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Biochemical Characterization of the Desmosome

      Before 1974, the importance of desmosomesin intercellular adhesionwas mere speculation based on theultrastructural likeness of these organellesto cellular “spot welds.” The firstbiochemical isolation of desmosomes, described by
      • Skerrow C.J.
      • Matoltsy A.G.
      Chemical characterization of isolated epidermal desmosomes.
      ,
      • Skerrow C.J.
      • Matoltsy A.G.
      Isolation of epidermal desmosomes.
      , is clearly a milestonethat marks a shift from the morphologicstudies published before thistime, and an emergence of studiesreporting expression cloning, cDNAsequencing, molecular characterizationand functional analysis of desmosomecomponents. More than 30 yearsafter their isolation technique wasdeveloped, it is now apparent that thisoriginal hypothesis was indeed accurate, and moreover that desmosomesmay play additional roles in tissuemorphogenesis that transcend theiradhesive functions.
      Desmosomes are highly insolublestructures resistant to usual homogenizationtechniques used for cell fractionation(i.e., hypotonic swelling orphysical shearing). Yet, more stringenttechniques such as the use of alkali orurea dissolve the structures withoutmaintaining native desmosome organizationor protein–protein interactions. In 1974, Skerrow and Matoltsyreported that by using citric acid–sodium citrate buffer to solubilizebovine epidermis and enrich epidermalfilaments, they could overcomethese limitations and obtain extractswhile maintaining desmosome structure(
      • Skerrow C.J.
      • Matoltsy A.G.
      Chemical characterization of isolated epidermal desmosomes.
      ). Following solubilization, they usedsucrose density gradient centrifugationto separate cell debris, organellesand membrane fragments, and founda fraction that contained many intactdesmosomes. In this preparation, largerintermediate filament (IF) bundleswere solubilized, but desmosomes, desmosome-associated IFs and mostmembrane structures were preserved, resulting in a desmosome-enrichedpreparation that allowed further biochemicalcharacterization. With thistechnique, Skerrow and Matoltsy wereable to separate the desmosome componentsby SDS-PAGE and showedthat several proteins were glycoproteins. They characterized the chemicalnature of the desmosome componentsby measuring the proportions of proteins, carbohydrates and lipids, nonpolaramino-acid content and sialicacid content. On measuring low levelsof sialic acid, they correctly deducedthat intercellular adhesion is not likelyto be attributable to carbohydrateinteractions but to other types of interactionsbetween the glycoproteins, contradicting a previous hypothesis onthe mechanism of calcium-dependentdesmosomal adhesion.
      Skerrow and Matoltsy’s work was followed by a series of studies from the Franke and Steinberg laboratories, characterizing and purifying desmosome components. Steinberg and colleagues further enriched the glycosylated transmembrane protein component in 1981 with modifications to Skerrow and Matoltsy’s isolation technique (
      • Gorbsky G.
      • Steinberg M.S.
      Isolation of the intercellular glycoproteins of desmosomes.
      ). Using monoclonal antibodies and proteolytic peptide mapping, they began to compare the sequence homology and carbohydrate composition between desmosome glycoproteins in 1983 (
      • Cohen S.M.
      • Gorbsky G.
      • Steinberg M.S.
      Immunochemical characterization of related families of glycoproteins in desmosomes.
      ) and began to isolate cytoplasmic plaque proteins in 1985 (
      • Gorbsky G.
      • Cohen S.M.
      • Shida H.
      • Giudice G.J.
      • Steinberg M.S.
      Isolation of the non-glycosylated proteins of desmosomes and immunolocalization of a third plaque protein: desmoplakin III.
      ).
      Werner Franke and colleagues took an inside-out approach and, in 1978, developed a desmosome isolation technique to enrich for IF cytoskeletons and studied the components and characteristics of IF–desmosome plaque complexes (
      • Drochmans P.
      • Freudenstein C.
      • Wanson J.C.
      • Laurent L.
      • Keenan T.W.
      • Stadler J.
      • et al.
      Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.
      ). In 1981 and 1983, Franke and colleagues made critical biochemical and immunochemical characterizations of the IF-binding protein desmoplakin (
      • Franke W.W.
      • Schmid E.
      • Grund C.
      • Muller H.
      • Engelbrecht I.
      • Moll R.
      • et al.
      Antibodies to high molecular weight polypeptides of desmosomes: specific localization of a class of junctional proteins in cells and tissue.
      ,
      • Mueller H.
      • Franke W.W.
      Biochemical and immunological characterization of desmoplakins I and II, the major polypeptides of the desmosomal plaque.
      ) and other desmosome plaque proteins (
      • Franke W.W.
      • Mueller H.
      • Mittnacht S.
      • Kapprell H.P.
      • Jorcano J.L.
      Significance of two desmosome plaque-associated polypeptides of molecular weights 75 000 and 83 000.
      ). Franke and colleagues have since made the major contribution towards the discovery and initial characterization of nearly all desmosome plaque proteins known to date. Franke’s early work on IF–plaque interactions provided the focus for subsequent studies demonstrating that anchoring of the IF cytoskeleton into the desmosomal plaque is essential for strengthening and maintaining desmosomal intercellular adhesion.
      While characterization of the chemical nature of desmosome proteins was being performed, Pam Cowin, David Garrod and colleagues made important observations regarding the biological significance of desmosomes. Cowin and Garrod generated specific antibodies against several desmosome components and used these for immunolocalization and immunoreactivity of desmosome preparations and tissue sections (
      • Cowin P.
      • Garrod D.R.
      Antibodies to epithelial desmosomes show wide tissue and species crossreactivity.
      ,
      • Cowin P.
      • Mattey D.
      • Garrod D.
      Distribution of desmosomal components in the tissues of vertebrates, studied by fluorescent antibody staining.
      ). They demonstrated that antibodies raised against epithelial desmosomes displayed crossreactivity between tissues and species, and observed that the distribution of desmosome components was similar in different tissues and between different vertebrate species. These observations highlight the importance of desmosomes as organelles whose structure and therefore function are highly conserved throughout vertebrate evolution.
      The groups mentioned here represent just a few among many others who have made significant contributions to the characterization of the structure and function of the desmosome. The biochemical tools developed by these pioneers constructed the foundation on which all subsequent desmosome researchers build.

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