Abstract
Purpose: :
Galectin-3 is a β-galactoside-binding lectin known to cluster cell surface glycoprotein receptors to modulate diverse cellular functions. At the ocular surface, galectin-3 maintains the integrity of the mucosal barrier by interacting with heavily O-glycosylated transmembrane mucins. The goal of this work was to determine whether galectin-3 binds additional glycoprotein partners on the apical cell surface glycocalyx of corneal epithelial cells.
Methods: :
In this study, binding partners of galectin-3 in the apical surface of stratified human cornea-limbal epithelial (HCLE) cells were investigated using affinity chromatography. Cell surface proteins on HCLE cells were labeled with biotin, purified with a Neutravidin® resin, and were chromatographed on a galectin-3-affinity column. and the column was sequentially eluted with 0.1M sucrose (non-competing sugar) and 0.1M lactose(competing sugar). Eluted proteins were separated on a 12% SDS-PAGE gel followed by tryptic digestion and mass spectrometry analyses. Criteria for positive identification included detection in duplicate experiments and known ocular surface localization.
Results: :
Two major low molecular weight bands at 37 and 50 kDa were visualized by SDS-PAGE after galectin-3 affinity chromatography. Mass spectrometry analyses of the bands revealed the presence of human basigin-2, ephrin-b1, galectin-3 binding protein, junctional adhesion molecule A and RON receptor tyrosine kinase. Interaction between galectin-3 and basigin-2, a transmembrane glycoprotein involved in cell-cell interactions and wound-healing, or ephrin b1, a cell surface tyrosine kinase involved in angiogenesis and axon guidance, have not been previously described in mammalian cells.
Conclusions: :
Galectin-3 has been shown to contribute to apical barrier function in corneal epithelial cells by interacting with MUC16 and MUC1. In this study, we were able to identify additional galectin-3 binding partners on the apical glycocalyx, suggesting that galectin-3 might regulate other important functions at the ocular surface.
Keywords: cornea: epithelium • glycoconjugates/glycoproteins • cornea: surface mucins