Abnormal Product of Corneal Explants From Patients ... - Europe PMC

Abnormal Product of Corneal Explants From Patients With Macular Corneal Dystrophy Gordon K. Klintworth, MD, PhD, and Clayton F. Smith, BS

The glycosaminoglycans synthesized by 5 comeal explants with macular corneal dystrophy were analyzed by sequential degradation using heparitinase (and/or butyl nitrite), chondroitin ABC lyase, chondroitin AC lyase, and keratan sulfate endo-f,-galactosidase. The tissue was first pulse-labeled by incubation in phosphate-buffered saline (pH 7.3) containing 100 ,uCi/ml 3H-glucosamine and approximately 1000 ,uCi/ml 'S-sulfate for 1 hour and chased with Eagle's minimum essential medium containing 10% fetal calf serum. Notable differences were detected between the products of these corneal explants with macular corneal dystrophy and controls that were prepared and analyzed in the same manner. As assayed by its susceptibility to keratan sulfate endo-f,-galactosidase degradation, corneal tissue with macular corneal dystrophy incorporated less 35S-sulfate, and usually less 3H-glucosamine, into keratan sufate than normal corneas. The comeal explants with macular corneal dystrophy also synthesized prominent fractions which eluted from DEAE-Sephacel columns in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 7.2) with 0.15 M and/or 0.25 M lithium chloride after pronase digestion. Since normal corneas produced small quantities of material with a similar elution profile after preparation and analysis under identical conditions it is possible that the fractions are normal synthetic products of the cornea. The significance of these findings in the pathogenesis of macular corneal dystrophy remains to be determined. (Am J Pathol 1980, 101:143-158)

FOR SEVERAL YEARS our laboratory has been investigating the pathogenesis of the inherited corneal disease macular corneal dystrophy, which is characterized by the accumulation of material with a marked affinity for histochemical stains for glycosaminoglycans within the fibroblasts and endothelial cells of the cornea.1-7 On the basis of morphologic and histochemical observations and the autosomal recessive mode of inheritance, we have postulated that the disease is a localized mucopolysaccharidosis restricted to the cornea and typified by the storage of corneal keratan sulfate (keratan sulfate type I). In view of this concept, we studied cultured corneal fibroblasts from patients with macular corneal dystrophy by methods yielding valuable data on systemic mucopolysaccharidoses.7 Unlike skin fibroblasts from individuals with systemic mucopolysaccharidoses, cultured corneal fibroblasts from individuals with macular corneal dystrophy do not accumulate 35S-sulfate and 3H-glucosamine-labeled From the Departments of Pathology and Ophthalmology, Duke University Medical Center, Durham, North Carolina. Supported in part by Research Grant IRO1-EY00146 from the National Eye Institute. Some of these observations were presented at the 1979 annual meetings of the Federation of the Societies of Experimental Biology (Dallas, Texas, April 1979) and the Association for Research in Vision and Ophthalmology (Sarasota, Florida, May 1979). Address reprint requests to Dr. Gordon K. Klintworth, Department of Pathology, Duke University Medical Center, Durham, NC 27710. 0002-9440/80/1008-0143$01 .00 143 © American Association of Pathologists

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glycosaminoglycans 4,6 or acridine orange particles.6 Nevertheless, these data remain consistent with the hypothesis that macular corneal dystrophy is a disorder of keratan sulfate catabolism, because the synthesis of keratan sulfate by corneal fibroblasts of man and other species diminishes markedly in culture.8'-2 Obviously cultured corneal fibroblasts that synthesize insufficient quantities of keratan sulfate will not accumulate this compound even if one or more critical degradative enzymes are absent. In view of the failure of cell culture techniques to shed light on the basic defect in macular corneal dystrophy, we compared the glycosaminoglycans synthesized by corneal explants from patients with this disease with those produced by normal corneas. This report describes notable differences between the products of corneal explants in macular corneal dystrophy and controls. Some of these observations have been presented in abstract form. 13,14 Materials and Methods Materials

