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P.N. Lewis, Y. Hayashida, R.D. Young, T.O. Akama, M.N. Fukada, B. Caterson, K. Nishida, A. Quantock; Sulfated Proteoglycans in the Corneal Stroma of Chst5–Null Mice . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2998.
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Chst5–null mice, which fail to express a carbohydrate GlcNAc 6–O sulfotransferase enzyme (GlcNAc6ST) essential for the the sulfation of keratan sulfate (KS) proteoglycan (PG), exhibit altered collagen fibril packing in the corneal stroma. Here we determine the structural architecture of sulfated PGs in the cornea of Chst5–null mice
Corneas were excised from Chst5 mice at 1.6,5.0 and 7.9 months of age, briefly fixed and incubated in buffer with or without PG–degrading enzymes (chondrotinase ABC (1U/ml) or keratanase I (5U/ml)). Sulfated PGs were visulised by transmission electron microscopy after contrasting with cuprolinic blue (0.05% w/v). Corneas from each age group were also fixed in 4% paraformaldehyde and embedded at low temprature in Lowicryl K4M resin for immunoelectron microscopy. Sections were labelled with antibody 5D4 against highly–sulfated KS, "anti–i" antibody against poly–N–acetyllactosamine (unsulfated KS) chain regions, or the lectin ECA, which binds N–actetyllactosamine (unsulfated KS).
As in wild type mice, cuprolinic blue–positive PGs in the Chst5–null mouse cornea comprised both fine collagen–associated filaments and long, branching tendrils with thread–like lateral extentions that seemed to interlace collagen fibrils within lamellae. The long PGs were keratanase resistant, but chondrotinase ABC sensitive and thus probably a form of CS/DS PG. They sometimes appeared more ornate and electron dense in Chst5–null corneas compared to wild type. Chondroitinase ABC removed all collagen–associated filamentous PGs in Chst5–null corneas, whereas some small filaments remained in wild type. Immunoelectron microscopy of sections from both nornal and Chst5–null corneas treated with antibody 5D4 to high sulfated KS revealed no reactivity."Anti–i" antibody and ECA confirmed the presence of unsulfated KS throughout the corneal stroma.
Differential enzyme digestion patterns indicate that the low sulfated KS PG filaments seen in wild type corneas are not present in Chst5–null corneas. Also, it appears as though the long PGs are larger in Chst5 corneas. These changes in PG structure induced by a Chst5 deletion reflect an important role for GlcNAc6ST in KS sulfation and in collagen fibril organisation.
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