Fractionation of proteoglycans from bovine corneal stroma.

Proteoglycans were extracted from bovine corneal stroma with 4M-guanidinum chloride, purified by DEAE-dellulose chromatography (Antonopoulos et al., 1974) and fractionated by precipitation with ethanol into three fractions of approximately equal weight. One of these fractions consisted of a proteoglycan that contained keratan sulphate as the only glycosaminoglycan. In the othertwo fractions proteoglycans that contained chondroitin sulphate, dermatan sulphate and keratan sulphate were present. Proteoglycans which had a more than tenfold excess of galactosaminoglycans over keratan sulphate could be obtianed by further subfractionation. The gel-chromatographic patterns of the glucosaminoglycans before and after digestion with chondroitinase AC differed for the fractions. The individual chondroitin sulphate chains seemed to be larger in cornea than in cartilage. Oligosaccharides, possibly covalently linked to the protein core of the proteoglycans, could be isolated from all fractions. The corneal proteoglycans were shown to have higher protein contents and to be of smaller molecular size than cartilage proteoglycans.     

Proteoglycan biosynthesis by human corneas from patients with types 1 and 2 macular corneal dystrophy

Corneal buttons were obtained from patients with types 1 and 2 macular corneal dystrophy (MCD) and from control patients with Fuchs' dystrophy or keratoconus. Buttons were incubated for 20 h in the presence of [3H]glucosamine or [2-3H]mannose. Radiolabeled proteoglycans and lactosaminoglycan-glycoproteins (L-GPs) were purified using chromatography on Q-Sepharose, Superose 6, and octyl-Sepharose. They were identified using chondroitinase ABC, keratanase or endo-beta-galactosidase digestion, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Superose 6 chromatography. This study confirms previous reports that type 1 MCD corneas synthesize a normal dermatan sulfate-proteoglycan (DS-PG) and an abnormal keratan sulfate-proteoglycan (KS-PG). The data indicate that typ 1 MCD corneas synthesize L-GP instead of KS-PG. This L-GP has a core protein of similar hydrophobicity (elution from octyl-Sepharose) and nearly similar mass (42 kDa) as the core protein of the KS-PG. It has identical glycoconjugates as those of the KS-PG except that they lack sulfate. Thus, type 1 MCD fails to synthesize keratan sulfate as a result of a defect in a sulfotransferase specific for sulfating lactosaminoglycans. Further, proteoglycans synthesized by a cornea from a patient with type 2 MCD were studied. This cornea synthesized a normal ratio of KS-PG to DS-PG although net synthesis of proteoglycans was approximately 30% below normal. The KS-PG appeared normal whereas the DS-PG had dermatan sulfate chains that were approximately 40% shorter than normal.     

Altered fine structures of corneal and skeletal keratan sulfate and chondroitin/dermatan sulfate in macular corneal dystrophy

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.     

Proteoglycan changes during restoration of transparency in corneal scars

Corneal scars generated in rabbits by penetrating wounds are initially opaque but become transparent within a year. Previous studies have shown that the corneal stroma consists of proteoglycans and collagen fibrils spaced at regular intervals and that the interfibrillar spaces, the presumed location of proteoglycans, are abnormally large in opaque scars. In the present study, the size and glycosaminoglycan composition of the corneal stromal proteoglycans were determined in corneal scars during the restoration of transparency. The results showed that initially opaque scars which contained the large interfibrillar spaces also contained unusually large chondroitin sulfate proteoglycans with glycosaminoglycan side chains of normal size. These opaque scars also lacked the keratan sulfate proteoglycan but did contain hyaluronic acid. In the 1-year-old scars there was a restoration of normal interfibrillar spacing, and a return to corneal stromal proteoglycans of normal size and composition. These correlations suggest that the corneal stromal proteoglycans may play a fundamental role in regulating corneal collagen fibril spacing.     

