Glycosaminoglycans of cat invertebral disc

The glycosaminoglycan contents of samples from cat intervertebral discs were examined by using cetylpyridinium chloride salt elution techniques. The values obtained related to the region of the vertebral column from which they were derived, to the area of the disc, and to water content. In wet tissue there was a significant difference between regions of the vertebral column and between areas of the disc and findings agreed with previous histological reports. The greater part of the glycosaminoglycans present consisted of chondroitin sulphate and dermatan sulphate with smaller amounts of hyaluronic acid; little keratan sulphate was found. The maximum amounts of chondroitin sulphate and dermatan sulphate occurred in the 0.5m-magnesium chloride fractions usually, but moved towards higher molar concentrations in samples derived from some sites, particularly in the lumbar region. Mean values for the water content of the areas of the disc were: nucleus pulposus, 82.4%; inner anulus, 65.6%; outer anulus, 50.5%. The water content was directly related to the amounts of chondroitin sulphate and dermatan sulphate.     

Regulated expression of keratan sulphate and peanut agglutinin binding sites during organogenesis in the developing chick

 Keratan sulphate proteoglycans are potentially important during development and are possible binding molecules for the lectin, peanut agglutinin, a marker for areas that are inhibitory for axonal growth in early embryos. The present study describes the spatiotemporal distributions of keratan sulphate epitopes and peanut agglutinin binding sites during organogenesis in the developing chick from E5 to hatching. The widespread distributions of these molecules did not often overlap but clearly delimited different carbohydrate compartments demonstrating that peanut agglutinin does not necessarily bind to keratan sulphate proteoglycans. These markers were mostly extracellular but keratan sulphate, in particular, was found within certain specific cells in cartilage, gonad, heart and pancreas, at certain ages. The presence of keratan sulphate in putative germ cells during their migrations and in the gonads may be of particular importance. Their distributions generally evoke modulation of adhesion allowing cell migrations or morphogenetic movements related to epitheliomesenchymal interactions, but may also suggest an involvement in axonal guidance in skin, cartilage, gut and possibly heart. Furthermore, in the kidney, peanut agglutinin binding sites seem to be related to the functional differentiation of the nephrons. Accepted: 23 February 1998     

Characterization of protein–polysaccharides of articular cartilage from mature and immature pigs

Protein-polysaccharides of femoral articular cartilage from pigs of ages 9 months and 5 weeks were compared after extraction at pH6.8 with iso-osmotic sodium acetate followed by 0.63m-calcium acetate. The cartilage from the younger animals had a higher moisture content and contained considerably larger amounts of protein-polysaccharide, but less than half as much collagen/g. dry weight, than cartilage from the older pigs. There was notably less keratan sulphate in the fractions from the less mature animals. After gel filtration on 6% agarose, elution profiles of the calcium acetate extracts were similar to those of the sodium acetate extracts of the same tissue. Chemical analyses, however, showed that in both age-groups the extraction procedure had achieved a sequential solubilization of protein-polysaccharides in that the initial extracts contained a higher proportion of keratan sulphate than those that were extracted subsequently. Both extracts from the older animals contained up to 25% of a relatively small protein-polysaccharide that was retarded on 6% agarose and that had a lower protein content and less keratan sulphate than the larger protein-polysaccharides. In contrast, in extracts from the less mature cartilage only about 5% of the protein-polysaccharides were small enough to be retarded by 6% agarose, suggesting that the small components may not be precursors of the larger. The average length of chondroitin sulphate chains, as calculated from the analytical data, was the same in the smaller protein-polysaccharides as in the larger.     

Studies of a monoclonal antibody to skeletal keratan sulphate. Importance of antibody valency.

