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Cell Surface Glycoproteins of Corneal Epithelium

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Cell Surface Glycoproteins of Corneal Epithelium Cell Surface Glycoproteins of Corneal Epithelium Noorjahan Panjwani, Sameer Ahmad, and Michael B. Raizman 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 (CP1, 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 glycopro- teins (GP10, 42 kd; GPU, 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. Invest Ophthalmol Vis Sci. 1995;36:355-363. After accidental injury to the cornea and various sur- Studies aimed at identifying individual membrane gical procedures, initial healing of the wound takes glycoproteins of corneal epithelium have shown that place by the migration of adjacent cells to the injured integrins, which are widely expressed transmembrane area.12 The failure of epithelium to migrate to the glycoproteins and are known for their role as recep- wound surface or the failure of migrated epithelium tors that mediate attachment of cells to extracellular to remain adherent to the substratum may result in matrix and cell-cell adhesion, are localized in the severe clinical problems, leading to the development cornea along the basal cell membrane of corneal epi- of persistent epithelial defects and corneal ulcer- thelium at the site of cell-substrate interactions and ation.3'4 Molecular events that mediate migration of cell-cell contact.14"17 Other known plasma membrane epithelium to the wound and adhesion of epithelium glycoproteins of corneal epithelium include bullous to the underlying substrata have not been fully eluci- pemphigoid antigens (BPAG 1+2), found along the dated, but a number of studies have suggested that basal membrane of corneal epithelium,'819 and uvo- plasma membrane glycoconjugates of corneal epithe- morvulin,20 also known as E-cadherin, which is a mem- lium play a central role in these events.5"13 ber of a family of cell adhesion molecules that regulate cell-cell contact. From the New England Eye Center,and the Department of Ophthalmology, Tufts It is well established that plasma membrane glyco- University School of Medicine, Boston, Massachusetts. conjugates of different cell types are diverse and cell- Supported by National Institutes of Health grants EY07088 and EY09349. Submitted for publication April 15, 1991; revised July 22, 1994; accepted October type specific. Integrins, BPAGs, and cadherins most 3, 1994. probably constitute only a portion of cell surface glyco- Proprietary interest category: N. Reprint requests: Noorjahan Panjwani, Department of Ophthalmology, Tufts proteins of corneal epithelium, and it is most likely University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. that other glycoproteins unrelated to any known com- Investigative Ophthalmology & Visual Scit ce, February 1995, Vol. 36, No. 2 Copyright © Association for Research in ^ sion and Ophthalmology 355 Downloaded from iovs.arvojournals.org on 09/24/2021 356 Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 3 ponents are also present and play an important role groups with NaB H4. Various reaction times and re- in the regulation of cell function in health and disease. agent concentrations were obtained from parameters To understand better the cell surface architecture determined by Panjwani and Baum.23 Cells were la- of the corneal epithelium with respect to glycoprotein beled directly on the monolayer in 100-mm dishes by constituents, two well-characterized cell surface carbo- sequential treatment with 45 /zmols of NaBH4 for 15 hydrate labeling techniques were used in this study to minutes at room temperature, NaIO4 at a final concen- identify the plasma membrane glycoproteins of pri- tration of 1 mM at 4°C for 10 minutes, and 2 or 3 mCi 3 mary cell cultures of rabbit corneal epithelium. To of NaB H4 (5 to 15 Ci/mmol) for 5 minutes at room identify the specific molecules that may be involved temperature. The cell membranes were solubilized by in cell-cell and cell-matrix interactions, electropho- incubating the labeled cells with 0.5% CHAPS or 0.5% retic patterns of plasma membrane glycoproteins of Triton X-100 in phosphate-buffered saline containing confluent-nonmigrating cell cultures were compared various protease inhibitors for 1 hour at 4°C with gen- with those of migrating cell cultures, and it was shown tle shaking. The solubilized membrane extracts were that during cell migration, levels of four 3-[(3-Cho then centrifuged at 12,000gfor 15 minutes in a Sorvall lamidopropyl) dimethylammonio]-l-propanesulfo- centrifuge to remove any insoluble material, the su- nate (CHAPS)-extractable glycoproteins are elevated, pernatants were transferred to Centricon-10 tubes and the level of one glycoprotein is markedly reduced. (Amicon, Beverly, MA) for further centrifugation at 6000g to remove detergent and salts, and the labeled glycoproteins were analyzed by sodium dodecyl sulfate MATERIALS AND METHODS (SDS) polyacrylamide gel electrophoresis. Control cells underwent the same labeling procedure except Preparation of Rabbit Corneal Epithelial Cell that the periodate step was omitted. Because similar Cultures results were obtained regardless of whether the mem- Rabbit corneal epithelial cells were grown in tissue branes were solubilized by CHAPS or Triton X-100, culture using eyes from Pel-Freeze Biologicals (Rog- with the exception of a few initial experiments, the ers, AR),)" as described by Jumblatt and Neufeld.21 membranes of the labeled cells were solubilized with Epithelial fragments from corneas were cultured in buffers containing CHAPS in all studies, mainly be- 100-mm dishes using supplemental hormonal epithe- cause it can be removed more efficiently than Triton lial medium. Within 3 days of starting the culture, X-100 by dialysis or centrifugation in Centricon tubes. approximately 30% to 50% of the dish was populated with cells migrating away from the explants. These Neuraminidase Treatment of Cell Surface cells were designated as migrating (M) corneal epithe- Sialoglycoproteins of Corneal Epithelial Cells lial cells. Within 10 to 12 days, 90% to 95% of the To ensure that the label was introduced specifically to dish was populated with tightly packed polygonal cells, cell surface sialic acid residues, membrane sialoglyco- designated as nonmigrating (N) corneal epithelial 3 proteins were radiolabeled with NaIO4/NaB H4 treat- cells. Migrating cells were also obtained by treating ment as described above and were then treated with monolayer, nonmigrating cultures with Dispase II (2 neuraminidase (1 U, Type VI from Clostridium per- mg/ml) (Boehringer Mannheim, Indianapolis, IN) fringens; Sigma, St. Louis, MO) or with enzyme buffer for 20 minutes at 37°C. Cells from one dish were re- alone for 1 hour at room temperature. After enzyme plated onto three dishes and were allowed to spread treatment, cell membranes were solubilized, as de- for 16 to 18 hours, at which time approximately 50% scribed above, using the CHAPS buffer and were elec- to 70% of the dish was populated with migrating cells. trophoresed. Radiolabeling of Plasma Membrane Affinity Chromatography of Membrane Sialoglycoproteins of Corneal Epithelial Cells Glycoproteins of Corneal Epithelial Cells on Sialic acid residues of epithelial membrane glycopro- Agarose-Bound Wheat Germ Agglutinin 3 teins were radiolabeled by sodium periodate (NaIO4)- To ensure further that the NaIO4/NaB H4 treatment 3 tritiated sodium borohydride (NaB H4) treatment us- specifically introduced the label to sialic acid residues, ing the Gahmberg and Andersson method,22 with an the radiolabeled sialoglycoproteins were chromato- additional prereduction step to minimize nonspecific graphed on a wheat germ agglutinin (WGA)-agarose labeling.23 This procedure is known to label externally column expected to bind specifically to glycoproteins exposed cell surface sialic acid residues specifically containing sialic acid and N-acetylglucosamine resi- and involves cleavage of carbons 8 and 9 of the sialic dues.24 For this, CHAPS-solubilized material from
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