Proc. Natl. Acad. Sci. USAVol. 83, pp. 7282-7286, October 1986Cell Biology

A cell surface desmosome-associated component: Identification of atissue-specific cell adhesion molecule

(autoantibodies/pemphigus/keratinocytes/desmosomes/celI adhesion)

JONATHAN C. R. JONES*, KAREN M. YOKOO, AND ROBERT D. GOLDMANDepartment of Cell Biology and Anatomy, Northwestern University Medical School, 303 East Chicago Avenue, Ward Building, Chicago, IL 60611

Communicated by E. Margoliash, June 9, 1986

ABSTRACT Autoantibodies in the serum of patients suf-fering the blistering skin disease pemphigus vulgaris recognizea 140-kDa glycoprotein (GP) present in enriched fractions ofbovine tongue epidermal desmosomes. Immunofluorescenceobservations of cryostat sections of bovine tongue epidermisreveal that afflinity-purified rabbit antibodies to the 140-kDaGP generate a punctate intercellular stain that is similar to thatgenerated by antibodies directed against a desmosome plaquecomponent (desmoplakin). In cultured mouse keratinocytes,the antibodies against 140-kDa GP recognize desmosomesalong areas of cell-cell contact. Double immunofluorescence ofcultured keratinocytes with these antibodies and a desmoplakinantiserum reveals that the antibodies against the 140-kDa GPstain a single fluorescent line along areas of cell-cell contact.This single fluorescent line lies between double fluorescent linesgenerated by the desmoplakin antiserum. Immunogoldultrastructural localization reveals that the 140-kDa antigen islocalized not only along the intercellular area of the desmosomebut also is found along the whole epidermal cell surface. Theantiiodies to the 140-kDa GP are able to induce a disruption ofcell-cell contact in cultured keratinocytes that possess desmo-somes. We propose that the 140-kDa GP is a cell adhesionmolecule (CAM). Furthermore we discuss the heterogeneity ofdesmosomes in the light of our findings that antibodies againstthe 140-kDa GP recognize specific stratified squamous epithe-lial tissues.

Desmosomes appear to play an important role in the adhesionof epithelial cells 1). Several components of the cytoplasmicplaque of the desmosome have been identified and charac-terized (2, 3). Other desmosome components have beenassigned to the extracellular portion of the desmosomebecause they are glycosylated and are enriched in desmo-somal "cores," which consist of the intercellular region ofthe desmosome in addition to plasma membrane and plaqueremnants (4, 5). Two of these glycoproteins, a 100-kDa anda 115-kDa polypeptide (the "desmocollins"), appear to beinvolved in adhesion (6).We have introduced another approach to studying

desmosomes through the analysis ofhuman autoantibodies inserum samples from patients suffering from pemphigus, ablistering disease of the skin (7). In this disease, epidermalcells undergo acantholysis (i.e., a loss of cell adhesionfollowed by cell death), and the patients possess autoanti-bodies that recognize desmosomes as detected in mouse skinpreparations and cultured mouse epidermal cells (7). We havespeculated that this loss of cell adhesion in pemphigus may bedue, at least in part, to an antibody-induced disruption ofdesmosomes (7). Thus, it is important to determine whetherany of the desmosomal antigens recognized by pemphigusautoantibodies are candidates for intercellular adhesion mol-

ecules. Indeed, Koulu et al. (8) have shown that autoanti-bodies in some, but not all, pemphigus foliaceus serumsamples recognize a 160-kDa glycoprotein (GP) (band 3 in ref.3) component of desmosomes. This same GP may be anintercellular desmosome component (4), although this hasbeen questioned by other authors (6).

