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

David Lewis, Yvonne Davies, Ian A. Nieduszynski, Fiona Lawrence, Andrew J. Quantock2, Richard Bonshek3 and Nigel J. Fullwood1

Department of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK, 2Department of Optometry and Vision Sciences, Cardiff University, Cardiff, UK, and 3University of Manchester and Royal Manchester Eye Hospital, Manchester, UK

Received on July 27, 1999; revised on August 24, 1999; accepted on August 24, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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.

Key words: antibody 5-D-4/cornea/electron microscopy/Erythrina cristagalli


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Corneal transparency is of fundamental importance to effective eyesight. It is maintained by the orderly arrangement of uniformly sized collagen fibrils in the stromal matrix, which accounts for ~90% of the corneal tissue (Maurice, 1957Go). Current evidence suggests that the regular collagen lattice of the cornea is sustained by several proteoglycans (PGs), binding to specific sites on the collagen fibrils (Scott and Haigh, 1988Go). These small leucine-rich PGs are glycosylated with keratan sulfate (KS) or chondroitin/dermatan sulfate (CS/DS) and these glycosaminoglycans (GAGs) are thought to be responsible for the correct interfibrillar spacing of the collagen fibrils.

Much work has centered around the characterization of the stromal KSPGs. Three KSPGs have been identified, lumican (Blochberger et al., 1992Go), keratocan (Corpuz et al., 1996Go), and mimecan (Funderburgh et al., 1997Go). Recent work involving lumican knockout mice (Chakravarti et al., 1998Go) has shown that corneal opacity results from the absence of this PG. Furthermore, it has also been shown in chick embryonic cornea (Funderburgh et al., 1986Go; Nakazawa et al., 1995Go) that the development of corneal transparency occurs simultaneously with the addition of KS to the lumican core protein, establishing that KS substitution of lumican is crucial for corneal transparency. The structure of the KS chains present in the corneal stroma have been well characterized (Oeben et al., 1987Go; Tai et al., 1996Go, 1997) and have been shown to consist of the repeating disaccharide N-acetyllactosamine which may be sulfated on C-6 of either or both the galactose and N-acetylglucosamine residues. In addition, the chains in human KS may be capped with NeuAc{alpha}(2–3)-, NeuAc{alpha}(2–6)-, and GalNAcß(1–3)- residues (Tai et al., 1997Go). The significance of such normal KS chains in the maintenance of corneal transparency is clearly evident in the consequences of the disease macular corneal dystrophy (MCD).

Macular corneal dystrophy is a very rare hereditary disorder involving the corneal stroma and endothelium (Klintworth and Vogel, 1964Go). Clinically, a patient suffers ongoing corneal clouding and periodic photophobia from the age of 10–12 years, resulting in severe visual impairment. Corneal transplantation is usually required when sufferers reach their twenties or thirties. Histologically, large opaque deposits accumulate in the keratocytes and endothelium and can be found located between stromal lamellae, particularly underneath the epithelium and in the posterial region of Descemet’s membrane, causing corneal opacity (Klintworth, 1994Go). It is known that MCD is essentially a disorder involving defective GAG synthesis, and the condition has so far been subdivided into three classes on the basis of the 5-D-4 antigenic KS expression in a MCD patient’s serum and cornea (Thonar et al., 1986Go; Yang et al., 1988Go). In MCD type I (MCD I), normal antigenic KS is absent from a patient’s cornea and blood serum (Yang et al., 1988Go). In MCD type IA (MCD IA), an immunophenotype recently confirmed in the Saudi population (Klintworth et al., 1997Go), normal antigenic KS is found only in the corneal keratocytes, with no antigenic KS found in the corneal stroma or serum. In MCD type II (MCD II) normal antigenic KS is found in both cornea and serum (Yang et al., 1988Go) but at levels which are lower than normal (Klintworth and Smith, 1983Go; Midura et al., 1990Go). Therefore, it is thought that some cases of MCD may involve an alternative defect to that which is expressed in MCD I. Recent work has shown that the genes responsible for MCD I and MCD II are colocalized to the same region of chromosome 16, and it is thought that the different types of MCD may be due to different mutations of the same gene (Liu et al., 1998Go). The most common form of the disease is MCD I and consequently most work has focused on this type.

