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(Investigative Ophthalmology and Visual Science. 2008;49:2424-2431.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1498
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Immunochemical Localization of Keratan Sulfate Proteoglycans in Cornea, Sclera, and Limbus Using a Keratanase-Generated Neoepitope Monoclonal Antibody

Saeed Akhtar,1,2,3 Briedgeen C. Kerr,3,4 Anthony J. Hayes,4 Clare E. Hughes,4 Keith M. Meek,1 and Bruce Caterson4

From the Schools of 1Optometry and Vision Sciences and 4Biosciences, Cardiff University, Cardiff, United Kingdom; and the 2Department of Optometry and Vision Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia.

PURPOSE. To evaluate the use of neoepitope monoclonal antibody BKS-1, which recognizes keratanase-generated keratan sulfate (KS) stubs on keratan sulfate proteoglycans in human cornea, limbus, and sclera.

METHODS. BKS-1 specifically recognizes a keratanase-generated neoepitope [N-acetyl-glucosamine-6-sulfate (GlcNAc-6-S)] at the nonreducing terminal of corneal and skeletal KS glycosaminoglycan chains. It was produced by using keratanase-digested KS peptides from bovine cartilage aggrecan as the immunizing antigen. BKS-1 was used in conjunction with 5D4 to analyze the KS distribution in human cornea, limbus, and sclera using Western blotting, immunohistochemistry, and electron microscopy.

RESULTS. 5D4 Western blot analysis displayed a diffuse staining pattern, and it was difficult to distinguish differences among cornea, sclera, and limbus. However, BKS-1 showed differences in KS levels, with higher levels in the cornea and lower levels in the limbus and sclera. Ultrastructural studies showed that the monoclonal antibody (mAb) BKS-1 neoepitope was not observed in the epithelium or basement membrane; however, 5D4 was present in these layers. Large quantities of both antibodies were present in Bowman’s layer, stroma, and Descemet’s membrane, but the quantity of 5D4 was significantly higher (P < 0.001) than the quantity of BKS-1 in all these layers of the cornea.

CONCLUSIONS. mAb 5D4 recognizes oversulfated structures within KS chains, whereas BKS-1 recognizes a single neoepitope on KS after keratanase digestion of monosulfated KS disaccharides. With the use of BKS-1, the authors identified a more clearly defined pattern for KS distribution in the cornea than was seen with 5D4. The presence of a large quantity of BKS-1 immunostaining in the cornea suggests that KS-substituted proteoglycans are more prevalent in the cornea than in the limbus or sclera.






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