Biochemical analysis of proteolytic fragments from desmosomal glycoproteins

Desmosomes are multi-component intercellular junctions found in most epithelial tissues. Desmosomal adhesion is mediated by two related molecules, desmosomal glycoproteins (dg) 2 & 3 (relative molecular mass (Mr) 116,000 and 107,000) whose structure is unknown. In this study part of their structure has been investigated by biochemical characterization of soluble fragments of dg 2 & 3 generated by digestion of whole isolated desmosomes with the proteolytic enzyme trypsin. As a result of these studies a model is proposed for the organization of their perimembrane regions. Four soluble fragments of dg 2 & 3 were identified by immunoblotting. These were fractionated by concanavalin (Con.) A affinity chromatography into two binding fragments (Mr32,000 and 17,000) and two non-binding fragments (Mr28,000 and 20,000). Anti-fragment antibodies were produced by affinity purification against each fragment of an anti-dg 2 & 3 antiserum. The reactivity pattern of the affinity purified antibodies showed that the two Con. A binding fragments are related, and that the two non-binding fragments are related to each other, to the 32,000, but not the 17,000. This suggested that these fragments are derived from the same region of dg 2 & 3. All four antibodies stained the outer surface of Madin Darby canine kidney (MDCK) cells indicating that these fragments originated, at least in part, from the extracellular domains of dg 2 & 3. Furthermore, the 28,000 fragment reacted with a monoclonal antibody to the cytoplasmic domain of dg 2 & 3. This indicates that the 28,000 fragment traverses the membane and that the four fragments encompass the perimembrane regions of dg 2 & 3. Endoglycosidase digestion reduced the Mr of the 32,000 and 17,000 fragments each by 1,500 and the intact molecules each by 5,000. This suggests that these fragments contain only part of the carbohydrate associated with whole dg 2 & 3. The 28,000 fragment contains a region stabilized by disulphide bonds. Also, the 28,000 and 20,000 fragments form a complex in solution of 66,000 which is stabilized by non-covalent interactions. Trypsinization of MDCK cells in the presence or absence of Ca2+, followed by staining with anti-fragment antibodies, indicated that Ca2+ protected the extracellular portions of the perimembrane regions of dg 2 & 3 from digestion. However, the soluble fragments of 2 & 3 showed no resistance to proteolysis. This indicates that the conformation of dg 2 & 3 is Ca2+ dependent such that tryptic sites in the perimembrane region are not exposed in the intact molecules on the cell surface in the presence of Ca2+ Tyrpsinization of whole isolated bovine epidermal desmosomes also yielded a 42,000 glycosylated fragment. Antibodies to this fragment precipitated a novel 140,000 glycoprotein from MDCK cells. It is not yet clear whether this is a true desmosomal component.