Macular corneal dystrophy I has long been known to involve the synthesis of an abnormal KS (Hassell et al., 1980Go). Previous work has indicated that the abnormal KS is produced in amounts equivalent to normal KS in normal cornea, and that antibodies to the core protein of normal KSPG react with the abnormal unsulfated KSPG found in MCD I (Hassell et al., 1982Go). Thus, it is suggested that KSPGs are produced with the apparent defect being a failure to sulfate the N-acetyllactosamine chains (Nakazawa et al., 1984Go). It has recently been suggested that this results from an inherited deficiency in the sulfotransferase specific for N-acetylglucosamine, upon which the sulfation of the galactose is also dependent (Hassell and Klintworth, 1997Go). The opaque deposits of MCD I have never been characterized, although it has been suggested that these deposits may contain the unsulfated KSPGs (Nakazawa et al., 1984Go). It is also unclear as to whether the unsulfated KSPGs are exported from the cells normally and if the unsulfated KSPGs are still able to associate normally with the a and c bands of the collagen fibrils. All of these questions have important implications for our understanding the exact role of normally sulfated KS in the secretion and conformation of the KSPG molecule and the role of normally sulfated KS in corneal transparency. Normal KS is recognized by the monoclonal antibody 5-D-4, which binds to hepta- or larger oligosaccharides of sulfated poly N-acetyllactosamine (Mehmet et al., 1986Go). However, there is no antibody available that is specific to unsulfated KS so it has proved difficult to localize this glycan structure. This investigation has used a lectin to localize the unsulfated KSPGs. Lectins have proved valuable in the study and localization of glycoconjugates in various tissues, including normal and diseased cornea (Panjwani et al., 1986Go; Brandon et al., 1988Go). The lectin Erythrina cristagalli agglutinin (ECA) is known to bind to the N-acetyllactosamine disaccharide (Iglesias et al., 1982Go) and it, therefore, has a very high affinity for the unsulfated form of KS (poly-N-acetyllactosamine). It is known that in MCD I KS is attached to its core protein (Midura et al., 1990Go) in which case ECA should display the distribution of the PG, which bears the unsulfated KS in MCD I.

In this study, both the lectin, ECA, which recognizes unsulfated KS and the antibody, 5-D-4, which recognizes normally sulfated KS have been used to investigate the distribution of unsulfated and sulfated KS in normal and MCD I cornea.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Normal cornea
The distribution of 5-D-4 antigenic KS in normal human cornea has been previously reported (Davies et al., 1999Go; Bairaktaris et al., 1998Go) but not subjected to statistical analysis. In the present study, the keratocytes labeled at low levels and the stroma labeled heavily for KS (Figure 1a). Low amounts of labeling were found in the epithelium and the endothelium, and high levels of labeling were evident in Bowman’s layer and Descemet’s membrane. The control nonspecific antibody gave negligible levels of labeling throughout the keratocytes and stroma (Figure 1b). Similarly, negligible levels of control antibody were also found in the epithelium and Bowman’s layer, and the endothelium and Descemet’s membrane. In all instances, the level of labeling in the various corneal regions showed a highly significant (where significance is denoted by P < 0.01) increase in the 5-D-4 labeled samples compared to the control (all regions P < 0.001, except the endothelium where P = 0.004) when subject to t-test.



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 1. Transmission electron micrographs showing immunolabeling (labeling appears as black dots) for sulfated KS in human corneal keratocytes (k) and stroma (s). (a) Normal cornea showing high levels of labeling with 5-D-4 in the stroma, and low levels in the keratocyte. (b) Normal cornea immunolabeled with negative control antibody showing negligible levels of labeling in both stroma and keratocyte. (c) MCD I cornea showing extremely low levels of labeling with 5-D-4 in the stroma, keratocytes, and the keratocyte deposits (*). Scale bar, 0.2 µm for all micrographs.

 
For normal cornea, ECA gave very low levels of labeling for the epithelium and Bowman’s layer, the stroma (Figure 2c), keratocytes (Figure 3c), Descemet’s membrane (Figure 4c), and the endothelium (Figure 5c). Predigestion of the sections with endo-ß-galactosidase did not influence the levels of labeling with ECA, however, the sugar pre-incubation control successfully removed ECA labeling. The amount of labeling observed was found to be not significant when compared with the ECA control: across the various regions of the cornea P values were between 0.52 and 0.046, with the exception of Descemet’s membrane (P = 0.002) where very low levels of labeling were found.



