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Desmosome-binding antibody KM48 recognises an extracellular antigen different from desmosomal cadherins Dsg 1-3 and Dsc 1-3

European Journal of Dermatology. Volume 15, Number 2, 80-4, March-April 2005, Investigative report

Summary

Author(s) : Stéphane Duhieu *, Céline Laperdrix *, Takashi Hashimoto, Marie-Aude Le Bitoux, Marek Haftek , Université Claude Bernard, Lyon 1, EA3732/CNRS, Department of Dermatology, Hôpital Edouard Herriot, Lyon, FranceFax: (+33) 4 72 11 02 90., Department of Dermatology, Kurume Univ. School of Medicine, Fukuoka, Japan.

Summary : Desmosomes are the most prominent and mechanically important epidermal intercellular junctions. Transmembrane proteins of desmosomes, desmogleins and desmocollins, are responsible for extracellular binding and, thus, are important for interkeratinocyte cohesion. We show here, using three different approaches, that the extracellular “cores” of epidermal desmosomes contain a highly glycosylated antigen, different from desmosomal cadherins. This protein, recognised by KM48 monoclonal antibody, is likely to be involved in the processes of keratinocyte differentiation, desmosome turnover and epidermal cohesion.

Keywords : adhesion molecules, desmosome, differentiation, keratinocyte

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ARTICLE

Auteur(s) :, Stéphane Duhieu*¹, Céline Laperdrix*¹, Takashi Hashimoto², Marie-Aude Le Bitoux¹, Marek Haftek^1,*

¹Université Claude Bernard, Lyon 1, EA3732/CNRS, Department of Dermatology, Hôpital Edouard Herriot, Lyon, FranceFax: (+33) 4 72 11 02 90.
²Department of Dermatology, Kurume Univ. School of Medicine, Fukuoka, Japan

accepté le 27 Novembre 2004

Desmosomes are intercellular junctions, typical of epithelial cells, which are associated with the keratin cytoskeleton. Particular forms of this type of cell-cell junction are expressed between the cardiac muscle cells, where they are associated with desmin, and in the lymph node follicle reticulum [1]. In the epidermis, desmosomes join together keratin cytoskeletons of individual keratinocytes into a supracellular structure resistant to mechanical stress [2]. Keratin filaments bind to the cytoplasmic plaques of desmosomes. These button-like structures are expressed symmetrically by two interacting cells at the sites of desmosome formation. Proteins composing the plaques attach to and stabilise the transmembrane molecules of desmosomes called desmosomal cadherins. Cadherins stick to one another in the intercellular spaces, thus binding together both desmosomal halves with the attached cytoskeleton filaments. Two families of desmosomal cadherins have been described: desmogleins (Dsg) and desmocollins (Dsc). Each family is basically composed of three members, although DSG4 gene has also been described recently [3] and linked to hair development [4]. Various desmosomal cadherins are differentially expressed by keratinocytes, according to the state of the cell differentiation. Thus, Dsg 3 and Dsc 3 are most abundant in the lower parts of the epidermis, whereas Dsg 1 and Dsc 1 are mainly encountered in the upper malpighian layers [5, 6]. This distribution explains the differences in the level of acantholytic split observed in various bullous diseases. In pemphigus vulgaris (Pv), where the auto-antibodies are directed against Dsg3, the bullae appear deeper in the epithelia and are more tense, whereas pemphigus foliaceus (Pf), characterised by more superficial blisters, is provoked by auto-antibodies to Dsg1. Also, Dsc 1 has been defined as an auto-antigen in the subcorneal pustular dermatosis type of IgA pemphigus [7] and Dsg 1 was found to be a target of bacterial proteases in Staphylococcal Scalded Skin Syndrome [8].Desmosomal cadherins interact in the intercellular space by displaying homophilic and heterophilic binding. Indeed, the strongest interactions have been observed between Dsg and Dsc molecules, whereas the homophilic Dsg – Dsg or Dsc – Dsc interactions remain rather weak [2]. Desmosomes are constantly recycled by keratinocytes evolving from the basal layer to the stratum corneum. This implies a controlled degradation of individual junctions and a constant re-expression of the desmosome molecules at the keratinocyte surface [9]. Little is known about this process, however, proteases may be involved and their action could be regulated by the level of glycosylation of the desmosomal extracellular domains [10].In the search for potential new components of desmosome junctions, we have developed a murine monoclonal antibody (MAb) which recognizes the extracellular parts of desmosomes in a differentiation-related manner [11, 12]. On epidermal cell suspensions, our KM48 MAb decorated exclusively split desmosomes and did not react with melanocytes or Langerhans cells. The quantity of the desmosome-bound KM48 antigen increased progressively from the basal to the granular layers, as defined with immunogold electron microscopy and flow cytometry. Because desmosomal cadherins are the only molecules described so far in the extracellular space of desmosomes, the aim of the present study was to verify whether our KM48 MAb recognizes one of the Dsg or Dsc present in the human epidermis.