Chondroitin ABC lyase (Proteus vulgaris), chondroitin AC lyase (Arthrobacter aurescens), endoglycosidase H, endoglycosidase D, mixed exoglycosidases, and disaccharide standards were purchased from Miles Laboratories, Elkhart, Indiana. Sephadex G-50 molecular sieve gels and DEAE-Sephacel were from Pharmacia Fine Chemicals Inc., Piscataway, New Jersey; tris(hydroxymethyl)aminomethane was from Sigma Chemical Company, St. Louis, Missouri; Aquasol-2 and 3S-sulfate were from New England Nuclear, Boston, Mass; Prorase was from Calbiochem-Behring Corp., La Jolla, Calif; Keratan sulfate endo-,8-galactosidase was a gift from Dr. K. Nakazawa, Department of Pharmaceutical Science, Meijo University, Showa, Nagoya 466, Japan, and heparitinase was a gift from Dr. A. Linker, Veterans Administration Hospital, Salt Lake City, Utah 84113, n-butyl nitrite was obtained from Kodak, Rochester, New York; 3H-glucosamine, 3H-fucose, '4Cmixed amino acids, and '4C-glucose were from Amersham, Arlington Heights, Illinois; lithium chloride was from Fisher Scientific Company, Fair Lawn, New Jersey; and Eagle's minimum essential medium was obtained from Grand Island Biological Company, Grand Island, New York. Source of Corneal Explants

Portions of corneal buttons removed from four patients with macular comeal dystrophy (MN family 3, NB family 14, MH family 85, and VC family 88) at the time of keratoplasty were pulse-labeled by incubation in phosphate buffered saline (pH7.3) containing approximately 100 microcuries per ml of 3H-glucosamine and approximately 1000 microcuries per ml of 35S sulfate for 1 hour and chased with Eagle's minimum essential medium containing 10% fetal calf serum, 10 mM HEPES buffer, and no antibiotics for 24 hours. The family numbers correspond to those in the registry of patients in the United States with macular corneal dystrophy maintained by the senior author. A second specimen from MN (family 3) was only labeled with 35S-sulfate. Control specimens were obtained from surgically enucleated eyes with normal corneas and from deceased persons within a few hours of death. Following pronase digestion the labeled material was passed through a Sephadex G-50 column and analyzed by a variety of techniques. These included an analysis of glycosaminoglycans by sequential degradation using heparitinase (and/or butyl nitrite),

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chondroitin ABC lyase, chondroitin AC lyase, and keratan sulfate endo-,B-galactosidase as described below. Samples were also eluted from DEAE-Sephacel with a linear gradient of lithium chloride. Analysis of Glycosaminoglycans by Sequential Enzymatic Degradation

Sequential degradation of the glycosaminoglycans by heparitinase (or butyl nitrite), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate endo-,l-galactosidase was performed by a modification of the method by Hart.'5 After incubation in radioactive medium specimens were frozen at -20 C until analyzed. For analysis specimens were thawed, heat-denatured in a boiling water bath for 10 minutes, incubated with pronase (1 mg/ml) for 24-48 hours in tris(hydroxymethyl)aminomethane buffer (pH 8) at 37 C, and treated with cold 5% trichloroacetic acid to precipitate proteins. The supernatant was neutralized with tris(hydroxymethyl)aminomethane and chromatographed on a Sephadex G-50 fine column (1.6 x 35 cm) to remove unincorporated isotope. The void volume, containing the high molecular weight glycosaminoglycans, was collected and lyophilized. This material was then incubated with 20 ,Ag of heparitinase in 0.1 M sodium acetate buffer (pH 7.0) at 40 C for 6 hours or treated with nitrous acid to selectively degrade N-sulfated glycosaminoglycans, like heparan sulfate and heparin.'1'7 Portions of labeled glycosaminoglycans in 2 ml of distilled water were mixed with 1 ml of 1 N hydrochloric acid and 1 ml of freshly prepared 20%o n-butyl nitrite (vol/vol) in absolute ethanol and allowed to react in an open vessel with gentle shaking for 2 hours at room temperature. The reaction was stopped by neutralizing with 1 ml of 1 N sodium hydroxide. This method completely degrades heparin and heparan sulfates by a single treatment.'8 Following treatment with heparitinase or nitrous acid, the specimens were dried under nitrogen, dissolved in distilled water, and passed through a 1.6 x 35-cm Sephadex G-50 column in 0.05M tris(hydroxymethyl) aminomethane buffer (pH 7.5). One-milliliter fractions were collected. As controls, samples of radioactive labeled material that had not undergone this treatment were similarly chromatographed. The percentage of degradation was determined by counting aliquots of the column fractions with Aquasol-2 in a Beckman LS-250 scintillation counter and calculating the percentage of shift from the void volume. The material not affected by heparitinase or nitrous acid was pooled, lyophilized, and redissolved in 3 ml of 0.01 M tris(hydroxymethyl)aminomethane buffer (pH 8.0) and was digested with chondroitin ABC lyase (EC 4.2.2.4, 0.133 U/ml) for 18 hours at 37 C. This material was rechromatographed on the same Sephadex column. The percentage of degradation was again determined by counting aliquots of column fractions, Material excluded from Sephadex G-50 after treatment with chondroitin ABC lyase was pooled and lyophilized. Specimens were dissolved in 1.0 ml 0.05 M tris(hydroxymethyl)aminomethane (pH 7.2), and the susceptibility of this material to degradation by keratan sulfate endo-,B-galactosidase was determined by adding 1.5 units of this enzyme in 0.01 ml of the same buffer and incubating it for 48 hours at 37 C. Fresh enzyme was added after 24 hours. This material was again chromatographed on the same Sephadex G-50 column, and the percentage of degradation was determined as before. Aliquots of solutions containing nondegraded labeled glycosaminoglycans from the first Sephadex G-50 column after pronase digestion were concentrated by lyophilization and analyzed by descending chromatography on Whatman 3MM or No. 1 paper in a solvent system of 1-butanol-acetic acid-1 N ammonia (2:3:1, vol/vol/vol) according to the method of Saito et al."' Specimens were chromatographed with and without prior treatment with chondroitin ABC lyase (EC 4.2.2.4) or chondroitin AC lyase (EC 4.2.2.5). Tento fifty-microliter samples were incubated in 0.1 unit of chondroitin ABC lyase, or 0.3 units of chondroitin AC lyase, for 18 hours at 37 C. The sulfated and nonsulfated disaccharides were readily separated in this system. Standard A-di-4S (2-acetamido-2deoxy-3-0-[D-gluco-4-enepyranosyluronic acid]-4-0 sulfo-D-galactose), A-di-65 (2-acetamido-2-deoxy-3-0-[D-gluco-4-enepyranosyluronic acid]-6-0-sulfo-D-galactose), and A-di-

