Morphology and proteoglycan content of the sulfotransferase- and glycosyltransferase-deficient mouse cornea.

Littlechild, Stacy 2016. Morphology and proteoglycan content of the sulfotransferase- and glycosyltransferase-deficient mouse cornea. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Keratan sulfate (KS) is an elongated glycosaminoglycan (GAG) chain found throughout the cornea, the clear tissue at the front of the eye. It is thought that KS plays a specialized role in maintaining the ordered spatial arrangement of collagen fibrils that comprise the thickest layer of the cornea, the stroma. Repeating N-­‐acetylglucosamine (GlcNAc) and galactose monosaccharides make up the fine structure of KS, and sulfate groups frequently modify their C-­‐6 positions. Recent studies have linked improper sulfation of GlcNAc residues to macular corneal dystrophy, a vision condition characterized by a progressive loss of corneal transparency. Since then, in vitro experiments have implicated four enzymes in highly sulfated KS biosynthesis. It is currently believed that β-­‐1,3-­‐N-­‐ acetylglucosaminyltransferase 7 (β3GnT7), corneal GlcNAc 6-­‐O sulfotransferase (CGn6ST), and β-­‐1,4-­‐ lactosyltransferase 4 (β4GalT4) sequentially catalyze the addition of GlcNAc, transfer sulfate to its C-­‐6 position, and link galactose to the growing KS backbone, respectively. The fourth enzyme, KS galactose 6-­‐O sulfotransferase (KSGal6ST) is thought to act last, sulfating galactose residues. Mutant mice deficient in CGn6ST, KSGal6ST, or β3GnT7 were recently developed to investigate the consequences of dysfunctional sulfo-­‐ or glycosyltransferases on corneal morphology. Electron microscopy and immunohistochemistry data in this thesis showed that the systemic absence of CGn6ST or β3GnT7 resulted in a corneal stroma devoid of KS, but mutation of only KSGal6ST led to a KS phenotype similar to that of wild type controls. Western blot analysis conducted on β3GnT7-­‐null corneas indicated that KS assumed an unusually truncated form, as compared to wild type controls. A secondary result evident in electron micrographs was that in cases where KS levels dropped below the detectable threshold (i.e. in CGn6ST and β3GnT7 mutant corneas), a concurrent appearance of atypically elongated GAGs was visible, suggesting a compensatory mechanism to preserve corneal organization. Since the unusual GAGs were susceptible to chondroitinase ABC digestion, it is thought they are comprised of chondroitin/dermatan sulfate (CS/DS). Studies using high performance liquid chromatography were also undertaken to establish protocols in which future work could quantify the change in CS/DS expression observed in CGn6ST and β3GnT7 mutant corneas.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Optometry and Vision Sciences
Subjects:	R Medicine > RE Ophthalmology
Uncontrolled Keywords:	Cornea, Proteoglycan, Glycosaminoglycan, Sulfotransferase, Glycosyltransferase, Keratan Sulphate, Chondroitin Sulphate, Dermatan Sulphate, N-acetylglucosamine, Galactose
Funders:	Sir Martin Evans President’s Research Scholarship, Cardiff University International Scholarship
Date of First Compliant Deposit:	9 February 2022
Last Modified:	09 Feb 2022 15:23
URI:	https://orca.cardiff.ac.uk/id/eprint/91230

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