A mouse monoclonal antibody (AN9P1) to keratan sulphate is described. In a competitive-inhibition solution-phase radioimmunoassay employing 125I-labelled intact proteoglycan, it reacts preferentially with keratan sulphate bound to the core protein of adult human articular-cartilage proteoglycan and to a much lesser degree with keratan sulphate purified from this proteoglycan. Proteolytic cleavage of the proteoglycan by pepsin and trypsin has little effect on antibody binding, but treatment with papain decreases binding considerably and more than does treatment with keratanase. An even greater decrease in binding is observed after treatment with alkaline borohydride. A comparison of binding of antibody AN9P1 with that of another previously described monoclonal antibody, 1/20/5-D-4, to keratan sulphate [Caterson, Christner & Baker (1983) J. Biol. Chem. 258, 8848-8854] revealed similar binding characteristics, both showing much diminished binding after papain digestion of proteoglycan and even less with purified skeletal keratan sulphate. Removal of the Fc piece of antibody AN9P1 had no significant effect on the differential binding of divalent F(ab')2 fragment to proteoglycan, to papain-digested proteoglycan and to keratan sulphate, although there was a small decrease in binding to papain-digested proteoglycan. Conversion of the antibody into univalent Fab fragment with removal of the Fc piece resulted in diminished binding to proteoglycan, compared with that observed with IgG, and in enhanced binding to free keratan sulphate and to papain-digested proteoglycan. These results suggest that close proximity of keratan sulphate chains on the core protein of proteoglycans favours preferential reactivity of bivalent antibody with these species through cross-bridging of chains by antibody. Conversely, much decreased binding to keratan sulphate on proteoglycan core-protein fragments and to free keratan sulphate results from a lack of close proximity of keratan sulphate. By using univalent Fab fragment in these assays these differences in binding are minimized by preventing cross-bridging and thereby enhancing detection of smaller fragments without sacrificing too much sensitivity of detection of larger proteoglycan species. The persistent preferential binding of Fab fragment to proteoglycan is probably in part the result of the increased epitope density in the intact molecule compared with keratan sulphate in a more disperse form.     

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.     

Keratan sulphate in the interglobular domain has a microstructure that is distinct from keratan sulphate elsewhere on pig aggrecan

The microstructure of keratan sulphate purified from the interglobular domain, the keratan sulphate-rich region and total aggrecan was compared using fluorophore-assisted-carbohydrate-electrophoresis. Keratan sulphate in the interglobular domain was substantially less sulphated than keratan sulphate elsewhere on aggrecan, based on the ratio of unsulphated: monosulphated disaccharides generated by endo-β-galactosidase digestion, and the ratio of monosulphated: disulphated disaccharides generated by keratanase II digestion. The ratio of unsulphated: monosulphated: disulphated disaccharides was 1:4:5 for keratan sulphate from total aggrecan and the keratan sulphate-rich region, but only 1:0.9:0.8 for the interglobular domain. These results show that keratan sulphate in the interglobular domain of pig aggrecan has a microstructure that is distinct from keratan sulphate in the keratan sulphate-rich region.     

The glycosaminoglycans of human tracheobronchial cartilage

1. The glycosaminoglycans of human tracheobronchial cartilages from subjects of various ages were liberated by proteolysis of the tissue and purified by ion-exchange chromatography. Purified glycosaminoglycans were fractionated on Dowex 1 resin and cetylpyridinium chloride was used to separate chondroitin sulphates and keratan sulphates occurring in the same fraction. 2. The total chondroitin sulphate content of the cartilages decreased linearly with increasing age. Age-dependent changes in the chemical heterogeneity of chondroitin sulphate were observed, a low-sulphated compound making up 25% of the total glycosaminoglycan at birth but rapidly diminishing in content during the first 6 months of life. Of the total chondroitin sulphate the 6-isomer became rather more prominent than the 4-isomer with increasing age. 3. The total keratan sulphate content of the cartilages increased from trace amounts only at birth to a plateau value by the beginning of the fifth decade. Of the total keratan sulphate approx. 70% was due to a high-molecular-weight compound with a sulphate/hexosamine ratio of 1.5-1.8: 1.0. The degree of sulphation varied between compounds isolated from different individuals. The remaining 30% of the keratan sulphate appeared to be intimately associated with chondroitin 6-sulphate and could only be separated from it after treatment with 0.45m-potassium hydroxide. The hybrid glycosaminoglycans were of lower molecular weight and had a lower sulphate/hexosamine ratio than the major keratan sulphate compound.     

The detection of substructures within proteoglycan molecules. Electron-microscopic immuno-localization with the use of Protein A-gold.

