N Panjwani1, S Ahmad, M B Raizman. 1. New England Eye Center, Tufts University School of Medicine, Boston, Massachusetts 02111.
Abstract
PURPOSE: To identify those plasma membrane glycoproteins of corneal epithelial cells that are synthesized in a higher amount or are downregulated during cell migration. METHODS: Primary cell cultures of rabbit corneal epithelium were used. Sialic acid and terminal galactose/N-acetylgalactosamine residues of plasma membrane glycoproteins of migrating and nonmigrating corneal epithelial cells were labeled using two well-characterized cell surface carbohydrate labeling techniques. The labeled glycoproteins were extracted in phosphate-buffered saline containing nonionic detergents and various protease inhibitors, and then they were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography. RESULTS: At least 12 to 13 radiolabeled components (molecular weight, 240 kd to 21 kd) were detected in the fluorographs of both sialic acid and galactose-labeled cells. Regardless of the labeling technique used, one sialoglycoprotein (GP1, 240 kd) was found in a higher amount in the extracts of nonmigrating cells than in migrating cells, and another glycoprotein (GP12, 28 kd) was present in a higher amount in migrating than in nonmigrating cells. Furthermore, one sialoglycoprotein (GP13, 21 kd) was detected only in migrating cells, and two glycoproteins (GP10, 42 kd; GP11, 32 kd) with terminal galactose/N-acetylgalactosamine residues were present in a higher amount in migrating than in nonmigrating cells. CONCLUSIONS: This study has demonstrated that during corneal epithelial cell migration, the level of one membrane glycoprotein is markedly reduced, and the levels of four membrane glycoproteins are elevated. Further characterization of these glycoproteins should contribute to a better understanding of the mechanisms that modulate corneal epithelial sheet migration and wound healing.
PURPOSE: To identify those plasma membrane glycoproteins of corneal epithelial cells that are synthesized in a higher amount or are downregulated during cell migration. METHODS: Primary cell cultures of rabbit corneal epithelium were used. Sialic acid and terminal galactose/N-acetylgalactosamine residues of plasma membrane glycoproteins of migrating and nonmigrating corneal epithelial cells were labeled using two well-characterized cell surface carbohydrate labeling techniques. The labeled glycoproteins were extracted in phosphate-buffered saline containing nonionic detergents and various protease inhibitors, and then they were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and fluorography. RESULTS: At least 12 to 13 radiolabeled components (molecular weight, 240 kd to 21 kd) were detected in the fluorographs of both sialic acid and galactose-labeled cells. Regardless of the labeling technique used, one sialoglycoprotein (GP1, 240 kd) was found in a higher amount in the extracts of nonmigrating cells than in migrating cells, and another glycoprotein (GP12, 28 kd) was present in a higher amount in migrating than in nonmigrating cells. Furthermore, one sialoglycoprotein (GP13, 21 kd) was detected only in migrating cells, and two glycoproteins (GP10, 42 kd; GP11, 32 kd) with terminal galactose/N-acetylgalactosamine residues were present in a higher amount in migrating than in nonmigrating cells. CONCLUSIONS: This study has demonstrated that during corneal epithelial cell migration, the level of one membrane glycoprotein is markedly reduced, and the levels of four membrane glycoproteins are elevated. Further characterization of these glycoproteins should contribute to a better understanding of the mechanisms that modulate corneal epithelial sheet migration and wound healing.