Structural and biochemical aspects of keratan sulphate in the cornea

Abstract

Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) in the cornea of the eye, where it exists in proteoglycan (PG) form. KS-PGs have long been thought to play a pivotal role in the establishment and maintenance of the array of regularly-spaced and uniformly- thin collagen fibrils which make up the corneal stroma. This characteristic arrangement of fibrils allows light to pass through the cornea. Indeed, perturbations to the synthesis of KS-PG core proteins in genetically altered mice lead to structural matrix alterations and corneal opacification. Similarly, mutations in enzymes responsible for the sulphation of KS-GAG chains are causative for the inherited human disease, macular corneal dystrophy, which is manifested clinically by progressive corneal cloudiness starting in young adulthood.

Fig. 1

Histologic image of a human cornea in sagittal section, after formalin fixation, paraffin wax embedding, and section staining with H&E; original magnification ×20. The stroma, which is about 500 μm thick, is bound superficially by the multicellular corneal epithelium and posteriorly by the endothelial monolayer and Descemet’s membrane, and forms the bulk of the tissue. Nuclei are stained blue, and splits in the stroma are artefacts of the preparative process. (Figure courtesy of Dr Hidetoshi Tanioka, Department of Ophthalmology, Kyoto Prefectural University of Medicine, Japan)

Fig. 2

A schematic depicting the generic structure of corneal KS and the actions of various KS-degrading enzymes. The disaccharide structure of KS can be either di-, mono- or unsulphated on the repeating disaccharide units. Disulphated structures occur towards the non-reducing terminal, monosulphated disaccharides towards the middle of the structure and unsulphated disaccharides towards the linkage region of the chain. Keratanase cleaves at β1-4 galactosidic linkages in which unsulphated galactose and sulphated N-acetyl glucosamine (GlcNAc) residues participate (red arrows). Keratanase II cleaves at β1-3 glucosaminidic linkages to galactose where the disaccharide structure can be either mono- or disulphated (yellow arrows). Endo-β-galactosidase cleaves at β1-4 galactosidic linkages where both the galactose and the GlcNAc residues are not sulphated (grey arrows). It can also cleave at the same sites as Keratanase but at a much lower reaction rate. (Figure courtesy of Dr Melody Liles, School of Optometry and Vision Sciences, Cardiff University, UK)

Fig. 3

Collagen fibrils in longitudinal section in the stroma of bovine cornea swollen in 0.15 M saline. The characteristic 65-nm D-periodic repeat along the collagen axis is clearly visible after contrasting with uranyl acetate. Gold particles, 10-nm in diameter, indicate sites of highly-sulphated KS-GAG labelled with monoclonal antibody 5D4. Bar 100 nm

Fig. 4

Stereo pair of transverse section of collagen fibrils (green) interconnected by histochemically stained KSPG filaments (purple). Reconstruction obtained from anterior central portion of bovine corneal stroma treated with chondroitinase ABC. Bar 50 nm. (Figure courtesy of Drs Carlo Knupp, Philip Lewis and Christian Pinali, School of Optometry and Vision Sciences, Cardiff University, Wales, UK)

Fig. 5

Diffuse structures, presumed to be PGs (arrowheads), are preserved between collagen fibrils in transverse section, in the absence of cationic staining. This micrograph is of mouse corneal stroma prepared by cryofixation using liquid nitrogen in a Leica EMPACT2 high pressure freezer, followed by freeze-substitution in acetone with osmium tetroxide, to better preserve native matrix architecture. Bar 100 nm