View larger version (69K):
[in this window]
[in a new window]
 
Fig. 2. Transmission electron micrographs showing labeling (labeling appears as black dots) with ECA in human corneal stroma. (a) MCD I cornea showing low levels of labeling. (b) MCD I cornea labeled with ECA pre-incubated with N-acetyllactosamine as a control, showing negligible levels of labeling. (c) Normal cornea showing extremely low levels of labeling. Scale bar, 0.2 µm for all micrographs.

 


View larger version (74K):
[in this window]
[in a new window]
 
Fig. 3. Transmission electron micrograph showing labeling (labeling appears as black dots) with ECA in human corneal keratocytes. (a) MCD I cornea showing low levels of labeling in the keratocyte cytoplasm (k) and very high levels of labeling in the keratocyte deposits (*). (b) MCD I cornea labeled with ECA preincubated with N-acetyllactosamine as a control, showing negligible levels of labeling in both the keratocyte cytoplasm (k) and the keratocyte deposits (*). (c) Normal cornea showing negligible levels of labeling. Scale bar, 0.2 µm for all micrographs.

 


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 4. Transmission electron micrographs showing labeling (labeling appears as black dots) with ECA in human corneal Descemet’s membrane. (a) MCD I cornea showing moderate/high levels of labeling. (b) MCD I cornea labeled with ECA preincubated with N-acetyllactosamine as a control, showing negligible levels of labeling. (c) Normal cornea showing extremely low levels of labeling. Scale bar, 0.2 µm for all micrographs.

 


View larger version (77K):
[in this window]
[in a new window]
 
Fig. 5. Transmission electron micrograph showing labeling (labeling appears as black dots) with ECA in human corneal endothelium. (a) MCD I cornea showing very low levels of labeling in the endothelial cytoplasm (e) and very high levels of labeling in the endothelial deposits (*). (b) MCD I cornea labeled with ECA pre-incubated with N-acetyllactosamine as a control, showing very low levels of labeling in both the endothelial cytoplasm (e) and the endothelial deposits (*). (c) Normal cornea showing negligible levels of labeling. Scale bar, 0.2 µm for all micrographs.

 
Figure 6a and b shows the results of the quantitative study carried out on the labeling of normal cornea with 5-D-4 and ECA, and illustrates the relative distribution of sulfated and unsulfated KS across normal cornea.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 6. Graphs illustrating the levels of labeling found throughout the various regions of cornea. (a) normal cornea labeled with 5-D-4 (with control) (b) normal cornea labeled with ECA (with control) (c) MCD I cornea labeled with 5-D-4 (with control) (d) MCD I cornea labeled with ECA (with control). Corneal regions are abbreviated as follows: epithelium (Epi), Bowman’s layer (Bow), stroma (Str), stromal deposits (MCD I cornea only) (SD), keratocytes (Ker), keratocyte deposits (MCD I cornea only) (KD), Descemet’s membrane (Des), endothelium (End), and endothelial deposits (MCD I cornea only) (ED). Error bars indicate SE.

 
Macular cornea
The antibody 5-D-4 revealed extremely low levels of labeling throughout the cornea including the keratocytes, providing immunohistochemical evidence that the cornea is MCD I (Figure 1c). The nonspecific antibody control for this reaction similarly produced negligible levels of labeling throughout the entire cornea. The 5-D-4 labeling observed in all the regions except the stromal deposits was deemed not significant compared to the control: t-testing produced values between 0.012 and 0.56 for the different regions of the cornea, except the stromal deposits which had significant (P = 0.004) but very low levels of labeling (Figure 1c).