Differential immunogold localisation of sulphated and unsulphated keratan sulphate proteoglycans in normal and macular dystrophy cornea using sulphation motif-specific antibodies

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the corneal stroma for tissue organisation and transparency. Macular corneal dystrophy (MCD) is a rare, autosomal recessive disease characterised by disturbances in KS expression. MCD is caused by mutations in CHST6, a gene encoding the enzyme responsible for KS sulphation. Sulphated KS is absent in type I disease causing corneal opacity and loss of vision. Genetic studies have highlighted the mutational heterogeneity in MCD, but supportive immunohistochemical studies on corneal KS have previously been limited by the availability of antibodies mostly reactive only with highly sulphated KS epitopes. In this study, we employed four antibodies against specific KS sulphation patterns, including one against unsulphated KS, to investigate their reactivity in a case of MCD compared with normal cornea using high-resolution immunogold electron microscopy. Mutation analysis indicated type I MCD with deletion of the entire open reading frame of CHST6. Contrast enhanced fixation revealed larger PG structures in MCD than normal. Unlike normal cornea, MCD cornea showed positive labelling with antibody to unsulphated KSPG, but was negative with antibodies to sulphated KSPG. These antibodies will thus facilitate high-resolution investigations of phenotypic heterogeneity in support of genetic studies in this disease.     

A comparative biochemical and ultrastructural study of proteoglycan-collagen interactions in corneal stroma. Functional and metabolic implications.

1. Corneas of mouse, rat, guinea pig, rabbit, sheep, cat, dog, pig and cow were quantitatively analysed for water, hydroxyproline, nucleic acid, total sulphated polyanion, chondroitin sulphate/dermatan sulphate and keratan sulphate, several samples or pools of tissue from each species being used. Ferret cornea was similarly analysed for water and hydroxyproline on one pool of eight corneas. Pooled frog (38) and ferret (eight) corneas and a single sample of human cornea were qualitatively examined for keratan sulphate and chondroitin sulphate/dermatan sulphate by electrophoresis on cellulose acetate membranes. Nine species (mouse, frog, rat, guinea pig, rabbit, sheep, cat, pig and cow) were examined by light microscopy and six (mouse, frog, rat, guinea pig, rabbit and cow) by electron microscopy, with the use of Alcian Blue or Cupromeronic Blue in critical-electrolyte-concentration (CEC) methods to stain proteoglycans. 2. Water (% of wet weight), hydroxyproline (mg/g dry wt.) and chondroitin sulphate (mg/g of hydroxyproline) contents were approximately constant across the species, except for mouse. 3. Keratan sulphate contents (mg/g of hydroxyproline) increased with corneal thickness, whereas dermatan sulphate contents decreased. The oversulphated domain of keratan sulphate was absent from mouse and frog corneas, increasing as percentage of total keratan sulphate with increasing corneal thickness. Sulphation of dermatan sulphate was essentially complete (i.e. one sulphate group per disaccharide unit). 4. Chondroitin sulphate/dermatan sulphate proteoglycans were present at the d bands of the collagen fibrils of all species examined, orthogonally arrayed, with high frequency, and occasionally at the e bands. Keratan sulphate proteoglycans were present at the a and c bands of all species examined, but with far higher frequency in the thicker corneas, where keratan sulphate contents were high. 5. Alcian Blue CEC staining showed much higher sulphation of keratan sulphate in thick corneas, e.g. that of cow, than in thin corneas, e.g. that of mouse, in keeping with biochemical analyses. 6. It is suggested that the constancy of interfibrillar volumes is regulated via the swelling and osmotic pressure of the interfibrillar polyanions, by adjustment of the extent of sulphation in two independent proteoglycan populations, to achieve an 'average sulphation' of the total polyanion similar to that of fully sulphated chondroitin sulphate/dermatan sulphate. 7. The balance of synthesis of the two kinds of proteoglycans may be determined by the O2 supply to the avascular cornea. O2 supply may also determine the conversion of chondroitin sulphate into dermatan sulphate.     

Characterization of the keratan sulphate proteoglycans from bovine corneal stroma.

The keratan sulphate proteoglycans that can be prepared from bovine corneal stroma [Axelsson & Heineg?rd (1975) Biochem. J. 145, 491-500] were characterized by gel chromatography, gel electrophoresis and analytical ultracentrifugation in associative (0.6 M-NaCl) and dissociative (6M-guanidinum chloride) solvents. The proteoglycans aggreagated at low salt concentrations and pH. The weight-average molecular weight of the monomer proteoglycans was established. Keratan sulphate peptides and oligosaccharide peptides were isolated after proteolysis. Their composition indicated that both are linked to protein via asparagine residues. A tentative model for corneal keratan sulphate proteoglycans is suggested.     

Heterogeneity of proteoglycans in monkey corneal stroma

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [³⁵S]sulfate and either [³H]serine or [³H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 m guanidine-HCl. After dialysis into 8 m urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-β-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.     