Recently we described autoantibodies in the serum ofpemphigus vulgaris patients that recognize a 140-kDa poly-peptide present in desmosomal fractions (9). In this study wedescribe affinity-purified rabbit autoantibodies directedagainst this 140-kDa desmosome-associated glycoprotein.These antibodies recognize desmosomes in bovine tongueepidermis and cultured epidermal cells as determined byimmunofluorescence and immunoblotting analyses. Further-more, ultrastructural immunolocalization of this 140-kDaantigen reveals that it is located in the intercellular region ofthe desmosome. The adhesive function of this 140-kDaantigen is discussed in the light of our finding that theantibodies against the 140-kDa GP induce a disruption ofintercellular contact in cultured mouse epidermal cells.

MATERIALS AND METHODSCell Culture. Primary mouse epidermal cells were prepared

and maintained in culture as reported elsewhere (10, 11).Antisera. A serum sample from one pemphigus vulgaris

patient obtained from John Stanley, Dermatology Branch,National Cancer Institute, National Institutes of Health, anda rabbit anti-desmoplakin antiserum (2, 7) were used in thisstudy.

Immunofluorescence. Tissue and cultured cells were pre-pared for indirect immunofluorescence as described (7).NaDodSO4/PAGE and Immunoblotting Procedure. NaDod-

S04/PAGE using 7.5% acrylamide slab gels was performedon bovine desmosome preparations (3, 12) and whole-mousekeratinocyte protein samples (9) as described by Laemmli(13). Separated polypeptides were transferred to nitrocellu-lose (14), and immunoblotting analyses were carried out asdescribed (9).The periodic acid-Schiffreagent (PAS) reaction for GP was

performed on NaDodSO4 gels of separated desmosomepolypeptides as reported elsewhere (15).

Production of the Chicken Anti-Bovine Desmoplakin Anti-serum and Purification of Rabbit Autoantibodies to the 140-kDa GP. The desmosome fraction was subjected to NaDod-S04/PAGE, and the desmoplakins were excised from the gel.These gel pieces were placed in Laemmli sample buffer on0.5-cm 4.5% Laemmli tube gels and electrophoresed at 4 mAinto dialysis tubing. The solution containing the desmoplakinpolypeptides was used to immunize a chicken. The antibodyagainst the 140-kDa GP was affinity-purified from a rabbitautoimmune serum sample by using the appropriate region of

Abbreviations: CAM, cell adhesion molecule(s); GP, glycoprotein.*To whom reprint requests should be addressed.

7282

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Proc. Natl. Acad. Sci. USA 83 (1986) 7283

nitrocellulose blots of bovine tongue desmosome prepara-tions as reported by Olmsted (16).

Ultrastructural Immunolocalization. Tongue epidermis wasfixed in 4% paraformaldehyde and embedded in LowicrylK4M as described (17). Thin sections were prepared, placedon nickel grids, and incubated for 30 min at room temperaturein a 1:20 dilution ofnormal goat serum in 20mM Tris-bufferedsaline (pH 8.2). The sections then were incubated overnightat room temperature with either the antibody to 140-kDa GPor the rabbit antidesmoplakin serum diluted 1:50 in 20 mMTris-buffered saline (pH 8.2) containing 0.5% bovine serumalbumin. The sections were rinsed in water and then incu-bated in 5-nm gold-conjugated goat anti-rabbit IgG (JanssenPharmaceutica, Beerse, Belgium) diluted 1:10 in albumin/saline buffer. The sections were washed in albumin/salinebuffer, stained for 15 min in 3% aqueous uranyl acetatefollowed by 1 min in lead citrate, and viewed in a JEOL 200CX electron microscope at 80 kV.