Proteoglycan monomers from pig laryngeal cartilage were examined by electron microscopy with benzyldimethylalkylammonium chloride as the spreading agent. The proteoglycans appeared as extended molecules with a beaded structure, representing the chondroitin sulphate chains collapsed around the protein core. Often a fine filamentous tail was present at one end. Substructures within proteoglycan molecules were localized by incubation with specific antibodies followed by Protein A-gold (diameter 4 nm). After the use of an anti-(binding region) serum the Protein A-gold (typically one to three particles) bound at the extreme end of the filamentous region. A small proportion of the labelled molecules (10-15%) showed the presence of gold particles at both ends. A monoclonal antibody specific for a keratan sulphate epitope (MZ15) localized a keratan sulphate-rich region at one end of the proteoglycan, but gold particles were not observed along the extended part of the protein core. This distribution was not changed by prior chondroitin AC lyase digestion of the proteoglycan. Localization with a different monoclonal antibody to keratan sulphate (5-D-4) caused a change in the spreading behaviour of a proportion (approx. 20%) of the proteoglycan monomers that lost their beaded structure and appeared with the chondroitin sulphate chains projecting from the protein core. In these molecules the Protein A-gold localized antibody (5-D-4) along the length of the protein core whereas in those molecules with a beaded appearance it labelled only at one end. Labelling with either of the monoclonal antibodies was specific, as it was inhibited by exogenously added keratan sulphate. The differential localization achieved may reflect structural differences within the proteoglycan population involving keratan sulphate and the protein core to which it is attached. The results showed that by this technique substructures within proteoglycan molecules can be identified by Protein A-gold labelling after the use of specific monoclonal or polyclonal antibodies.     

Degradation of keratan sulphate by beta-N-acetylhexosaminidases A and B.

Enzymic cleavage of beta-N-acetylglucosamine residues of keratan sulphate was studied in vitro by using substrate a [3H]glucosamine-labelled desulphated keratan sulphate with N-acetylglucosamine residues at the non-reducing end. Both lysosomal beta-N-acetylhexosaminidases A and B are proposed to participate in the degradation of keratan sulphate on the basis of the following observations. Homogenates of fibroblasts from patients with Sandhoff disease, but not those from patients with Tay--Sachs disease, were unable to release significant amounts of N-acetyl[3H]glucosamine. On isoelectric focusing of beta-N-acetylhexosaminidase from human liver the peaks of keratan sulphate-degrading activity coincided with the activity towards p-nitrophenyl beta-N-acetylglucosaminide. A monospecific antibody against the human enzyme reacted with both enzyme forms and precipitated the keratan sulphate-degrading activity. Both isoenzymes had the same apparent Km of 4mM, but the B form was approximately twice as active as the A form when compared with the activity towards a chromogenic substrate. Differences were noted in the pH--activity profiles of both isoenzymes. Thermal inactivation of isoenzyme B was less pronounced towards the polymeric substrate than towards the p-nitrophenyl derivative.     

Maturation-related changes in proteoglycans of fetal articular cartilage

Proteoglycans of the articulating and growing zones of maximum and minimum contact of bovine fetal articular cartilage were studied and compared to proteoglycans of immature calf and adult steer. During fetal maturation, localized changes were observed as early as the second trimester of fetal life but were restricted to the most superficial zones. Proteoglycans extracted from the growing zones were purified by density-gradient ultracentrifugation. The majority of proteoglycan monomers were able to interact with endogenous hyaluronate to form aggregates. Monomers had, at all fetal stages, similar elution profiles on Sepharose 2B and similar ratios of chondroitin sulfate chains/keratan sulfate chains/O-glycosidically linked oligosaccharides. Keratan sulfate chains were of similar size at all stages, but chondroitin sulfate chain size decreased markedly with fetal maturation. In the first and second trimesters of fetal life, the proteoglycans were poorly substituted with glycosaminoglycans. A major increase in the absolute number of glycosaminoglycans and oligosaccharides attached to core protein was detected during the third trimester of fetal life. No further changes in substitution occurred in early postnatal life. Enzymatic digestion of proteoglycan monomer demonstrated that the increase in substitution with keratan sulfate occurred to the same extent in the main polysaccharide attachment region and in the keratan sulfate-rich region.     

