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M. Liles, C. Hughes, B. Kerr, A. Harris, B. Caterson, A. J. Quantock; Immunochemical Studies of Differentially Sulfated Keratan Sulfate in the Developing Chick Cornea. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1483.
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© ARVO (1962-2015); The Authors (2016-present)
During the final week of embryonic development, the chick cornea thins and becomes transparent. Proteoglycans (PGs) have been implicated in refinements to corneal structure during this time; indeed fluctuations in PG sulfation mirror an increase in collagen content and organisation. The aim of this study is to investigate keratan sulfate (KS) PG sulfation during matrix compaction at the onset of corneal transparency.
X-ray diffraction was used to deduce mean collagen fibril spacing and local order at specific timepoints in a group of 158 developing chick corneas. These same individual corneas were quantitatively analysed by ELISA, using 5D4 and 1B4 for highly and lower sulfated KS chains respectively, or BKS-1 to measure keratanase generated neoepitope KS-stubs. Finally, hydroxyproline assays were used to determine collagen content.
The ratio of 5D4 (measuring highly sulfated KS chains) to hydroxyproline (total collagen) in the developing chick cornea (±SE) was 36.21±10.65 (day 12), rising to 148.71±16.22 (day 18); a four-fold increase. The ratio of 1B4 (lower sulfated KS chains) to hydroxyproline in these same corneas (±SE) rose from 6.01±2.39 (day 12) to 8.51±1.69 (day 18), a 1.39-fold increase. This rise in KS epitopes follows a fall in mean collagen fibril spacing from 63.7nm (day 12) to 52.7nm (day 18), which occurs mainly after day 15. For the data set of 71 corneas there was an inverse correlation between collagen interfibrillar spacing and the quantified sulfation patterns of KS chains, as measured by 5D4 and 1B4. A parallel study of over 80 corneas again showed collagen fibril compaction (from 64.9nm (day 12) to 57.4nm (day 18)), but with no significant change in keratanase generated KS stub quantification using BKS-1 over the same time period.
The compaction of chick corneal collagen is accompanied by an increase in tissue levels of sulfated KS. Both highly and lower sulfated KS were found to increase significantly as fibrils condense and collagen deposition increases. However, the consistent nature of keratanase sensitive KS detection suggests a possible working hypothesis of no change in the number of keratanase sensitive KS sites with embryonic growth, with net sulfation increasing towards the non-reducing terminal over time. Remodelling of the collagen fibril arrangement may be related to a switch towards highly sulfated KS production, with a role for KS in the structural "fine tuning" of the fibrillar array.
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