Monoclonal antibodies and N-terminal peptide antisera in a study of the structure of desmosomal glycoproteins 2 and 3

Holton, Janice Lesley (1991) Monoclonal antibodies and N-terminal peptide antisera in a study of the structure of desmosomal glycoproteins 2 and 3. University of Southampton, Doctoral Thesis.

Abstract

The adhesion of the intercellular junctions known as desmosomes is thought to be mediated by desmosomal glycoproteins 2 and 3 (dg2 & dg3). It was proposed to use monoclonal antibodies against dg2 & dg3 to inhibit desmosomal adhesion by cultured cells. Although no inhibitory antibodies were found, two antibodies with interesting characteristics were obtained. These antibodies only stained bovine tissues, each recognising different tissues. This demonstrates the presence of bovine tissue specific dg2 & dg3 variants. One antibody (07-4G) stained three stratified epithelia including all the layers of the epidermis. The second (07-4D) stained meninges and the supra-basal epidermal strata showing that there are differentiation related dg2 & dg3 variants. The molecular nature of these variants was investigated. 07-4D was found to recognise an epitope in the desmosomal intercellular space and a 19kd N-terminal proteolytic fragment of both dg2 and dg3 which is not glycosylated. This shows that variants of dg2 & dg3 can be related to differences in the protein sequence or conformation rather than glycosylation. The putative amino acid sequence of the N-terminus of a mixture of dg2 & dg3 was made available. This suggested that these glycoproteins may be related to the calcium-dependent cell adhesion molecules (cadherins). Peptides corresponding to this sequence were synthesised and antisera raised against them. The antisera demonstrated that the sequence is derived from dg2 & dg3 and that their N-termini are the same or closely related. Immuno-localisation of the N-termini showed that, like the cadherins, they are extracellular. Neither the N-terminal peptides nor the antisera raised against them inhibited desmosome formation suggesting that the adhesion sites probably lie outside the immediate N-terminal domains. The structures of dg2 & dg3 were investigated by one-dimensional peptide mapping and immunoblotting. The proteolytic fragments derived from each molecule appeared to be identical. Antibodies to extracellular epitopes recognised fragments of the same size from each molecule but variations between dg2 & dg3 were found using antibodies against cytoplasmic epitopes. This suggests that the extracellular domains of dg2 & dg3 may be similar and is consistent with the conclusions of other workers that the cytoplasmic domains are different.

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