For ECA, a distinct pattern of labeling was observed throughout the cornea. The epithelium and Bowman’s layer gave low levels of labeling. The band of very large deposits found just under Bowman’s layer and the other stromal deposits, labeled heavily for ECA, but the stroma itself revealed a low concentration of labeling (Figure 2a). The cytoplasm of the keratocytes also showed low levels of labeling, but the inclusions found within the keratocytes (termed here as keratocyte deposits) labeled heavily for ECA (Figure 3a). The anterior region of Descemet’s membrane showed only low levels of labeling and was morphologically unchanged. The posterior region of Descemet’s membrane revealed moderate/high levels of labeling (Figure 4a) and exhibited the honeycombed morphology previously described (Quantock et al., 1997Go). The cytoplasm of the endothelial cells gave low levels of labeling, and the inclusions that are found within the endothelial cells (here termed endothelial deposits) labeled very heavily for ECA (Figure 5a). Although predigestion of the sections with endo-ß-galactosidase did not influence labeling with ECA, labeling was successfully reduced by the sugar preincubation control across the various regions of the cornea (Figures 2b–5b). In all regions where the labeling found was higher than that of the control, highly significant differences in the amounts of labeling (P < 0.001) were observed, the only exception being the distribution of labeling in the stroma which was not significant (P = 0.089) when compared to the control.

Inhibition-ELISA carried out on blood serum from the MCD patient produced negligible inhibition of the 5-D-4 antibody, while a serum from a normal age matched control produced a characteristic inhibition curve (Figure 7). This confirms that the MCD cornea is type I.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7. Inhibition-ELISA carried out on normal, age-matched, human blood serum (solid circles) shows that it produces inhibition of binding by 5-D-4, demonstrating the presence of KS. By contrast, blood serum from the MCD patient (solid squares) produces negligible inhibition, confirming the disease as MCD type I.

 
Figure 6c,d summarizes the quantitative study carried out on the labeling of MCD I cornea with 5-D-4 and ECA, and therefore illustrates the relative distribution of sulfated and unsulfated KS across MCD I cornea.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The results of this study provide the first quantitative histochemical comparison between sulfated and unsulfated KS in normal and MCD I age-matched cornea. The combined 5-D-4 immunohistochemistry and ELISA results confirms the tissue as MCD I cornea (immunohistochemistry is essential in order to distinguish type I from type IA). By using the lectin ECA to label N-acetyllactosamine, these results map the distribution of N-acetyllactosamine (unsulfated KS) in MCD I cornea, specifically, that it is localized to the characteristic deposits of this condition. This work also suggests that unsulfated KS is not present to any significant degree in normal cornea other than Descemet’s membrane where it is present only at very low levels.

It is important to note that although ECA has a high affinity for N-acetyllactosamine (Iglesias et al., 1982Go), lectins are not as selective as antibodies and it is possible that ECA is binding to other sugars for example galactose and N-acetylgalactosamine (De Boeck et al., 1984Go), for which it has a low affinity. However, there is good reason to believe that ECA is labeling the N-acetyllactosamine present in abnormal KS of MCD I. Macular corneal dystrophy type I is known to produce large amounts of unsulfated KS chains (Nakazawa et al., 1984Go; Midura et al., 1990Go), which is not found in normal cornea (Hassell et al., 1980Go). As the only known biochemical difference between normal cornea and MCD I cornea is the presence of abnormal unsulfated poly-N-acetyllactosamine chains on the KSPG (Nakazawa et al., 1984Go; Midura et al., 1990Go), this strongly suggests that the high levels of ECA labeling are due to binding to the abnormal N-acetyllactosamine in the MCD I corneal deposits. The removal of labeling on the control sections by pre-incubation with N-acetyllactosamine further supports the assumption that ECA is indeed labeling poly-N-acetyllactosamine chains.

It is also necessary to comment on the inability of the enzyme digestion control to remove labeling on the sections. Previous work has shown that enzyme digestion controls are effective in solution, for example, keratanase and endo-ß-galactosidase removing KS from the endothelial cell surface of corneal whole mounts (Davies et al., 1997Go). However, our experience has shown that keratanase and endo-ß-galactosidase are not effective when used on resin embedded sections. Consequently, the endo-ß-galactosidase digest controls are ineffective in digesting unsulfated KS in this case because the sample is embedded, unlike the case of Midura et al., 1990Go where the sample was in solution (Midura et al., 1990Go).