Materials and methods

Antibodies

Murine monoclonal KM48 antibody of IgM class was obtained after immunization of mice with a suspension of normal human epidermal keratinocytes [12]. Maja 7, a monoclonal IgM antibody of undefined specificity, not reacting with human epidermis on Western blot or on immunofluorescence, was used as a control in all experiments. Pemphigus vulgaris and pemphigus foliaceus sera (antibody titer 160) were collected from patients in the acute phase of disease and controlled for reactivity with Dsg 3 and Dsg 1, respectively, using indirect immunofluorescence and Western blot. For immunofluorescence studies, KM48 MAb was used undiluted and the sera were employed at 1:50 dilution, resulting in a specific intercellular staining of intensity comparable to the monoclonal.

Immunofluorescence assays

Human skin specimens were obtained from plastic surgery. For immunohistochemical studies, small tissue fragments were snap-frozen in liquid nitrogen, embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, Holland), and stored at –20 °C. Five mμ-thick cryosections collected on glass slides were used for sequential immunolabelling. Briefly, the sections were re-hydrated in PBS with 10% bovine serum albumin, incubated with KM48 MAb (undiluted culture supernatant), washed in PBS (3 × 5 min.), incubated with human Pv or Pf sera diluted 1:50, washed again, and revealed with a mixture of fluorescent conjugates: rhodamine (TRITC)-conjugated F(ab’)2 fragment goat anti-mouse IgM and FITC-conjugated F(ab’)2 fragment goat anti-human IgG (diluted 1:100; Jackson ImmunoResearch Labs., West Grove, PA). Alternatively, the sequence of incubations with the primary antibodies was inversed or they were mixed and incubated simultaneously. Labelling with only one primary antibody at a time served as positive controls, whereas incubations with the fluorescent conjugates alone were used to determine the background. The results were observed and photographed in an epifluorescent microscope (Nikon, Japan) using appropriate filters.

Immunoblot

Protein extracts were obtained from normal human epidermis, dissociated from the dermis by heat (1 min. at 60 °C). The epidermal sheets were minced and sonicated in the presence of antiproteases (Complete mini inhibitor of proteases, Boehringer-Manneheim, Manneheim, Germany) in 10 mM Tris HCl buffer, pH 7.4, supplemented with 150 mM NaCl, 0.5% nonidet P40 and 1% Triton X100 (Sigma, L’Isle-d’Abeau, France). The protein samples were separated by electrophoresis on 8% polyacrylamide gels (SDS-PAGE) and electro-transferred onto nitrocellulose membranes (Protran BA85; Schleicher & Schuell, Dassel, Germany). Antibodies used for the Western blot detection: DG3.10 MAb to Dsg1 and 2, anti-Dsc 1 and anti-Dsc 3 (1:500; Progen, Heidelberg, Germany).

Enzyme-linked immunosorbent assay

ELISA test using recombinant human Dsg 1 and Dsg 3 was prepared and applied as described previously [13]. KM48 and control Maja7 MAb were used at 1:100 to 1:1000 dilutions, whereas the secondary goat anti-mouse IgM immunoperoxidase conjugate (Zymed, San Francisco, CA) was applied at 1:5000 to 1:25000.

Surface immunofluorescence of transfected COS-7 cells

COS-7 cells transfected with cDNA of human desmocollins 1, 2 and 3, expressing on their surface extracellular portions of the recombinant human desmocollins were used for immunolabelling with KM48 MAb according to the protocol of Hashimoto et al. [7]. The positive controls included anti- Dsc 1, 2 and 3 MAb and a human IgA pemphigus serum. The appropriate secondary antibodies were FITC-conjugated and the results were read in an epifluorescence microscope.

