CHARACTERIZATION OF A NOVEL HUMAN-IGG ANTIBODY-REACTIVE WITH A CA2-SENSITIVE CELL-CELL ADHESION EPITOPE OF PTK2 EPITHELIAL-CELLS()

We have characterized a human IgG antibody present in the serum of a p atient with an autoimmune undifferentiated connective tissue disease a nd reactive with PtK2 epithelial cell-cell adhesions. The fluorescent staining pattern is observed only at cell-cell contacts whether cells are permeabilized or not. The serum reacts with polypeptides of 90, 48 and 45 kD by immunoblotting. Ige affinity-purified from these bands F ailed to reproduce the original immunofluorescence staining pattern. T reatment with cycloheximide did not abolishe the staining pattern sugg esting that the recognized antigen is not a newly expressed protein. H owever, when EGTA was used for chelating calcium ions in the culture m edium, the original staining pattern observed at cell-cell adhesions w as affected although some fluorescence was still present at cell perip hery. This was reversible when cells were reincubated with fresh mediu m containing Ca2+. The recognized antigen colocalizes at cell-cell adh esions with actin, the microflament-associated proteins vinculin, a-ac tinin and myosin light chain, and with Triton-insoluble uvomorulin (E- cadherin) material. We conclude that the antibody reacts with, at leas t, an extracellular portion of a Ca2+-dependent PtK2 antigen. The char acterization of this antibody based on (1) its localization at cell-ce ll adhesions, (2) its sensitivity to EGTA-treatment and (3) its coloca lization with the epithelial cellular adhesion molecule (CAM) uvomorul in, strongly suggest that the recognized Ag is a CAM or a CAM-associat ed protein.