Matrix Biology 22 (2003) 467–475 0945-053X/03/$30.00  2003 Elsevier B.V. yInternational Society of Matrix Biology. All rights reserved. doi:10.1016/S0945-053XŽ03.00081-7 An X-ray scattering investigation of corneal structure in keratocan- deficient mice Keith M. Meek *, Andrew J. Quantock , Craig Boote , Chia Yang Liu , Winston W.-Y. Kao a, a a b,c b Structural Biophysics Group, Cardiff School of Optometry and Vision Sciences, Cardiff University, King Edward VII Avenue, Cardiff, a CF10 3NB, UK Department of Ophthalmology, University of Cincinnati, Cincinnati, OH, 45627-0527, USA b Bascom Palmer Eye Institute, University of Miami School of Medicine, MacKnight Eye Research Building, 1638 N.W. 10th Avenue 621, c Miami, FL 33136, USA Received 26 August 2003; received in revised form 26 September 2003; accepted 29 September 2003 Abstract The transparency of the cornea has been closely linked with the characteristic size and arrangement of its constituent collagen fibrils. This arrangement, in turn, is thought to depend on interactions with intervening matrix proteoglycans. The purpose of this investigation was to examine fibrillar collagen organisation in the corneas of mice homozygous for a null mutation in keratocan, a keratan sulfate-containing proteoglycan. Low-angle synchrotron X-ray scattering techniques were used. We found that keratocan- deficient mice had corneal collagen fibrils with significantly larger diameters than those in wild-type littermates. Furthermore, there was an increase in the centre-to-centre spacing of the collagen fibrils that was accompanied by a decrease in nearest- neighbour fibrillar order. We hypothesise that a lack of keratocan might lower the number of keratan sulfate proteoglycans that associate with collagen, leading to alterations in their diameters and spatial arrangements. Alternatively, it might change the osmotic balance between the inside and outside of fibrils, causing them to swell and move further apart.  2003 Elsevier B.V.yInternational Society of Matrix Biology. All rights reserved. Keywords: Cornea; Collagen; Proteoglycans; Keratocan-deficient; Glycosaminoglycans 1. Introduction The cornea of the eye is a uniquely transparent, predominantly collagenous connective tissue bound internally and externally by endothelial and epithelial cell layers. In between these layers of cells is the corneal stroma, which makes up approximately 90% of the tissue’s thickness, and is composed of stacked belts or lamellae containing parallel collagen fibrils that are regularly spaced and of equal diameter. Corneal trans- parency depends on the maintenance of short-range order in the array of collagen fibrils (Farrell and Mc- Cally, 2000), and it has long been supposed that the interfibrillar proteoglycans play an important role in both the regulation of fibril diameter and the mainte- nance of fibril order (Borcherding et al., 1975; Hassell et al., 1983; Hahn and Birk, 1992). The core proteins *Corresponding author. Tel: q44-0-2920876317; fax: q44-0- 2920874859. E-mail address: Meekkm@cf.ac.uk (K.M. Meek). of proteoglycans have the capacity to bind to the collagen fibrils, and appear to do so at specific sites (Meek et al., 1986; Scott and Haigh, 1988; Neame et al., 2000) whereas the highly charged glycosaminogly- can component can surround andyor space apart the collagen fibrils (Scott, 1992), and space-filling roles have been envisaged even though it is not known how glycosaminoglycans in the corneal stroma interact with one another. There are two groups of small leucine-rich proteogly- cans (SLRP) in the cornea, one containing dermatany chondroitin sulfate glycosaminoglycans and the other containing keratan sulfate glycosaminoglycans. This lat- ter group consists of three distinct molecules, keratocan (Corpuz et al., 1996; Liu et al., 1998), lumican (Bloch- berger et al., 1992) and mimecan (sometimes called osteoglycin)(Funderburgh et al., 1997). Each has a unique core protein but they share approximately 35% amino acid identity and a have a number of structural features in common (Funderburgh et al., 1991). Lumican