Immunochemical localization of keratan sulfate proteoglycans in cornea, sclera, and limbus using a keratanase-generated neoepitope monoclonal antibody.
OBJECTIVE
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.
Akhtar
Saeed
S
School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom. akhtars@ksu.edu.sa
Kerr
Briedgeen C
BC
Hayes
Anthony J
AJ
Hughes
Clare E
CE
Meek
Keith M
KM
Caterson
Bruce
B
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000276:immunology; D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002417:Cattle; D003315:Cornea / Q000378:metabolism* / Q000648:ultrastructure; D000939:Epitopes / Q000276:immunology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007632:Keratan Sulfate / Q000378:metabolism* / Q000648:ultrastructure; D016850:Limbus Corneae / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D008875:Middle Aged; D011509:Proteoglycans / Q000378:metabolism* / Q000648:ultrastructure; D012590:Sclera / Q000378:metabolism* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011509:Proteoglycans; C033137:N-acetylglucosamine-6-sulfate; D007632:Keratan Sulfate; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D000117:Acetylglucosamine