Results

Labelling of epidermal sections with the mixture of KM48 MAb and Pv or Pf sera resulted only in a slight decrease of respective immunofluorescence intensities, when compared to the mono-specific staining. The initial application of KM48 MAb did not prevent the subsequent binding of Pv and Pf sera but only attenuated it. In the same manner, when the pemphigus sera containing antibodies to desmosomal cadherins were applied in the first place, their binding only partially interfered with the following KM48 MAb attachment (figures 1 and 2). Reduction of the KM48-related immunofluorescence was most pronounced in the intermediate layers of the stratum spinosum pre-treated with the Pv serum ( (figure 2) ).

To test the hypothesis that KM48 MAb recognizes one of the desmosomal cadherins, we used the antibody in ELISA with recombinant human Dsg 1 and 3 and in indirect immunofluorescence studies on COS-7 cells expressing on their surface recombinant human Dsc 1, 2 and 3. Both assays revealed no specific binding of KM48 MAb when compared to the control, non-relevant IgM antibody.

These results were also confirmed by immunoblot analysis ( (figure 3) ). KM48 MAb detected a whole spectrum of epidermal proteins presenting as a smear ranging from 250 to 110 kDa, whereas antibodies to desmosomal cadherins revealed only individual bands of apparent molecular weight characteristic of each molecule. Additionally, a huge polydispersity of molecules reactive with KM48 MAb, visible on the Western blot, indicates a highly glycosylated nature of the recognized antigen.

Discussion

Double labelling studies with KM48 MAb and pemphigus sera have demonstrated that the epidermal epitopes recognized by these immune probes were not identical. The attenuation of the signal belonging to the antibody applied in the second position was mostly visible in the tissue regions expressing lower levels of the corresponding antigen. Such changes induced by the preincubation of skin sections with the Pv serum before KM48 MAb, or by the pre-treatment with the monoclonal before the Pf serum, occurring in the lower epidermis which expresses high levels of Dsg3 but less KM48 or Dsg1, indicated that the KM48 epitope was located close enough to the desmosomal cadherins to provoke interference. However, it was impossible to establish with this technique whether these epitopes belonged to the same protein or to different, closely packed molecules.

The fact that on Western blot KM48 MAb reacted with a wide range of differentially glycosylated proteins, instead of detecting the individual protein bands typical for the commercially available antibodies to desmosomal cadherins, indicated that the recognized antigens were not identical.

The use of recombinant desmosomal cadherins definitively solved this question, clearly demonstrating that KM48 MAb was unable to detect the extracellular moieties of human desmogleins 1 and 3 and desmocollins 1-3. We can, therefore, conclude that the extracellular parts of desmosomes do contain an antigen different from these desmosomal cadherins.

Recent reports indicate that at least one additional desmoglein, Dsg 4, is expressed in human skin and provide evidence that this molecule is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation [3, 4]. Although, based on our results, we can not formally exclude the possibility that KM48 MAb recognises Dsg 2, Dsg 4, or a new member of the family of desmosomal cadherins, we consider this unlikely. Indeed, the level of glycosylation of the antigen recognised by KM48 MAb, observed on immunoblot, is clearly unusual for a cadherin [14] and the well individualised bands detected with the commercially available antibodies support our reasoning.

The presence of a new, highly glycosylated protein in the desmosome “cores” may be functionally important, since sugars have been demonstrated to play a protective role against the proteolytic degradation of the junctions [10].

KM48 antigen is expressed in desmosomes of all living epidermal layers, with a gradient of expression proportional to the degree of keratinocyte differentiation [12]. Desmosomes from the upper epidermal layers, known to be more resistant to the mechanical stress, are more numerous and express higher amounts of the KM48 protein. Moreover, the antigen disappears from the highly de-differentiated epithelial tumours like squamous cell carcinomas, which is paralleled by the disappearance of desmosomes [15]. During the process of acantholysis, whether autoimmune, e.g., Pv, or related to hereditary problems of calcium redistribution, e.g., Hailey-Hailey disease, the KM48 antigen remains at the surface of freshly dissociated keratinocytes and disappears only with the complete dissipation of desmosomes [16]. These observations suggest that functional studies of the new intercellular component of desmosomes will be important for a better understanding of normal and pathological keratinocyte differentiation. Further biochemical characterisation of the protein recognised by KM48 MAb is under way.

Acknowledgements

The authors are indebted to Dr Masayuki Amagai from the Department of Dermatology, Keio University School of Medicine, Tokyo, Japan, for performing the ELISA tests.

References

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