Distribution of Neoepitope Bks–1 in Normal and Keratoconus Cornea

Abstract

Purpose: : Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) of the corneal stroma. In this study we investigated the precise distribution of KS–chains in normal and keratoconus corneas by using the neoepitope monoclonal antibody (MAb) BKS–1.

Methods: : Four normal and four severe keratoconus corneas were used for the study. The distribution of KS–GAGs was investigated by using a MAb (BKS–1) that specifically recognises a keratanase–generated neoepitope [N–acetyl–glucosamine–6–sulphate (GlcNAc–6–S)] at the non–reducing terminal of both corneal and skeletal KS– GAG chains. It was produced by using keratanase–digested "KS–peptides" from bovine cartilage aggrecan and was used with Western blotting and immunogold electron microscopy to quantify the labelling of KS–GAG chains in different layers of normal and keratoconus corneas.

Results: : Western blot studies showed only a small difference between normal and pathological cornea. Immunogold microscopy showed that BKS–1 was present in Bowman’s layer, stroma and Descemet’s membrane but not in the epithelium and basement membrane in normal and keratoconus cornea. The labelling of KS–GAG in the stroma of keratoconus was significantly (p>0.01) less compared to the normal cornea in all layers except in the middle stroma..

Conclusions: : MAb BKS–1 recognises a single neoepitope on KS after keratanase digestion, and therefore allows better quantitative determination of the number of KS GAGs than other GAG antibodies. The results suggest that there are more KS GAGs in the normal cornea than in keratoconus cornea. Keratan sulphate plays an important role in stromal collagen fibril assembly and a dysregulation of KS synthesis in keratoconus could explain changes in collagen fibril spacing and diameter, which we have reported elsewhere.

Keywords: pathology: human • cornea: stroma and keratocytes • immunohistochemistry