filtration chromatography revealed a Stokes radius of about 5.0 nm, from which an Mr of about 170,000 was estimated. By using isoelectric focusing under non-denaturing conditions, soluble approximately equal to 7-S plakoglobin had an isoelectric point at about pH 5.3. The plaque-bound and the soluble form of plakoglobin were indistinguishable by electrical charge and molecular mass, regardless of the source, indicating molecular identity. Cross-linking of soluble proteins with cupric 1,10-phenanthroline resulted in the formaton of a cross-linked product of plakoglobin with similar physical properties as the native approximately equal to 7-S particle, which is compatible with the interpretation that the soluble plakoglobin particle is a dimer. While a major proportion of the plakoglobin in the desmosomal plaque was resistant to various extraction procedures, plakoglobin present in the plaques of non-desmosome-containing cells and tissues was readily extractable under low and high salt conditions. This indicates that differences exist in the binding of plakoglobin to desmosomal plaques and the plaques of non-demosomal junctions.

The desmosomal plaque and the cytoskeleton.

Two major plasma membrane domains are involved in the architectural organization of the cytoskeleton. Both are junctions of the adherens category characterized by the presence of dense plaques associated with the cytoplasmic surface of their membranes. The plaques serve as specific anchorage structures for two different types of cytoplasmic filaments. Intermediate-sized filaments (IF) of several types, i.e. cytokeratin IF in epithelial cells, desmin IF in cardiac myocytes and vimentin IF in arachnoidal cells of meninges, meningiomas and several other cells, attach to the desmosomal plaques, whereas actin-containing microfilaments associate with non-desmosomal adhering junctions such as the zonula adherens, fascia adherens and punctum adherens. The plaques of both kinds of adhering junctions contain a common acidic polypeptide of Mr 83,000 identical to 'band 5 protein' of bovine snout epidermal desmosomes. However, other plaque components are mutually exclusive to one of the two subclasses of adhering junctions. The desmosomal plaque structure, which does not contain vinculin and alpha-actinin, comprises representatives of cytoplasmic, non-membrane-integrated proteins such as desmoplakin(s) and the cytoplasmic portions of transmembrane glycoproteins such as 'band 3 glycoprotein'. The analysis of both categories of junction-associated plaques should provide a basis for understanding the establishment and the dynamics of junction-cytoskeleton interaction.

Detection of specific human immunodeficiency virus IgM antibodies.

This study was done to demonstrate whether the use of the antigen-sandwich human immunodeficiency virus (HIV) antibody-screening assays (3rd generation assays), which detect all classes of anti-HIV immunoglobulins, leads to an earlier detection of HIV IgM compared to the 2nd generation HIV antibody-screening assays. We tested sequential bleeds of three donors obtained from commercially available seroconversion panels. Anti-HIV testing was done before and after high-performance liquid chromatography separation of IgG and IgM fractions. The positive result of the first bleedings from all three panels was linked to the IgM fraction, while at that time the IgG fraction was still negative. For subsequent samples drawn 5-9 days later, a positive signal was obtained with the IgG fraction in addition to a stronger positive signal obtained with the IgM fraction. We conclude that an assay capable of simultaneously detecting different immunoglobulin classes, including IgM, will help to narrow the "window period" for serological detection of seroconversion to HIV by detecting anti-HIV IgM-containing samples earlier than conventional assays using only anti-human IgG enzyme conjugates (indirect anti-HIV-screening assay, 2nd generation assays).

The role of autolysis in activity of the Ca2+-dependent proteinases (mu-calpain and m-calpain).

A recent hypothesis suggests that proteolytic activity of the micromolar and millimolar Ca2+-requiring forms of the Ca2+-dependent proteinases (mu- and m-calpain, respectively) is regulated in vivo by their association with a phosphatidylinositol-containing site on the plasma membrane followed by autolysis of the proteinases. Phosphatidylinositol association lowers the Ca2+ concentration needed for autolysis, and autolysis, in turn, lowers the Ca2+ concentration needed for proteolytic activity. To test this hypothesis, we have compared the Ca2+ concentrations needed for autolysis and for proteolytic activity of the calpains both in the presence and the absence of phosphatidylinositol. Bovine skeletal muscle mu-calpain required 40-50 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 140-150 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 190-210 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. Consequently, mu-calpain is an active proteinase and does not require autolysis for activation. Bovine skeletal muscle m-calpain required 700-740 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 370-400 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 740-780 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. These results are consistent with the idea that m-calpain functions in its autolyzed form, but the results do not demonstrate that unautolyzed m-calpain is inactive. 80 microM phosphatidylinositol had no effect on the Ca2+ requirement of the autolyzed forms of either mu- or m-calpain but lowered the specific activity of mu-calpain to 20% of its activity in the absence of phosphatidylinositol. Of the four forms of the calpains, unautolyzed m-calpain, autolyzed m-calpain, and unautolyzed mu-calpain would not be proteolytically active at the free Ca2+ concentrations of 300-1200 nM present inside normal cells, and neither mu- nor m-calpain would undergo autolysis at these Ca2+ concentrations, even in the presence of phosphatidylinositol. Cells must contain a mechanism other than or in addition to membrane association and autolysis to activate the calpains.