Electron tomography reveals multiple self-association of chondroitin sulphate/dermatan sulphate proteoglycans in Chst5-null mouse corneas

The spatial distribution of collagen fibrils in the corneal stroma is essential for corneal transparency and is primarily regulated by extrafibrillar proteoglycans, which are multi-functional polymers that interact with hybrid type I/V collagen fibrils. In order to understand more about proteoglycan organisation and collagen associations in the cornea, three-dimensional electron microscopy reconstructions of collagen-proteoglycan interactions in the anterior, mid and posterior stroma from a Chst5 knockout mouse, which lacks a keratan sulphate sulphotransferase, were obtained. Both longitudinal and transverse section show sinuous, oversized proteoglycans with near-periodic, orthogonal off-shoots. In many cases, these proteoglycans traverse over 400nm of interfibrillar space interconnecting over 10 collagen fibrils. The reconstructions suggest that multiple chondroitin sulphate/dermatan sulphate proteoglycans have aggregated laterally and, possibly, end-to-end, with orthogonal extensions protruding from the main electron-dense stained filament. We suggest possible mechanisms as to how sulphation differences may lead to this increase in aggregation of proteoglycans in the Chst5-null mouse corneal stroma and how this relates to proteoglycan packing in healthy corneas.     

Human corneal GlcNac 6-O-sulfotransferase and mouse intestinal GlcNac 6-O-sulfotransferase both produce keratan sulfate

Human corneal N-acetylglucosamine 6-O-sulfotransferase (hCGn6ST) has been identified by the positional candidate approach as the gene responsible for macular corneal dystrophy (MCD). Because of its high homology to carbohydrate sulfotransferases and the presence of mutations of this gene in MCD patients who lack sulfated keratan sulfate in the cornea and serum, hCGn6ST protein is thought to be a sulfotransferase that catalyzes sulfation of GlcNAc in keratan sulfate. In this report, we analyzed the enzymatic activity of hCGn6ST by expressing it in cultured cells. A lysate prepared from HeLa cells transfected with an intact form of hCGn6ST cDNA or culture medium from cells transfected with a secreted form of hCGn6ST cDNA showed an activity of transferring sulfate to C-6 of GlcNAc of synthetic oligosaccharide substrates in vitro. When hCGn6ST was expressed together with human keratan sulfate Gal-6-sulfotransferase (hKSG6ST), HeLa cells produced highly sulfated carbohydrate detected by an anti-keratan sulfate antibody 5D4. These results indicate that hCGn6ST transfers sulfate to C-6 of GlcNAc in keratan sulfate. Amino acid substitutions in hCGn6ST identical to changes resulting from missense mutations found in MCD patients abolished enzymatic activity. Moreover, mouse intestinal GlcNAc 6-O-sulfotransferase had the same activity as hCGn6ST. This observation suggests that mouse intestinal GlcNAc 6-O-sulfotransferase is the orthologue of hCGn6ST and functions as a sulfotransferase to produce keratan sulfate in the cornea.     

Roles of lumican and keratocan on corneal transparency

Lumican and keratocan are members of the small leucine-rich proteoglycan (SLRP) family, and are the major keratan sulfate (KS) proteoglycans in corneal stroma. Both lumican and keratocan are essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. This is attributed to their bi-functional characteristic (protein moiety binding collagen fibrils to regulate collagen fibril diameters, and highly charged glycosaminoglycan (GAG) chains extending out to regulate interfibrillar spacings) that contributes to their regulatory role in extracellular matrix assembly. The absence of lumican leads to formation of cloudy corneas in homozygous knockout mice due to altered collagenous matrix characterized by larger fibril diameters and disorganized fibril spacing. In contrast, keratocan knockout mice exhibit thin but clear cornea with insignificant alteration of stromal collaegenous matrix. Mutations of keratocan cause cornea plana in human, which is often associated with glaucoma. These observations suggest that lumican and keratocan have different roles in regulating formation of stromal extracellular matrix. Experimental evidence indicates that lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and tumorgenesis, besides regulating collagen fibrillogenesis. Published in 2003.     

