Ultrastructural quantitation of desmosome and differentiation-related keratinocyte membrane antigen

Abstract

The keratinocyte membrane antigen KM 48 was defined by a new monoclonal antibody obtained after mouse immunization with normal human epidermal cell suspension. Specific reactivity of the antibody with desmosomal regions of keratinocyte cell membrane was demonstrated by immunoelectronmicroscopy. Langerhans cells, melanocytes, and indeterminate cells did not express the KM 48 antigen. Immunogold labelling permitted ultrastructural quantitation of KM 48 antibody binding on keratinocytes from various epidermal layers. A gradual increase in desmosome-related KM 48 antigen expression accompanied differentiation of keratinocytes during their transit from basal to granular layer. Distribution of the antigen on individual cells was uneven. The upper pole of a keratinocyte facing overlying more differentiated cells was always found to be laden with an immunogold marker about twice that of the opposite, lower surface of the cell. The results support the previous reports on gradual development of desmosomes during epidermal cell maturation and open up new possibilities for keratinocyte differentiation studies. They also underline the virtues of the immunogold-labelling method used for cell-surface antigen tracing and quantitation.

Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies

“…This presumption was further reinforced by the detection of desmosealin within the desmoglea. The terminal differentiation‐dependent expression of this latter protein has been well documented in our previous studies (12,16).…”

“…KM48 monoclonal IgM to desmosealin, a new epidermal proteoglycan incorporated into the desmoglea, was obtained after immunization of mice with a suspension of normal human keratinocytes and harvested as a hybridoma culture supernatant produced in our laboratory (12). Primary antibodies to the extracellular part of desmoglein‐1 (Dsg1‐P23), to desmogleins‐1 and ‐2 (DG‐3.10), to desmocollin‐1 (Dsc1‐U100), to desmocollin‐3 (Dsc3‐U114), desmoplakins‐I and ‐II (DP‐2.15) and to plakoglobin (PG‐5.1) were purchased from Progen (Heidelberg, Germany).…”

In vitro reconstructed normal human epidermis expresses differentiation-related proteoglycans

“…regulate cell proliferation, migration or differentiation. We have previously described a new epidermal PG, desmosealin, present between keratinocytes and integrated into the extracellular parts of desmosomes [1,2]. Differentiation-related expression of desmosealin in normal human epidermis could be demonstrated with the post-embedding immunogold labelling [1].…”

“…We have previously described a new epidermal PG, desmosealin, present between keratinocytes and integrated into the extracellular parts of desmosomes [1,2]. Differentiation-related expression of desmosealin in normal human epidermis could be demonstrated with the post-embedding immunogold labelling [1]. Because the ''outside-in'' regulation of the keratinocyte behaviour may be mediated by extracellular matrix molecules [3,4], we have studied the expression of desmosealin and of some of the other epidermal PGs in two commercially available air-exposed keratinocyte culture systems, Episkin 1 and SkinEthic 1 (SkinEthic Laboratories, Nice, France).…”

“…The following antibodies were employed: mouse monoclonals anti-CD44v3/epican (clone VFF-327V3; at 1:30; Abcys SA, Paris, France), anti-CD44 H-CAM (clone DF1485, recognizing all forms of CD44; 1:30; Santa Cruz Biotechnology, USA), anti-syndecan-4 (clone 5G9; 1:100; Santa Cruz Biotechnology), anti-desmosealin (KM48 hybridoma supernatant; Lyon [1]), 7C1 antibody to another chondroitin/ dermatan sulphate PG (a generous gift of Prof. H. Suzuki [2,5]), and rabbit polyclonal antibodies H174 anti-syndecan-1, M140 anti-syndecan-2 (both at 1:100; Santa Cruz Biotechnology). Localization of the respective antigens was revealed with the LSAB2 kit (Dako) and an AEC peroxidase substrate (Invitrogen).…”