Abstract
The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.
Highlights
The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis
Following our recent development of fluorophore-assisted carbohydrate electrophoresis (FACE) technology for glycosaminoglycan quantitation and sulfation analyses [25,26,27,28,29], we report a detailed study of the fine structural characteristics of both keratan sulfate and chondroitin/dermatan sulfate chains in cornea and cartilage from individuals affected by MCD type I and in corneas of individuals affected by MCD type II
FACE Analyses of KS Sulfation in Normal and MCD Corneas—Glycopeptides prepared from normal and diseased corneas were digested with keratanase II and endo--galactosidase under conditions previously shown to completely depolymerize unsulfated, mono, or disulfated and fucosylated regions of KS chains [29], and all digestion products were separated by FACE
Summary
The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Chondroitin/ dermatan sulfate chain concentrations were increased by 60 –70% in types I and II corneas Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Mutations in the sulfotransferase gene in subtype II patients are deletions and/or replacements in the untranslated regions of the gene, where regulatory elements may be localized These defects may lead to a reduced transcription of the enzyme and possibly result in an enzyme deficiency limited to the corneas, explaining the published studies that reported normal levels of sulfated keratan sulfate in the serum of the subtype II patients. There is no reported chemical analyses of keratan sulfate chain fine structure, accumulated in vivo by cells harboring the MCD types I and II mutations in C-GlcNAc6ST activity, nor has there been any examination of the effect of impaired keratan sulfate syn-
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