Ultrastructural localization of sulfated and unsulfated keratan sulfate in normal and macular corneal dystrophy type I

Keratan sulfate (KS) proteoglycans are of importance for the maintenance of corneal transparency as evidenced in the condition macular corneal dystrophy type I (MCD I), a disorder involving the absence of KS sulfation, in which the cornea becomes opaque. In this transmission electron microscope study quantitative immuno- and histochemical methods have been used to examine a normal and MCD I cornea. The monoclonal antibody, 5-D-4, has been used to localize sulfated KS and the lectin Erythrina cristagalli agglutinin (ECA) to localize poly N -acetyllactosamine (unsulfated KS). In normal cornea high levels of sulfated KS were detected in the stroma, Bowman's layer, and Descemet's membrane and low levels in the keratocytes, epithelium and endothelium. Furthermore, in normal cornea, negligible levels of labeling were found for N -acetyllactosamine (unsulfated KS). In the MCD I cornea sulfated KS was not detected anywhere, but a specific distribution of N -acetyllactosamine (unsulfated KS) was evident: deposits found in the stroma, keratocytes, and endothelium labeled heavily as did the disrupted posterior region of Descemet's membrane. However, the actual cytoplasm of cells and the undisrupted regions of stroma revealed low levels of labeling. In conclusion, little or no unsulfated KS is present in normal cornea, but in MCD I cornea the abnormal unsulfated KS was localized in deposits and did not associate with the collagen fibrils of the corneal stroma. This study has also shown that ECA is an effective probe for unsulfated KS.     

Ultrastructural demonstration of proteoglycans in adult rat cornea

Proteoglycans in the adult rat cornea were demonstrated at the electron microscope level using two approaches: (a) staining with cuprolinic blue dye in the presence of 0.3 MgCl2, and (b) immunocytochemical localization of glycosaminoglycans with monoclonal antibodies and protein A-gold complexes. In the stroma two kinds of cuprolinic blue-induced filaments were morphologically differentiated and characterized according to their sensitivity to enzymatic degradations as keratan sulphate-rich and chondroitin-dermatan sulphate-rich proteoglycans respectively. Both types were mostly associated with collagen fibres, occupying the whole stroma except in certain areas whose significance is discussed. By immunocytochemistry, anterior and posterior regions of the stroma were found to be richer in chondroitin sulphate than the middle part, whereas keratan sulphate showed an homogeneous distribution throughout the stroma. Glycosaminoglycans were also detected in corneal basement membranes, epithelium and endothelium. The latter localizations are discussed in the light of what is known at present about the production of glycosaminoglycans by corneal cells.     

The structure of keratan sulphates from various sources.

Quantitative structural comparisons were made between keratan sulphates isolated from various sources, namely pig nucleus pulposus, bovine cornea, and the costal cartilages of children, a young adult with Marfan syndrome and of old human autopsies. In human costal cartilage the amount of keratan sulphate increases markedly with age, although total mucopolysaccharide decreases to some extent, concomitant with a decrease in chondroitin 4-sulphate and an increase in chondroitin 6-sulphate. Comparison of molecular weights estimated by gel chromatography with those calculated from the molar ratio of galactose to mannose indicates that keratan sulphates of human costal cartilages of children and of a young adult with Marfan syndrome, and of pig nucleus pulposus, contain one mannose residue per chain, whereas keratan sulphates of old human costal cartilage and of bovine cornea contain one to two, and two, per chain respectively. After mild acid-catalysed desulphation of pig nucleus pulposus keratan sulphate, approx. 12% of the mucopolysaccharide aggregates irreversibly once the water is removed from the polysaccharide. The following conclusions have been drawn from a methylation analysis of keratan sulphates of various sources, aided by g.l.c.-mass spectrometry. (1) Fucose and N-acetylneuraminic acid are non-reducing terminal residues and the sialic acid is linked to the 3-position of galactose residues. (2) Pig nucleus pulposus keratan sulphate has approximately 4 non-reducing terminal groups per molecule and appears to be slightly less branched than the costal-cartilage keratan sulphate of children. The branching in human costal-cartilage keratan sulphates decreases with age. Bovine corneal keratan sulphate appears to be unbranched. (3) Mannose residues are linked by 3 different substituents in human costal-cartilage and bovine corneal keratan sulphates, and by two different substituents in pig nucleus pulposus keratan sulphate. (4) The sulphate ester groups are all on the 6-position of N-acetyl-glucosamine and galactose residues. The degree of sulphation increases with age in costal keratan sulphates with the increase mainly of the galactose 6-sulphate residues.     

Characterization and biosynthesis of proteoglycans of corneal stroma from rhesus monkey.

