Wheat germ agglutinin and concanavalin a binding during epithelial wound healing in the cornea

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Abstract
It has been hypothesized that there are differences between membrane-associated glycoconjugates of wounded (migrating) epithelium and those of nonwounded (stationary) epithelium. To test this hypothesis, wheat germ agglutinin-ferritin (WGA-Fe) and concanavalin A-ferritin (Con A-Fe) binding to apical membranes of wounded and nonwounded rabbit corneal epithelia were compared. Epithelial abrasions of the superior half of the cornea were allowed to heal in vivo for six hours. Fixed corneas were then incubated with lectin-ferritin and prepared for electron microscopy. Measurements (ferritin particles per linear um of membrane) of WGA-Fe indicated that binding to leading cells (40.7 particles/um), to areas 20 to 35 cells behind the leading edge (46.5 particles/um) and to nonwounded epithelium (45.1 particles/um) from contralateral eyes were not significantly different. A competitive inhibitor of WGA, 0.2M N-acetylglucosamine, however, blocked 94 percent of WGA binding on leading cells (2.3 particles/um), while binding persisted in areas behind the leading edge (39.5 particles/um) and on nonwounded epithelium (43.6 particles/um). This indicates that leading cell surfaces have a weak affinity for WGA. Unlike WGA, Con A showed a distinct preference for leading-edge cells (33.9 particles/um) compared to nonwounded epithelium (9 particles/um). In areas 20-35 cells behind the leading edge, Con A binding was intermediate to these two extremes. This differential in Con A binding to the cells in wounded epithelium compared to nonwounded epithelium suggests that wounding induces a change in the composition of membrane-associated glycoconjugates. Evidence that Con A was binding primarily to simple oligosaccharides in each test area was obtained when endoglycosidase H, an enzyme which hydrolyzes high mannose glycoconjugates at their di-N-acetylchitobiose core, reduced binding by at least 60% in each test area. Taken together these data support the contention that there are differences in lectin receptor sites on wounded and nonwounded corneal epithelia.