It is known that MCD I involves defective synthesis of KS, producing a KS that has a lack of sulfate groups on the poly-N-acetyllactosamine chains (Hassell et al., 1980Go; Nakazawa et al., 1984Go). It is thought that this abnormal unsulfated KS is localized in the deposits found throughout the stroma and endothelium of MCD I corneas, although it has proved difficult to test this idea. Characterization of these deposits is important since it is the accumulation of these deposits which causes light scattering and hence results in corneal opacity. This study has provided convincing evidence that the deposits are composed of unsulfated KS. The majority of ECA labeling (80.5%) was confined to the characteristic deposits found in the stroma, keratocytes and the endothelium (the labeling in Descemet’s membrane accounted for 13.1% of the sampled labeling, and all other regions collectively accounted for the remaining 6.4%). There is a clear distinction between labeling found within the deposits and that found within the surrounding medium, i.e., the stroma, keratocyte, cytoplasm, and endothelial cytoplasm. In all three cases this clear difference in labeling proved highly significant (P < 0.001) when subjected to t-test. This suggests that the characteristic deposits in MCD I do contain the unsulfated KS.

Previous work has shown that normal cornea contains stromal KSPGs that bind to the a and c bands of collagen fibrils, with CS/DSPGs binding to the d and e bands (Scott and Haigh, 1988Go). For MCD I cornea it has been found that CS/DSPGs are present within the stroma but normal KSPGs are absent and the unsulfated form is produced instead. Why is this unsulfated KS apparently localized within the characteristic deposits found within the cornea, and not with the collagen fibrils of the stroma? It would appear there is a problem with the export or the solubility of this molecule. It is likely that the absence of sulfate groups along the KS chains would dramatically decrease solubility causing the molecules to precipitate. Therefore in the cells that manufacture KS, i.e., the keratocytes and the endothelial cells, this unsulfated KS is possibly precipitating out, forming insoluble deposits in the cell’s cytoplasm or within organelles such as the endoplasmic reticulum (Klintworth and Vogel, 1964Go). The extracellular stromal deposits may be unsulfated KS that is exported from the cell as an insoluble deposit. Alternatively, after the cell dies the deposits that have accumulated within keratocytes may be resistant to breakdown and may be left to accumulate in the stroma.

Macular corneal dystrophy is also characterized by disruption to Descemet’s membrane (Quantock et al., 1997Go). Previous workers have reported labeling for KS in normal corneal endothelium and Descemet’s membrane and suggest that KS is synthesized by the endothelium and secreted into Descemet’s membrane (Fullwood et al., 1996Go; Davies et al., 1999Go). The results from this investigation of MCD I show no labeling for normal KS in these regions, but significant ECA labeling in Descemet’s membrane and the deposits within corneal endothelial cells. This supports the hypothesis that sulfated KS is produced by endothelial cells and plays an important structural role in Descemet’s membrane in normal cornea.

These results have provided the first histochemical study of poly-N-acetyllactosamine in MCD I cornea, revealing its occurrence within the characteristic deposits of this condition. This study has also shown that ECA can be used successfully as a probe for poly-N-acetyllactosamine, and for unsulfated KS.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Specimens
A cornea and a blood sample from a 43-year-old patient suffering from macular corneal dystrophy were obtained from the Manchester Royal Eye Hospital (Manchester, UK) during routine keratoplasty. This cornea was placed into fixative immediately after excision and the blood serum, obtained by centrifugation, was stored at –20°C. Blood serum was similarly obtained from a 43-year-old normal control subject. A normal human cornea was obtained from Manchester Eye Bank (Manchester, UK). The donor was a 52 year-old male with no history of ocular disease. Both normal and MCD I corneas were stored in 4% formaldehyde, 0.1% glutaraldehyde in phosphate buffered saline (PBS).

Transmission electron microscopic histochemistry
Histochemicals.
The biotinylated lectin Erythrina cristagalli agglutinin (ECA) was obtained from Sigma (Poole, UK). The monoclonal anti-keratan sulfate antibody 5-D-4 was obtained from ICN Biomedicals (Thame, UK). The respective secondary antibodies, goat anti-biotin and goat anti-mouse, were both 5nm gold conjugated and obtained from British Biocell International (Cardiff, UK).

