Evidence that Nectin-3 is the soybean agglutinin binding protein on rat corneal endothelium cell surfaces

Abstract

The means by which the lectin soybean agglutinin (SBA) binds to the corneal endothelium cell surface following explantation into organ culture was investigated using Sprague-Dawley rats. SBA binding does not occur in freshly isolated and fixed rat corneal endothelium. However, after 48 h in organ culture, SBA binding occurs in a punctate pattern that clearly outlines all endothelial cells of the tissue monolayer. To determine what cell surface component was responsible for this binding, a series of experiments were employed that focused on the possibility that SBA bound to a nectin molecule(s). To this extent we performed a series of immunocytochemical localizations using antibodies against either nectin-2, nectin-3 or nectin-4. Of these, only nectin-3 bound to the endothelium in a manner that mimicked SBA binding. To further verify that nectin-3 bound SBA, displacement experiments employing non-labeled SBA were undertaken. Following a 48 h organ culture, tissues were fixed and incubated with SBA followed by exposure to nectin-3 antibody. No subsequent immunofluorescence could be detected, indicating that anti-nectin-3 binding was prevented. Likewise, when organ-cultured tissues were fixed and incubated in anti-nectin-3 antibody, followed by SBA exposure, no SBA binding could be detected. These results suggest that stresses accompanying explantation of the tissue into organ culture promote the appearance of nectin-3 around the cell periphery. The emergence of nectin-3 along the peripheral endothelial cell membrane in organ culture may imply a necessary role for this molecule in maintaining monolayer integrity and barrier function during either a pathologic condition, wound repair, or in organ storage.