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Title: Role of keratan sulphate proteoglycans in the maintenance of mouse corneal stromal ultrastructure
Author: Beecher, Nicola
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2006
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Abstract:
The cornea is a remarkable connective tissue that is transparent to visible light as a consequence of the regularly arranged, uniformly-sized collagen fibrils that constitute it. Evidence suggests that this intricate collagen fibril architecture is maintained by the presence of keratan sulphate-carrying proteoglycans (KSPGs) within the corneal stroma. The KSPGs, lumican, keratocan, and mimecan are believed to be involved in the regulation of collagen fibril diameter and fibril spacing. KSPGs have recently been investigated using transgenic technology, which allows the manipulation of gene expression in order to determine the significance of a gene product in a biological system. Thus, to ascertain the role of KSPGs in this thesis transgenic mice were used. Two methodologies were employed to investigate the structural organisation of collagen within normal and mutant mouse corneas small angle X-ray diffraction (XRD) provided quantitative information on collagen fibril spacing, collagen fibril diameter and degree of local order in the fibrillar array averaged throughout the tissue, and transmission electron microscopy (TEM) afforded a view of collagen fibril morphology within the tissue. Previous research has shown that a homozygous-null mutation for lumican affects corneal collagen fibril architecture. But no information exists about whether these structural changes develop in adulthood or start early in life. Thus, the role of lumican during neonatal corneal stromal development between days 8 and 14 was explored. Collagen fibril spacing is considerably higher in lumican-null corneas until day 14, and fibril diameter is, on average, smaller-than-normal. TEM provided evidence of stromal disorder in mutants and fibril fusion at day 14, indicating that lumican plays a key role in development of the neonate cornea. Next, the effect of a cysteine-serine substitution in the N-terminal region of lumican was investigated to determine the importance of this region in lumican-collagen interaction. Mutant collagen fibrils are appreciably larger in diameter, signifying failure of lumican to bind collagen and regulate fibril growth. This chapter also investigated the consequences of lumican over-expression, to reveal that excess lumican has no significant effect on mutant corneal stromal ultrastructure. The role of mimecan in the corneal ECM via ablation of gene expression was explored. The extent of ultrastructural alteration was minimal with mutants having collagen fibril spacing and fibril diameter that were essentially unchanged. This work indicated that mimecan plays a minor role in the maintenance of matrix structure in the cornea. Finally, to better understand what structural motifs contribute to KSPG effectivity, the significance of a carbohydrate sulphotransferase gene, ChstS, in the mouse corneal stroma was investigated. The ablation of ChstS, responsible for the production of a carbohydrate sulphotransferase enzyme, revealed that smaller fibril spacing and fibril diameters, and stromal disorganisation are the result. KSPGs are structurally and functionally distinctive. Mimecan appears to have little influence over matrix morphogenesis, and it is possible that absence of mimecan is compensated for by the upregulation of other PGs. In contrast, lumican is an important component of the ECM the structure of lumican bestows this molecule the ability to regulate matrix morphogenesis, as exemplified by absence of lumican and also N-terminal mutation of the core protein. Indeed, the sulphated form of KS is required within cornea, as the absence of a carbohydrate sulphotransferase gene renders the corneal stroma structurally altered.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.583892  DOI: Not available
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