Labeling process.
Specimens were embedded in Unicryl resin (British Biocell Ltd.) and the ultrathin sections that were taken from the samples underwent a two-step labeling process, as previously described (Fullwood et al., 1996Go). In this case, the 5-D-4 labeling was performed over a 2 h period at a 1:50 concentration, and was visualized by the anti-IgG secondary antibody. Sections to be labeled with ECA were first washed in droplets of 0.1M glycine in PBS buffer for 6 min and then in 0.1% bovine serum albumin (BSA) in PBS buffer for 2 min. Sections were subsequently incubated with the lectin in 0.1% BSA in PBS for 18 h at a concentration of 1:25. They were then washed for 8 min in buffer (0.1% BSA in PBS) before being incubated with the secondary anti-biotin IgG and continued through the process described previously (Fullwood et al., 1996Go). Incubations were carried out at room temperature in a standard grid box with the wells loaded with 15 µl of labeling solution. Sections were finally counterstained in aqueous uranyl acetate before being examined by a JEOL 100cx transmission electron microscope.

Controls.
For the ECA labeling the following controls were used. (1) Enzyme digestion of the N-acetyllactosamine chains on the MCD I sections that were carried out prior to the labeling procedure. Samples were digested in 1 U per milliliter of endo-ß-galactosidase (ICN Biomedicals Inc., USA) in 50 mM sodium acetate buffer at pH 5.8, for 24 h at 35°C. (2) Preincubation of lectin with 0.1M N-acetyllactosamine (ICN Biomedicals Inc., USA) for 20 min at room temperature, before addition to the sections. (3) Normal corneal sections showed negligible levels of labeling with ECA and thus acted as negative control tissue sections. For the 5-D-4 immunolabeling, the following controls were used. (1) The 5-D-4 antibody was replaced with a negative control mouse IgG (Serotec, Oxford, UK) at an equivalent dilution. (2) The MCD corneal sections revealed negligible levels of labeling for 5-D-4 and thus acted as negative control tissue sections.

Statistical analysis
For the quantification of the labeled samples, photographs were taken of the different regions of the cornea. For normal cornea these were the epithelium, Bowman’s layer, the stroma, keratocytes, Descemet’s membrane, and the endothelium. For the MCD I cornea, these included the above with micrographs of the deposits that are found in the stroma, within the keratocytes and within the endothelium. An area calculated to be 0.5 µm2 was randomly selected on a micrograph of a particular region of the cornea and the number of gold particles within that area was counted. For each region of the cornea, at least 18 different areas were counted. People independent to the project carried out counting. The data were then subjected to analysis by independent t-test using the statistical package SPSS 8.00 for Windows. To determine if labeling were significant the levels of labeling of 5-D-4 were compared to labeling with the control antibody. Levels of labeling of ECA were compared to levels of labeling with ECA after it had been incubated with its control sugar.

Inhibition enzyme-linked immunoabsorbent assay (ELISA) for KS detection
Inhibition ELISAs, using 5-D-4, were carried out on blood serum from the MCD I patient and an age-matched normal control subject to determine whether KS was present or not. The method described previously (Fullwood et al., 1996Go; Davies et al., 1997Go) was followed and the absorbance was read automatically at a wavelength of 450 nm by a Labsystem Multiskan RC.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Ruth Berry for technical assistance, Gavin Brown for helpful discussion and Steven Lewis for assistance with the statistical analysis. This project was supported by the Royal National Institute for the Blind.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BSA, bovine serum albumin; CS, chondroitin sulfate; DS, dermatan sulfate; ECA, Erythrina cristagalli agglutinin; ELISA, enzyme-linked immunosorbent assay; GAG, glycosaminoglycan; KS, keratan sulfate; MCD, macular corneal dystrophy; MCD I, macular corneal dystrophy type I; MCD IA, macular corneal dystrophy type IA; MCD II, macular corneal dystrophy type II; PBS, phosphate-buffered saline; PG, proteoglycan.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bairaktaris,G., Lewis,D., Fullwood,N.J., Nieduszynski,I.A., Marcyniuk,B., Quantock,A.J. and Ridgway,A.E.A. (1998) An ultrastructural investigation into proteoglycan distribution in human corneas. Cornea, 17, 396–402.[ISI][Medline]

Blochberger,T.C., Vergnes,J.P., Hempel,J. and Hassell,J.R. (1992) cDNA to chick lumican (corneal keratan sulfate proteoglycan) reveals homology to the small interstitial proteoglycan gene family and expression in muscle and intestine. J. Biol. Chem., 267, 347–352.[Abstract/Free Full Text]

Brandon,D.M., Nayak,S.K. and Binder,P.S. (1988) Lectin binding patterns of the human cornea. Cornea, 7, 257–266.[ISI][Medline]

