Matrix Alterations in the Corneas of Chst5 Null Mice

Abstract

Abstract: : Purpose: In corneal stroma keratan sulphate (KS) glycosaminoglycans substituted on proteoglycans (PGs) influence matrix ultrastructure. Corneas of mice lacking the KSPGs lumican, keratocan, or mimecan show structural matrix changes. To assess the role of KS sulphation we generated mice with null mutations in Chst5, a gene encoding an N–acetylglucosamine–6–O–sulphotransferase (GlcNAc6ST) that is integral to the sulphation of KS chains. This gene is an ortholog of CHST6, which in humans encodes for a GlcNAc6ST and is causative for macular corneal dystrophy (MCD: Akama, Nishida, et al. Nat Genet 2000). Methods: Chst5^+/– ES cells were generated by homologous recombination using a target vector that contained a genomic DNA fragment of Chst5 with a neo expression cassette which replaced a protein encoding exon of the gene. Chst5 null mice were generated by intercrossing Chst5^+/– mice originated from the targeted ES cells. The collagen fibrillar architecture of corneas from 21 mature (5–10 month) mice was studied by synchrotron x–ray fibre diffraction. Results: Corneas of Chst5 null mice were optically clear, and even in old age showed no evidence of a murine form of MCD. X–ray diffraction showed that average collagen fibril diameters in heterozygous (35.7nm ± 0.6nm; n=11), homozygous (34.9nm ± 0.7nm; n=18), and wild type (36.4nm ± 0.9nm; n=12) corneas were not greatly different. Average centre–to–centre fibril spacing in corneas of homozygous mutants (42.5nm ± 3.4nm; n=18), on the other hand, was considerably lower than that in wild type (47.8nm ± 3.5nm; n=12) or heterozygous corneas (48.3nm ± 2.2nm; n=11), and these differences were highly significant (p<0.001). Close packing of collagen fibrils is also a feature of human corneas with MCD (Quantock et al., Curr Eye Res 1990). Local order in the fibrillar array, as indicated by the coherence distance, was lower in corneas of homozygous mutants (184nm ± 19nm; n=18) than heterozygous mutants (236nm ± 13nm; n=11) or wild types (247nm ± 18nm; n=12). Conclusions: Despite the fact that KS in mouse cornea has less highly sulphated epitopes than KS in the corneas of other, larger mammals (Young RD, et al., ARVO 2004), our discovery of structural matrix changes in homozygous Chst5 null mice points to a functional role for KS sulphation in mouse cornea.

Keywords: proteoglycans/glycosaminoglycans • cornea: stroma and keratocytes • extracellular matrix