RESULTSAn enriched fraction ofbovine tongue desmosomes was usedin immunoblotting assays to determine the immunoreactivityof the chicken and rabbit desmoplakin antisera and thepemphigus vulgaris serum sample (Fig. 1). Both of thedesmoplakin antisera recognized two polypeptides of250 and200 kDa (desmoplakins 1 and 2) present in the preparation(Fig. 1). The serum sample from the patient with pemphigusvulgaris contained antibodies that react with a 140-kDadesmosome-associated polypeptide (9) that is not recognizedby the desmoplakin antisera (Fig. 1). Periodic acid/Schiffreagent staining of a NaDodSO4/gel of the desmosomefraction suggested that this 140-kDa polypeptide is a glyco-protein (Fig. 2).We wondered, therefore, whether the 140-kDa polypeptide

recognized by the pemphigus vulgaris antibodies was acomponent of desmosomes or whether it was a contaminantof our desmosome fraction. We affinity-purified rabbit auto-antibodies that recognized the 140-kDa GP by using thescheme of Olmsted (16) (Figs. 1 and 3). These antibodies

P 140 D DD1-D2 --

165/f160 -140 -

1 2 3 4 5

FIG. 1. An enriched fraction of bovine tongue epidermaldesmosomes was subjected to NaDodSO4/PAGE and transferred tonitrocellulose. Lanes: 1, an amido black stain of transferred proteins.[Note the two high molecular mass polypeptides D1 and D2 (des-moplakins 1 and 2)]; 2, an itnmunoblot using the serum of a

pemphigus vulgaris patient (autoantibodies in this serum samplerecognize a 140-kDa polypeptide); 3, an immunoblot of affinity-purified rabbit autoantibodies against the 140-kDa GP; 4 and 5,desmoplakin antibodies from rabbit (lane 4) and chicken (lane 5),which recognize both D1 and D2 in the tongue desmosome-enrichedpreparation.

CB PAS

U-:~% 14

f,..'' ': :.. ..

...: '' . . '*. ;',.

.:.: :...

*. R,2 ;'

1 2

-165 160140

FIG. 2. A bovine tongue epi-dermal desmosome preparationwas subjected to NaDodSO4/PAGE. Lanes: 1, Coomassieblue (CB) stain; 2, GP detectionwith the periodic acid/Schiffreagent (PAS) reaction. Notethat there are a number of GPs inthe preparation. These include the160- and 165-kDa polypeptidesand the 140-kDa polypeptide.

recognized a 140-kDa polypeptide present in an enrichedfraction of bovine tongue desmosomes. However, the anti-bodies to the 140-kDa GP did not recognize any polypeptidesin an enriched fraction of bovine muzzle desmosomes (Fig.3). Furthermore, these cross-reacted with a polypeptide of-130 kDa in whole-cell protein prepared from culturedmouse keratinocytes (Fig. 3).

Cryostat sections of bovine tongue epidermis were pro-cessed for indirect immunofluorescence by using either thepurified antibodies to the 140-kDa GP or the chicken anti-desmoplakin antiserum (Fig. 4). Both generated similarpunctate intercellular staining patterns typical for cells con-taining numerous desmosomes (ref. 2; Fig. 4). No staining ofbovine muzzle epidermis or nonstratified squamous epithelialtissues (e.g., the intestinal epithelium) prepared for indirectimmunofluorescence using the antibody against 140-kDa GP

D2 I I

U.;,

" I$C

1 2 3

FIG. 3. Enriched fractionsof bovine tongue desmosomes(lanes 1 and 4), muzzle epider-mal desmosomes (lanes 2 and 5),

4 140 and whole-mouse keratinocyte30 protein extract (lanes 3 and 6)

were subjected to NaDodSO4/PAGE and transferred to nitro-cellulose. Lanes: 1-3, amidoblack stains of transferred pro-teins; 4-6, immunoblots usingthe desmosome affinity-purifiedrabbit antibodies to the 140-kDaGP. These antibodies recognizethe 140-kDa polypeptide in thetongue desmosome fraction(lane 4). They do not appear torecognize polypeptides in themuzzle desmosome fraction(lane 5). They recognize a poly-peptide of -130 kDa in thewhole-mouse keratinocyte cellextract (lane 6). D1 and D2,

4 5 6 desmoplakins 1 and 2.