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OS (2-acetamido-2-deoxy-3-O[D-gluco-4-enepyranosyluronic acid]-D-galactose) were chromatographed along with the labeled samples. The locations of the standards were observed under ultraviolet light and after spraying the chromatograms with aniline hydrogen phthalate reagent and heating at 110 C.' The positions of the labeled disaccharides derived from the corneal explants and the nutrient media were established by cutting the strips into segments and measuring the radioactivity of each strip using liquid scintillation counting. The position of the spots was also elicited in some cases by prior autoradiography of the chromatograms after keeping them in the dark in direct contact with Ultrofilm 3H (LKB, Rockville, Md). Determinations of Glycosaminoglycan Percentages

The percentages of each type of glycosaminoglycan were determined as follows: By Sephadex G-50 chromatography after each degradation procedure, the percentages degradable by heparitinase (or butyl-nitrite), chondroitin ABC lyase, and keratan sulfate endo-,B-galactosidase were determined to give four classes to total 100%: heparan sulfate, chondroitin ABC lyase degradable, keratan sulfate, and unidentified (nondegradable). The chondroitin ABC lyase degradable material was subdivided into: dermatan-4-sulfate, dermatan-6-sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, hyaluronic acid, chondroitin, and unidentified fractions based on the characteristics of this material on descending paper chromatography after chondroitin ABC lyase and chondroitin AC lyase digestion. The formulas used to determine the percentages of these compounds are based on certain assumptions about the substrates of these enzymes: 1) chondroitin ABC lyase completely degrades dermatan-4-sulfate, dermatan-6-sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, hyaluronic acid, chondroitin and no other substrate; 2) chondroitin AC lyase does not degrade dermatan-4-sulfate and dermatan-6-sulfate but completely degrades all of the other substrates of chondroitin ABC lyase and no other compounds. Ion Exchange Chromatography on DEAE-Sephacel

After being passed through a Sephadex G-50 column the high-molecular weight pronase-digested material was applied to a DEAE Sephacel column (1.6 X 4 cm) in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 7.2) and eluted with a linear gradient of lithium chloride from zero to 1.0 M. Fractions of 2.9 ml were collected. An equal volume of Aquasol-2 was added to each fraction for counting in a Beckman LS-250 scintillation counter. Analyses on DEAE-Sephacel Peaks In order to learn more about the nature of the material that was produced by the cor-

neal explants with macular corneal dystrophy and that seemed to be synthesized by the normal cornea (see Results), we incubated aliquots of fraction II produced by the corneal explant from MH (family 85) with butyl nitrite and a variety of enzymes, including heparitinase, chondroitin ABC lyase, and keratan sulfate endo-f,-galactosidase, endoglycosidase D, endoglycosidase H, and mixed exoglycosidases isolated from the marine gastropod Charonia lampus (a-mannosidase, f8-mannosidase, a-glucosidaes, fl-glucosidase, a-galactosidase, fi-galactosidase, a-L-fucosidase, a-D-fucosidase, f8-D-fucosidase, f?-xylosidase, aN-acetylglucosaminidase, ,B-N-acetylglucosaminidase, a-N-acetylgalactosaminidase, fl-Nacetylgalactosaminidase, ,B-L-arabinosidase, fl-D-arabinosidase, sialidase). To learn more about these fractions which were produced by the cornea, we incubated normal corneas with 3H-fucose and "'C-labeled mixed amino acids or 3H-fucose and "GC-labeled glucose and analyzed them by ion exchange chromatography on DEAE Sephacel as described above.

