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.
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Abstract |
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Key words: antibody 5-D-4/cornea/electron microscopy/Erythrina cristagalli
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Introduction |
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Much work has centered around the characterization of the stromal KSPGs. Three KSPGs have been identified, lumican (Blochberger et al., 1992), keratocan (Corpuz et al., 1996
), and mimecan (Funderburgh et al., 1997
). Recent work involving lumican knockout mice (Chakravarti et al., 1998
) 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., 1986
; Nakazawa et al., 1995
) 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., 1987
; Tai et al., 1996
, 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
(23)-, NeuAc
(26)-, and GalNAcß(13)- residues (Tai et al., 1997
). 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, 1964). Clinically, a patient suffers ongoing corneal clouding and periodic photophobia from the age of 1012 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 Descemets membrane, causing corneal opacity (Klintworth, 1994
). 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 patients serum and cornea (Thonar et al., 1986
; Yang et al., 1988
). In MCD type I (MCD I), normal antigenic KS is absent from a patients cornea and blood serum (Yang et al., 1988
). In MCD type IA (MCD IA), an immunophenotype recently confirmed in the Saudi population (Klintworth et al., 1997
), 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., 1988
) but at levels which are lower than normal (Klintworth and Smith, 1983
; Midura et al., 1990
). 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., 1998
). 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., 1980). 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., 1982
). Thus, it is suggested that KSPGs are produced with the apparent defect being a failure to sulfate the N-acetyllactosamine chains (Nakazawa et al., 1984
). 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, 1997
). 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., 1984
). 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., 1986
). 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., 1986
; Brandon et al., 1988
). The lectin Erythrina cristagalli agglutinin (ECA) is known to bind to the N-acetyllactosamine disaccharide (Iglesias et al., 1982
) 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., 1990
) 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.
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Results |
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For ECA, a distinct pattern of labeling was observed throughout the cornea. The epithelium and Bowmans layer gave low levels of labeling. The band of very large deposits found just under Bowmans 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 Descemets membrane showed only low levels of labeling and was morphologically unchanged. The posterior region of Descemets membrane revealed moderate/high levels of labeling (Figure 4a) and exhibited the honeycombed morphology previously described (Quantock et al., 1997). 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 2b5b). 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.
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Discussion |
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It is important to note that although ECA has a high affinity for N-acetyllactosamine (Iglesias et al., 1982), 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., 1984
), 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., 1984
; Midura et al., 1990
), which is not found in normal cornea (Hassell et al., 1980
). 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., 1984
; Midura et al., 1990
), 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., 1997). 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., 1990
where the sample was in solution (Midura et al., 1990
).
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., 1980; Nakazawa et al., 1984
). 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 Descemets 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, 1988). 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 cells cytoplasm or within organelles such as the endoplasmic reticulum (Klintworth and Vogel, 1964
). 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 Descemets membrane (Quantock et al., 1997). Previous workers have reported labeling for KS in normal corneal endothelium and Descemets membrane and suggest that KS is synthesized by the endothelium and secreted into Descemets membrane (Fullwood et al., 1996
; Davies et al., 1999
). The results from this investigation of MCD I show no labeling for normal KS in these regions, but significant ECA labeling in Descemets 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 Descemets 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.
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Materials and methods |
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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., 1996). 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., 1996
). 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, Bowmans layer, the stroma, keratocytes, Descemets 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., 1996; Davies et al., 1997
) was followed and the absorbance was read automatically at a wavelength of 450 nm by a Labsystem Multiskan RC.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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