Cell Biology: Jones et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

FIG. 4. Cryostat sections of bovine tongue epidermis wereprocessed for indirect immunofluorescence using antibodies againstthe 140-kDa GP (a) or chicken desmoplakin antiserum (b). Theantibodies in a generate a punctate intercellular stain in bovinetongue epidermis. This pattern is similar to that observed using thedesmoplakin antibodies (b). Note, however, that in some instancesa double line is observed along an area of cell contact whendesmoplakin antibodies are used. (Inset) Enlargement of area ar-rowed in b. (a and b, x390; Inset, x800.)

was detected (results not shown). However, in cryostatsections of bovine muzzle and intestinal epithelium, a typicaldesmosome pattern could be seen with the chicken anddesmoplakin serum samples (results not shown).

Cultured keratinocytes possessing desmosomes showedintense, intermittent surface staining only in areas of cell-cellcontact with the anti-140-kDa GP antibody preparation (Fig.5). However, no staining of keratinocytes maintained in lowlevels of Ca2l (i.e., before induction of desmosome forma-tion) could be observed. Keratinocytes also were processedfor double indirect immunofluorescence using the anti-140-kDa GP antibody preparation and the chicken anti-desmo-plakin serum (Fig. 6). The desmoplakin antibodies can beseen to stain a double line at sites of keratinocyte cell-cellcontact (9) (Fig. 6a). Along areas of cell contact, the antibodyto the 140-kDa GP stained a single line (Fig. 6b), whichappeared to lie in the nonfluorescent area between the doublelines stained by the desmoplakin antiserum (Fig. 6). Theantibodies to the GP also generated some less intensefluorescence along the cell surface in between desmosomes(Fig. 6).Thin sections of Lowicryl K4M-embedded bovine tongue

epidermis were processed for indirect "immunogold" local-ization by using the antibodies to the 140-kDa GP and thedesmoplakin antiserum (18). In the latter case, gold particles

FIG. 5. Mouse keratinocytes that possess desmosomes were

processed for indirect immunofluorescence using antibodies againstthe 140-kDa GP. The antibodies stain intermittently along areas of

cell-cell contact (arrow in a). Indeed, the antibodies against the

140-kDa GP appear to recognize desmosomes viewed as phasedensities in the corresponding phase-contrast micrograph (arrow in

b). (x2050.)

74- t*Am's . .'''> .'S, ';.t',.';:FIG. 6. Mouse keratinocytes prepared for double-label indirect

immunofluorescence using the desmoplakin antiserum (a) and theantibodies against the 140-kDa GP (b). Note that the desmoplakmnantibodies stain a double line along an area of cell-cell contact (arrowin a). The antibodies against the 140-kDa GP stain a single line alongthis same area (arrow in b). The single line appears to localize to thenonfluorescent band between the double line stained by thedesmoplakin antibodies (arrows in a and b). Note that both thedesmoplakin antiserum and the antibodies against the 140kDa GPstain areas that correspond to desmosomes, viewed as phase den-sities in c. The antibodies against the 140-kDa GP generate someless-intense fluorescence between desmosomes (small arrows in b).(x 1750.)

appeared primarily over the area of the desmosomal plaqueto which intermediate filaments appeared to be attached (Fig.7a). Gold particles were located over the intercellular regionof the desmosome when the antibody to the 140-kDa GP wasused (Fig. 7b) [1.34 + 0.18 particles per 100 nm ofdesmosome(n = 8)]. Gold particles could be seen also along thenondesmosomal regions of the epidermal cell surface [0.56 +0.13 particles per 100 nm of nondesmosomal cell surface (n =9)] (Fig. 7b). Thus, where two cell surfaces adhered to forma desmosome, there were =1.3 particles per 100 nm, whilealong a nondesmosomal surface (i.e., half of the surfacepresent in a desmosome), there were approximately half asmany gold particles (i.e., 0.56 particle per 100 nm).We also determined whether the rabbit antibodies directed