The proteoglycans of the Rhesus monkey corneal stroma were characterized by analyzing both radiolabeled proteoglycans synthesized by corneas in organ culture and native corneal proteoglycans obtained by large scale preparations. The analyses indicate that the proteoglycans synthesized in organ culture were similar to, if not identical with, their counterparts in the stroma although they are synthesized in different prportions in vitro than they acumulate in vivo. The corneal stroma contains two proteoglycans. The chondroitin-dermatan sulfate proteoglycan consists of approximately 70% protein and has a Mr = approximately 100,000 to 150,000. It contains one chondroitin-dermatan sulfate side chain of Mr = approximately 55,000. The keratan sulfate proteoglycan consists of approximately 74% protein and has a Mr = approximately -40,000 to 70,000. It contains one or two keratan sulfate side chains with a Mr = approximately 7,000 each. Radiolabeling indicates that both proteoglycans contain glycoprotein-type oligosaccharides as part of their structure.     

Interactions between different corneal proteoglycans.

Proteoglycans were extracted from bovine cornea with 4M-guanidinium chloride and purified by CsCl-density-gradient centrifugation. Under associative conditions two fractions were found: one capable of forming assemblies of high molecular weight and another lacking this property. The heavier fraction (density 1.59 g/ml) was eluted as a single retarded peak from Sepharose 2B, but on DEAE-Sephadex chromatography, gave two peaks: the first (eluted with 0.75 M-NaCl) contained mainly proteochondroitin sulphate and the second (eluted with 1.25 M-NaCl) mainly proteokeratan sulphate. Each of these proteoglycans was more retarded on Sepharose 2B than was the original sample from density-gradient centrifugation. Re-aggregation was obtained by recombination of the two fractions. The lighter fraction (density 1.44 g/ml), containing predominantly keratan sulphate chains, was eluted from DEAE-Sephadex as a single peak with 1.25 M-NaCl and was retarded on Sepharose 2B: this fraction was not able to form aggregates with proteochondroitin sulphate. Chemical analyses of the carbohydrate and protein moieties of the proteoglycans from DEAE-Sephadex confirmed that, in the cornea, different subunits are present with characteristic aggregation properties and hydrodynamic volumes.     

Proteoglycans of the intervertebral disc. Homology of structure with laryngeal proteoglycans.

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.     

Proteoglycan distribution in developing rabbit cornea

We used a staining procedure specific for sulfated glycosaminoglycans, cuprolinic blue dye (CBD), and immunohistochemical techniques to determine the histological distribution and ultrastructural organization of proteoglycans in developing rabbit cornea. We found several types of CBD-stained structures located throughout the corneal stroma, indicative of the distribution and perhaps the chemical heterogeneity of proteoglycans in this tissue. Keratan sulfate-specific immunohistochemical evidence supports our cytochemical findings. Our results suggest that low-sulfated keratan sulfate proteoglycans are found throughout most of the developing stroma, with the exception of the posterior margin of this tissue. Highly sulfated keratan sulfate proteoglycans in young fetal corneas, initially restricted to the subepithelial stroma, progressively extend to deeper portions of the stroma with development. Dermatan sulfate proteoglycans are located throughout the stroma, including the posterior margin. Invoking a recently published "oxygen-lack hypothesis" and correlating the tissue location of proteoglycans with the source of oxygen, we hypothesize that the distribution of proteoglycans in the developing rabbit cornea is related to the selective synthesis of keratan sulfate glycosaminoglycans under hypoxic conditions.     

An X-ray scattering investigation of corneal structure in keratocan-deficient mice.

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.     

The structure and composition of cartilage keratan sulphate

Keratan sulphate was isolated from bovine intervertebral disc and bovine nasal septum after hydrolysis with proteinases and treatment with dilute alkali. Each preparation was found to contain, per keratan sulphate chain: (a) 1 residue of mannose; (b) 3 residues of N-acetylneuraminic acid (2 residues after alkali treatment); (c) 1 residue of N-acetylgalactosamine (lost after alkali treatment); (d) 1 residue or less of fucose. N-Acetyl-neuraminic acid residues were at non-reducing termini and were bonded to keratan sulphate through galactose residues. Evidence is presented for two different types of linkage between skeletal keratan sulphate and protein. Consideration of molecular parameters and compositions leads to a proposed structure for keratan sulphate-protein as found in skeletal proteoglycans.