Chakravarti,S., Magnuson,T., Lass,J.H., Jepson,K.J., LaMantia,C. and Carroll,H. (1998) Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican. J. Cell Biol., 141, 1277–1286.[Abstract/Free Full Text]

Corpuz,L.M., Funderburgh,J.L., Funderburgh,M.L., Bottomley,G.S., Prakash,S. and Conrad,G.W. (1996) Molecular cloning and tissue distribution of keratocan. Bovine corneal keratan sulfate proteoglycan 37A. J. Biol. Chem., 271, 9759–9763.[Abstract/Free Full Text]

Davies,Y., Fullwood,N.J., Marcyniuk,B., Bonshek,R., Tullo,A. and Nieduszynski,I.A. (1997) Keratan sulfate in the trabecular meshwork and cornea. Curr. Eye Res., 16, 677–686.[ISI][Medline]

Davies,Y., Lewis,D., Fullwood,N.J., Nieduszynski,I.A., Marcyniuk,B., Albon,J. and Tullo,A. (1999) Proteoglycans on normal and migrating human corneal endothelium. Exp. Eye Res., 68, 303–311.[ISI][Medline]

De Boeck,H., Loontiens,F.G., Lis,H. and Sharon,N. (1984) Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand. Arch. Biochem. Biophys., 234, 297–304.[ISI][Medline]

Fullwood,N.J., Davies,Y., Nieduszynski,I.A., Marcyniuk,B., Ridgway,A.E.A. and Quantock,A.J. (1996) Cell surface-associated keratan sulfate on normal and migrating corneal endothelium. Invest. Ophthalmol. Vis. Sci., 37, 1256–1270.[Abstract]

Funderburgh,J.L., Caterson,B. and Conrad,G.W. (1986) Keratan sulfate proteoglycan during embryonic development of the chicken cornea. Dev. Biol., 116, 267–277.[ISI][Medline]

Funderburgh,J.L., Corpuz,L.M., Roth,M.R., Funderburgh,M.L., Tasheva,E.S. and Conrad,G.W. (1997) Mimecan, the 25kDa corneal keratan sulfate proteoglycan, is a product of the gene producing osteoglycin. J. Biol. Chem., 272, 28089–28095.[Abstract/Free Full Text]

Hassell,J.R. and Klintworth,G.K. (1997) Serum sulfotransferase levels in patients with macular corneal dystrophy type I. Arch. Opthalmol., 115, 1419–1421.[Abstract]

Hassell,J.R., Newsome,D.A., Krachmer,J.H. and Rodrigues,M.M. (1980) Macular corneal dystrophy: failure to synthesise a mature keratan sulfate proteoglycan. Proc. Natl. Acad. Sci. USA, 77, 3705–3709.[Abstract]

Hassell,J.R., Newsome,D.A., Nakazawa,K., Rodrigues,M.M. and Krachmer,J.H. (1982) Defective conversion of a glycoprotein precursor to keratan sulfate proteoglycan in macular corneal dystrophy. In Hacks,S. and Wang,J. (eds.), Extracellular Matrix. Academic Press, New York, pp. 397–406.

Iglesias,J.L., Lis,H. and Sharon,N. (1982) Purification and properties of a D-galactose/N-acetyl-D-galactosamine-specific lectin from Erythrina cristagalli. Eur. J. Biochem., 123, 247–252.[ISI][Medline]

Klintworth,G.K. and Vogel,F.S. (1964) Macular corneal dystrophy: an inherited acid mucopolysaccharide storage disease of the corneal fibroblast. Am. J. Pathol., 45, 565–586.[ISI][Medline]

Klintworth,G.K. (1994) Disorders of glycosaminoglycans (mucopolysaccharides) and proteoglycans. In Garner,A. and Klintworth,G.K. (eds.), Pathobiology of Ocular Disease: A Dynamic Approach. Marcel Dekker, New York, Chapter 28, pp. 855–892.