against the 140-kDa component were able to induce aseparation of epidermal cells maintained in culture. Affinity-purified antibody to the 140-kDa GP was diluted into culturemedium containing normal levels of Ca2+ at =0.5 mg/mi.Mouse epidermal cells containing desmosomes in areas ofcell-cell contact were placed in the medium containing theantibody. Within 1-2 hr of the addition ot the antibody, thekeratinocytes lost their adhesion and detached from eachother as determined by phase-ccontrast optics (Fig. 8). Insome instances the cells remained connected by fine cyto-plasmic strands, whereas in others, no cell connectionsappear to be retained and cells rounded up. In controlexperiments, in which mouse epidermal cells were incubatedwith normal or anti-desmoplakin immunoglobulin fractions,no effects on cell-cell contacts were seen. Furthermore,when mouse epidermal cells were incubated with antibodiesto the 140-kDa GP in medium containing low levels of Ca2+[i.e., under conditions when they do not possess desmosomes(7, 11)], the antibodies appeared to have no effect on cellmorphology (Fig. 9).

DISCUSSION

We have identified a 140-kDa GP component of bovinetongue desmosomes that is recognized by autoantibodies inthe serum of pemphigus vulgaris patients (9). This protein isdistinct from certain other desmosomal GPs ofapproximately160-kDa designated as band 3 (3) and desmoglein I1(5), whichhave been described by other authors. These latter proteinsare recognized by autoantibodies in some, but not all, serumsamples from patients with pemphigus foliaceus (8, 9). In

7284 Cell Biology: Jones et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

.' ..X,.'- %'''.,,,lksi *,w,. .;

, , S ;', .ev *

Bl,,: 6. +K

1

;,lw.. b

f .'

bFIG. 7. Thin sections of Lowicryl K4M embedded bovine tongue epidermis were prepared for indirect immunogold localization by using

the desmoplakin antiserum (a) and the antibodies against the 140-kDa GP (b). Note that in a, the desmoplakin antibodies recognize the areaof the desmosomal plaque to which IF bundles appear to attach (arrow). In b the antibodies against the 140-kDa GP recognize an intercellulardesmosome component located between the two adjacent membranes of the desmosome (long arrow in b). Note also that gold particles arelocated over nondesmosomal cell surfaces (short arrows in b). (x40,000.)

order to determine whether the 140-kDa GP is a desmosomecomponent, we have affinity-purified antibodies to the 140-kDa GP from a rabbit autoimmune serum, and these anti-bodies recognize a 130-kDa polypeptide in whole-cell proteinextracts of keratinocytes. This mouse keratinocyte polypep-tide may be the same 130-kDa cell surface GP that can beimmunoprecipitated from mouse and human keratinocytesby pemphigus autoantibodies (18). However, this 130-kDaGP has not until this point been shown to be associated withdesmosomes. By immunofluorescence microscopy, the an-tibodies to the 140-kDa GP recognize the desmosomes ofbovine tongue epidermis and cultured mouse epidermal cellsin a fashion similar to that observed using serum samplesfrom pemphigus vulgaris patients (9) and antisera directedagainst known desmosome components (7).

Double-label immunofluorescence observations of cul-tured mouse epidermal cells reveal that the antibodies to the140-kDa GP stain a single line that is coincident with thenonfluorescent region between the double fluorescent linesstained by the desmoplakin antibodies. This suggested thatthe 140-kDa GP may lie within the intercellular cementingregion of the desmosome, and we confirmed this by immu-noelectron microscopy. These ultrastructural studies alsosuggest that this GP is not only located within the intercellularregion of the desmosome but also is found elsewhere on thesurface of epidermal cells. Indeed, morphometric analyses ofour gold localization of the antibodies to the 140-kDa GPsuggest that the 140-kDa antigen is dispersed equally over thewhole cell surface (see Fig. 7b). Therefore, it appears to beboth a desmosome and a cell surface component. However,immunofluorescence observations with the antibodies to the140-kDa GP appear to contradict this latter finding; althoughthe antibodies generate a punctate fluorescent staining pat-tern similar to desmoplakin, a desmosomal plaque protein, inbovine tongue epidermis and cultured cells (2), in someinstances, some interdesmosomal staining could be detected(see Fig. 6). One explanation for this apparent contradictionmay lie in the stability of desmosomes to extraction proce-

dures used for immunofluorescence (19). The desmosomeand the 140-kDa GP appear to be resistant to acetoneextraction and, therefore, are visualized by immunofluores-cence, whereas the 140-kDa GP present on other regions of