Stromelysin-1 (MMP-3) in forming enamel and predentine in rat incisor-coordinated distribution with proteoglycans suggests a functional role

Stromelysin-1 (matrix metalloproteinase-3) or proteoglycanase was visualized by light and electron microscopy immunolabelling in the forming zone of rat incisors. In predentine, labelling was more dense at the transition zone between the inner proximal third and the two outer thirds. Odontoblast processes were also positively stained, mostly in predentine and to a lesser degree in dentine. The dentine–enamel junction was intensely labelled, whereas dentine and forming enamel were only faintly stained. Gold–antibodies complexes were seen inside secretory ameloblasts and odontoblasts in cytosolic locations. The distribution of stromelysin-1 was compared with the distribution of 2-B-6 epitope, an antibody recognizing chondroitin-4-sulphate/dermatan sulphate and which showed a decreasing gradient from the proximal zone to the distal part of predentine. In contrast, both 5-D-4, an anti-keratan sulphate antibody and an anti-lumican antibody displayed a reversed distribution, with an increase seen from the proximal and central thirds to the distal part of predentine. This coordinated distribution suggests that stromelysin-1 may have a functional role, being implicated in predentine in the degradation of chondroitin-4-sulphate/dermatan sulphate-containing proteoglycans, and consequently allowing keratan sulphate proteoglycan concentration to increase near the border where mineralization is initiated.     

O-linked oligosaccharides of human articular cartilage proteoglycan

O-linked oligosaccharides and keratan sulphate chains have been isolated from the proteoglycan subunits of human articular cartilage. The oligosaccharides possessed a size and chemical composition similar to the equivalent moieties present in the proteoglycan submits of the Swarm rat chondrosarcoma. Futhermore, the size and chemical composition of th oligosaccharides showed little change with the age of the individual from whom the proteoglycan was obtained. In contrast, the keratan sulphate chains appeared to increase in chain lenght with increased age of the individual. The total number of keratan sulphate and oligosaccharide chains per core protien decreased with age, but it was not clear whether there was any change in the ration of the two components with respect to one another.     

Stromelysin-1 (MMP-3) in Forming Enamel and Predentine in Rat Incisor – Coordinated Distribution with Proteoglycans Suggests a Functional Role

Stromelysin-1 (matrix metalloproteinase-3) or proteoglycanase was visualized by light and electron microscopy immunolabelling in the forming zone of rat incisors. In predentine, labelling was more dense at the transition zone between the inner proximal third and the two outer thirds. Odontoblast processes were also positively stained, mostly in predentine and to a lesser degree in dentine. The dentine–enamel junction was intensely labelled, whereas dentine and forming enamel were only faintly stained. Gold–antibodies complexes were seen inside secretory ameloblasts and odontoblasts in cytosolic locations. The distribution of stromelysin-1 was compared with the distribution of 2-B-6 epitope, an antibody recognizing chondroitin-4-sulphate/dermatan sulphate and which showed a decreasing gradient from the proximal zone to the distal part of predentine. In contrast, both 5-D-4, an anti-keratan sulphate antibody and an anti-lumican antibody displayed a reversed distribution, with an increase seen from the proximal and central thirds to the distal part of predentine. This coordinated distribution suggests that stromelysin-1 may have a functional role, being implicated in predentine in the degradation of chondroitin-4-sulphate/dermatan sulphate-containing proteoglycans, and consequently allowing keratan sulphate proteoglycan concentration to increase near the border where mineralization is initiated.     

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.     

The proteoglycans and glycosaminoglycan chains of rabbit synovium

The synovial lining of joint capsules is important because it controls the flow of fluid into and out of the joint cavity. Physiological studies have shown that the glycosaminoglycans, particularly hyaluronan, have an important role in the control of fluid flow. The distribution of the glycosaminoglycans and proteoglycans in the synovium and subsynovium of rabbits (approximately 12 weeks old) was, therefore, determined immunohistochemically. Hyaluronan, chondroitin-4- and chondroitin-6-sulphates and keratan sulphate are present in the synovium and subsynovium; chondroitin-4-sulphate is at higher concentrations than chondroitin-6-sulphate. The core proteins of the chondroitin sulphate proteoglycans, biglycan and decorin, and of the keratan sulphate proteoglycan, fibromodulin, are also present. To date, fibromodulin has not been located in other synovial linings, and its presence corroborates that of keratan sulphate.     

