Characterization of a Novel Human IgG Antibody Reactive with a Ca²⁺-Sensitive Cell-Cell Adhesion Epitope of PtK2 Epithelial Cells

Abstract

We have characterized a human IgG antibody present in the serum of a patient with an autoimmune undifferentiated connective tissue disease and 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. IgG affinity-purified from these bands failed to reproduce the original immunofluorescence staining pattern. Treatment with cycloheximide did not abolishe the staining pattern suggesting that the recognized antigen is not a newly expressed protein. However, when EGTA was used for chelating calcium ions in the culture medium, the original staining pattern observed at cell-cell adhesions was affected although some fluorescence was still present at cell periphery. This was reversible when cells were reincubated with fresh medium containing Ca²⁺. The recognized antigen colocalizes at cell-cell adhesions with actin, the microfilament-associated proteins vinculin, α-actinin and myosin light chain, and with Triton-insoluble uvomorulin (E-cadherin) material. We conclude that the antibody reacts with, at least, an extracellular portion of a Ca²⁺-dependent PtK2 antigen. The characterization of this antibody based on (1) its localization at cell-cell adhesions, (2) its sensitivity to EGTA-treatment and (3) its colocalization with the epithelial cellular adhesion molecule (CAM) uvomorulin, strongly suggest that the recognized Ag is a CAM or a CAM-associated protein.

Key Words:

• Autoimmunity
• antibody
• cell-cell adhesions
• uvomorulin (E-cadherin)
• PtK2 cells