-..4F X

C',

FIG. 8. Living keratinocytes containing desmosomes at areas ofcell-cell contact were incubated in medium containing normal levelsof Ca2l and, in addition, 0.5 mg of antibody against the 140-kDa GPper ml. (a) Five minutes after addition of the antibody, cells havemaintained their adhesions to their neighbors. Within 45 min of theaddition of the antibody (b), the cells begin to lose their adhesion. At90 min, an obvious loss of cell-cell contact has occurred (c). (x500.)

Cell Biology: Jones et al. 7285

Proc. Natl. Acad. Sci. USA 83 (1986)

(f~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A~~~~~~~~~

Ke<4,V.a

FIG. 9. Living keratinocytes that do not possess desmosomeswere incubated in medium containing low levels of Ca2l and, inaddition, 0.5 mg of antibody against the 140-kDa GP per ml. (a) Fiveminutes after addition of the antibody. (b) Two hundred and fortyminutes after the addition of the antibody. Note that the antibody hasno effect on the morphology of the cells. (x550.)

the cell surface may be extracted or antigenically alteredunder the same conditions. It should be of interest todetermine whether other desmosome GPs like the 140-kDaGP are found at cell surfaces in locations other than atdesmosomes.Our results reveal that affinity-purified antibodies to the

140-kDa GP are able to induce a disruption of cell contacts incultured keratinocytes. Indeed, the 140-kDa polypeptidepossesses all of the characteristics of a cell-cell adhesionmolecule (CAM) (20), i.e., the 140-kDa polypeptide is a GPthat is found at the cell surface, and antibodies directedagainst it perturb cellular adhesion. Other authors havedescribed epithelial cell-specific CAMs whose antibodiesdisrupt cell-cell adhesion of tissue-cultured epithelial cells-i.e., uvomorulin (21, 22), cadherin (23), and CAM 120/80(24). All of these molecules have approximately the samemolecular mass (i.e., around 120-140 kDa). The relationshipbetween the 140-kDa GP that we have analyzed and thesethree other molecules remains to be determined. One impor-tant difference between the 140-kDa desmosome-associatedCAM and these other CAMs lies in its tissue specificity. Theantibodies to the 140-kDa GP do not recognize componentsof nonstratified squamous epithelial cells. Moreover, the140-kDa GP appears to be specifically localized only incertain stratified squamous epithelial tissues (i.e., both im-munofluorescence and immunoblotting analyses suggest thatit is either missing or in reduced quantity in bovine muzzleepidermis). Indeed, from our recent work with pemphigusautoantibodies as desmosome markers, we have suggestedthat desmosome components vary both between tissues andmay even vary within one tissue type (9). Therefore, the140-kDa polypeptide may be a useful marker for desmosomediversity.

Our original interest in 140-kDa polypeptide began becauseof our finding that certain pemphigus autoantibodies recog-nize this molecule (9). Serum samples from patients withpemphigus also possess autoantibodies that appear to berelated to an induction of a disruption of keratinocyte cellcontact (7, 25). Whether the human autoantibodies to the140-kDa GP are those that are the causative factors in thedysadhesion of keratinocytes, leading to acantholysis andblister formation-a process that characterizes pemphigus,remains to be determined.We thank Drs. James Arnn and Andrew Straehelin for the gift of

the rabbit anti-desmoplakin serum, Dr. Kathleen Green for helpfuldiscussion, and Ms. Laura Davis for her considerable patience intyping the manuscript. This work was supported by National CancerInstitute grants to R.D.G.

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