The effect of benzyl beta-D-xyloside on keratan sulphate synthesis in ox articular cartilage

Cartilage from adult bovine hock joints was incubated with [3H]galactose or [35S]sulphate in the presence of benzyl beta-D-xyloside. Radioisotope incorporation into proteoglycan was inhibited by the xyloside; the magnitude of this inhibition depended on the concentration of xyloside used. With 0.2mM xyloside radioisotope incorporation into keratan sulphate was not altered but inhibition was observed at xyloside concentrations of 1.0mM or higher. The decrease in radioisotope incorporation into keratan sulphate in the presence of 1.0mM benzyl beta-xyloside was directly related to a reduction in the average length of the keratan sulphate chains. This effect of beta-xyloside on keratan sulphate biosynthesis was markedly different from its effect on chondroitin sulphate biosynthesis.     

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.     

Comparisons between extracted and residual proteoglycans on the glycosaminoglycan level and changes with ageing

Sheep nasal cartilage from animals of five different ages were studied. Significant variations in the composition and on the extractability of the tissue occur with ageing. The ratio chondroitin sulphate to keratan sulphate in extracted and residual proteoglycans does not change in the same manner with ageing. The relative distribution of molecular sizes of keratan sulphate differs between extracted and residual proteoglycans. Hyaluronic acid and chondroitin sulphate appear homogeneous on the gel chromatography for all ages both in extracted and residual proteoglycans.     

Sequential distribution of keratan sulphate and chondroitin sulphate epitopes during ameloblast differentiation

Proteoglycans are complex macromolecules containing one or more glycosaminoglycan chains and exhibiting a variety of biological functions in connective tissues. The aim of the present study was to immunolocalize the distribution of keratan sulphate and chondroitin sulphate epitopes during initial enamel formation in order to study temporo-spatial expression patterns of these macromolecules. Third molars of four-months-old pigs were used for immunolocalization of keratan sulphate and chondroitin sulphate epitopes in the developing enamel layer. Tooth organs were prepared for paraffin sections in order to perform indirect immunohistochemistry. The results demonstrated a mutually exclusive positioning between these two epitopes. Keratan sulphate epitopes were observed in pre-secretory pre-ameloblasts and adjacent stratum intermedium while chondroitin sulphate epitopes were demonstrated in secretory ameloblasts and adjacent stratum intermedium. Our findings suggest that proteoglycans containing glycosaminoglycan chains may play a regulatory role during enamel mineralization.     

Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly-N-acetyllactosamine backbones. An immunochemical and chromatographic study of keratan sulphate oligosaccharides after desulphation or nitrosation

Conditions were established for desulphation of hexa-, octa-, deca- and larger oligosaccharides derived from corneal keratan sulphate after treatment with endo-beta-galactosidase. The antigenicities of the desulphated oligosaccharides were compared with those of the native oligosaccharides in chromatogram binding, plastic-plate binding or inhibition of binding assays using a novel microimmunochemical approach with oligosaccharide-lipid conjugates (neoglycolipids). The results clearly show that sulphate residues are essential components of the antigenic determinant(s) recognised by three monoclonal antibodies to keratan sulphate, 5-D-4, 1-B-4 and MZ15, but they mask the i antigen activity of the linear poly-(N-acetyllactosamine) backbones of this glycosaminoglycan. Immunochemical assays, before and after beta-N-acetylglucosaminidase treatment of desulphated linear hexa-, octa- and decasaccharides derived from keratan sulphate, indicate that for reaction with one anti-i antibody, Den, there is an absolute requirement for the non-reducing beta-galactosyl residue of the i antigen structure to be in the terminal position, but with a second anti-i antibody, Tho, there is in addition some reactivity with the i antigen structure having an N-acetylglucosamine residue at the non-reducing end. The chromatographic properties after desulphation or nitrosation of a minor keratan sulphate oligosaccharide (a dodecasaccharide), which reacts especially well with antibody 5-D-4, have provided the first evidence for the presence of glucosamine residues that may be N-sulphated in corneal keratan sulphate.