Plakoglobin: a protein common to different kinds of intercellular adhering junctions.

We have established, by means of a monoclonal antibody and a cDNA clone, that a desmosomal polypeptide of Mr 83,000 also occurs at the plaques of other types of adhering junctions, including the vinculin-actin-associated intercellular junctions, e.g., the zonula adhaerens of epithelial cells and the endothelial, lens, and Sertoli cell junctions. This is the first component found in common among otherwise biochemically distinct plaque domains. Despite its concentration at these intercellular junctions, it is absent from the respective cell-substratum contact sites. In addition, it appears in a globular soluble 7S form in the cytoplasm. We discuss the significance of this protein, for which the name plakoglobin is proposed, in terms of its interaction with such biochemically diverse membrane domains and their different types of associated cytoskeletal filaments.

Significance of two desmosome plaque-associated polypeptides of molecular weights 75 000 and 83 000.

Isolated desmosomes from bovine epidermis contain two major polypeptides of mol. wts. 75 000 (D6) and 83 000 (D5) which, like the desmoplakins of mol. wt. greater than 200 000, are associated with the insoluble desmosomal plaque structure. We have characterized these two polypeptides and examined their significance by peptide map comparisons and translation of bovine epidermal mRNA in vitro. Polypeptide D5 is different from polypeptide D6 by its apparent mol. wt., its isoelectric pH (approximately 6.35, whereas D6 is a basic polypeptide isoelectric at pH approximately 8.5) and its peptide map. By all these criteria desmosomal polypeptides D5 and D6 are also different from cytokeratins, desmoplakins and the glycosylated desmosomal proteins. Both polypeptides are synthesized from different mRNAs separable by gel electrophoresis on agarose: mRNA coding for polypeptide D5 is approximately 3500 nucleotides long, that for D6 is significantly shorter (estimated to 3050 nucleotides), and both contain relatively large proportions of non-coding sequences. The translational products of these mRNAs co-migrate, on two-dimensional gel electrophoresis, with the specific polypeptides from bovine epidermis, indicating that they are genuine polypeptides and are not the result of considerable post-translational processing or modification of precursor molecules. The cell and tissue distribution of these two cytoskeletal proteins and possible functions are discussed.

Seroepidemiology of hepatitis E virus in the Egyptian Nile Delta.

The seroendemicity of hepatitis E virus (HEV) in an entire village population located in the Egyptain Nile Delta is described. Serum specimens were obtained from 68% of the total population of 1,850 villagers. The lack of serum specimen was greatest in the youngest age group (< 5). Commercially available enzyme immunoassays (EIA) for antibody to hepatitis A virus (anti-HAV), to hepatitis B virus core antigen (anti-HBc), to second-generation hepatitis C virus (anti-HCV) core and nonstructural antigen, and to hepatitis E virus (HEV) were used. Only repeated reactive sera were coded as positive. Stool specimens were examined for Schistosoma mansoni by the Kato method and standard methods for the examination of the liver and spleen by ultrasonography were used. Unadjusted for nonrespone, the seroprevalence of anti-HEV was 17.2% (SE +/- 1.1). Anti-HEV seroprevalence increased by age and was not associated statistically with any of the other viral markers including HCV. Anti-HAV seroprevalence was consistently > 95%, even in the youngest age group (< 5). The overall sero-endemicity of HEV was higher than reported elsewhere and appears not to have been introduced into the village population recently.

Hepatitis E virus antibodies among patients with hemophilia, blood donors, and hepatitis patients.

The presence of antibodies to hepatitis E virus (HEV) was studied among hemophiliacs, blood donors, and hepatitis patients. Four of 296 (1.4%) hemophiliacs and 5 of 1,275 (0.4%) donors were confirmed as positive for HEV antibodies (difference was not significant: P = 0.07). Parenteral transmission of HEV to hemophiliacs was thus rare or nonexistent. Seven of 187 hepatitis patients were found with HEV antibodies (IgG and IgM). Six persons fell ill shortly after arriving from HEV-endemic countries. The seventh patient, without a history of travel, represents a case of nontropical hepatitis E. Consequently, hepatitis E should be considered in patients suffering from acute non-ABC hepatitis, even in industrialized countries.