Klintworth,G.K. and Smith,C.F. (1983) Abnormalities of proteoglycans synthesised by corneal organ cultures derived from patients with macular corneal dystrophy. Lab. Invest., 48, 603–612.[ISI][Medline]

Klintworth,G.K., Oshima,E., Al-Rajhi,A., Al-Salif,A., Thonar,E.J. and Karcioglu,Z.A. (1997) Macular corneal dystrophy in Saudi Arabia: A study of 56 cases and recognition of a new immunophenotype. Am. J. Ophthalmol., 124, 9–18.[ISI][Medline]

Liu,N.P., Baldwin,J., Lennon,F., Stajich,J.M., Thonar,E.J., Pericak-Vance,M.A., Klintworth,G.K. and Vance,J.M. (1998) Coexistence of macular corneal dystrophy types I and II in a single sibship. Br. J. Ophthalmol., 82, 241–244.[Abstract/Free Full Text]

Maurice,D.M. (1957) The structure and transparency of the cornea. J. Physiol., 136, 263–286.[ISI]

Mehmet,H., Scudder,P., Tang,P.W., Hounsell,E.F., Caterson,B. and Feizi,T. (1986) The antigenic determinants recognised by three monoclonal antibodies to keratan sulfate involve sulfated hepta- or larger oligosaccharides of the poly (N-acetyllactosamine) series. Eur. J. Biochem., 157, 385–391.[Abstract]

Midura,R.J., Hascall,V.C., MacCallum,D.K., Meyer,R.F., Thonar,E.J., Hassell,J.R., Smith,C.F. and Klintworth,G.K. (1990) Proteoglycan biosynthesis by human corneas from patients with types 1 and 2 macular corneal dystrophy. J. Biol. Chem., 265, 15947–15955.[Abstract/Free Full Text]

Nakazawa,K., Hassell,J.R., Hascall,V.C., Lohmander,S., Newsome,D.A. and Krachmer,J. (1984) Defective processing of keratan sulfate in macular corneal dystrophy. J. Biol. Chem., 259, 13751–13757.[Abstract/Free Full Text]

Nakazawa,K., Suzuki,S., Wada,K. and Nakazawa,K. (1995) Proteoglycan synthesis by corneal explants from developing embryonic chicken. J. Biochem., 117, 707–718.[Abstract]

Oeben,M., Keller,R., Stuhlsatz,H.W. and Greiling,H. (1987) Constant and variable domains of different disaccharide structure in corneal keratan sulfate chains. Biochem. J., 248, 85–93.[ISI][Medline]

Panjwani,N., Rodrigues,M.M., Alroy,J., Albert,D. and Baum,J. (1986) Alterations in stromal glycoconjugates in macular corneal dystrophy. Invest. Ophthalmol. Vis. Sci., 27, 1211–1216.[Abstract]

Quantock,A.J., Fullwood,N.J., Thonar,E.J., Waltman,S.R., Capel,M.S., Mitsutoshi,I., Verity,S.M. and Schanzlin,D.J. (1997) Macular corneal dystrophy type II: multiple studies on a cornea with low levels of sulfated keratan sulfate. Eye, 11, 57–67.[ISI][Medline]

Scott,J.E. and Haigh,M. (1988) Identification of specific binding sites for keratan sulfate proteoglycans and chondroitin-dermatan sulfate proteoglycans on collagen fibrils in cornea by the use of cupromeronic blue in ‘critical-electrolyte-concentration’ techniques. Biochem. J., 253, 607–610.[ISI][Medline]

Tai,G.-H., Huckerby,T.N. and Nieduszynski,I.A. (1996) Multiple non-reducing chain termini isolated from bovine corneal keratan sulfates. J. Biol. Chem., 271, 23535–23546.[Abstract/Free Full Text]

Tai,G.-H., Nieduszynski,I.A., Fullwood,N.J. and Huckerby,T.N. (1997) Human corneal keratan sulfates. J. Biol. Chem., 272, 28227–28231.[Abstract/Free Full Text]

Thonar,E.J., Meyer,R.F., Dennis,R.F., Lenz,M.E., Maldonado,B., Hassell,J.R., Hewitt,A.T., Stark,W.J., Stock,E.L., Kuettner,K.E. and Klintworth,G.K. (1986) Absence of normal keratan sulfate in the blood of patients with macular corneal dystrophy. Am. J. Ophthalmol., 102, 561–569.[ISI][Medline]

Yang,C.J., SunderRaj,N., Thonar,E.J. and Klintworth,G.K. (1988) Immunohistochemical evidence of heterogeneity in macular corneal dystrophy. Am. J. Ophthalmol., 106, 65–71.[ISI][Medline]