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Results Glycosaminoglycan Synthesis by Comeal Explants

As assayed by its susceptibility to keratan sulfate endo-,B-galactosidase degradation, all 5 corneal explants with macular corneal dystrophy incorporated less 35S-sulfate, and usually less 3H-glucosamine, into keratan sulfate than normal (Table 1). Chromatography

on

DEAE-Sephacel

An unexpected observation was made when the pronase-resistant macromolecules synthesized by corneal explants were eluted from DEAESephacel with a linear gradient of lithium chloride (Text-figures 1-4). Normal corneas synthesized a considerable amount of 3S-sulfate and 3Hglucosamine-labeled material that eluted over a wide range of lithium chloride concentrations. The 4 corneal specimens with macular corneal dystrophy analyzed in this way differed markedly from the control specimens and from each other. The elution profile from Case 1 (MN, a 54year-old individual) had a peak elution at a lithium chloride concentration of 0.15 M (Fraction I) and a small later peak at about 0.25 M lithium chloride (Fraction II) (Text-figure 1). The cornea of Case 2 (MH, a 34-year-old patient) synthesized a prominent amount of 3H-glucosamine-labeled Fraction II (Text-figure 2). The corneas of Case 3 (NB, a 41-year-old patient) Table 1 -Percentages of Keratan Sulfate Synthesized by Corneal Explants Labeled keratan sulfate

35S-Sulfate 3H-Glucosamine Macular corneal dystrophy MH (34 years) 0.0 (0.3)% 3.5 (3.5%) NB (41 years) 0.3(1.5)% 7.7(11.4)% MN (52 years) 8.0% MN (54 years) 11.6 (21.5)% 36.6 (45.1)% VC (69 years) 2.8 (1.9%) 8.2 (8.7)% Controls Enucleated eyes JE(1 year) 41.5% AD (2 years) 41.4% 19.4% WP (2 years) 33.3 (34.6)% 14.7 (16.3)% LB (54 years) 36.7% 19.3% Postmortem eyes DB (18 years) 54.9 (63.5%) 21.9 (24.2)% MB (60 years) 3.7 (4.0)% 7.4 (7.7)% * Percentages given are based on sequential degradation, including the use of butyl nitrite. Figures in parentheses are based on the use of heparitinase instead of butyl nitrite.



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-j

FRACTION NUMBER TEXT-FIGURE 1-Profile of pronase-resistant macromolecules synthesized by a corneal explant from a case of macular comeal dystrophy (MN) as eluted from DEAE-Sephacel with a linear gradient of lithium chloride. In contrast to control specimens, this specimen contains an excessive amount of material that elutes at a lithium chloride concentration of 0.15 M (Fraction I) and some at 0.25 M (Fraction

II).


0L

c

u

5 10

20

30

40

50

60

70

80

90

FRACTION NUMBER TEXT-FIGURE 2-Profile of pronase-resistant macromolecules synthesized by a corneal explant from a case of macular comeal dystrophy (MH) as eluted from DEAESephacel with a linear gradient of lithium chloride. In contrast to control specimens, this specimen contains excessive amounts of material that elutes at a lithium chloride concentration of 0.25 M (Fraction II).



MACULAR CORNEAL DYSTROPHY 600

500

u

0

400

!8

300

.6 U

200

.4

100

.2

1-

5 10

20

30 40 50 60 70 80 90 100 FRACTION NUMBER TEXT-FIGURE 3-Profile of pronase-resistant macromolecules synthesized by a corneal explant from a case of macular corneal dystrophy (VC) as eluted from DEAE-Sephacel with a linear gradient of lithium chloride. In contrast to control specimens, this specimen contains excessive amounts of material that elutes at lithium chloride concentrations of 0.15 M (Fraction I) and 0.25 M (Fraction II).


E

u

FRACTION NUMBER

TEXT-FiGURE 4-Profile of pronase-resistant macromolecules synthesized by corneal explants from a case of macular corneal dystrophy (NB) as eluted from DEAE-Sephacel with a linear gradient of lithium chloride. In contrast to control specimens, this specimen contains excessive amounts of material that elutes at lithium chloride concentrations of 0.15 M (Fraction I) and 0.25 M (Fraction II).

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150



incorporated 3H-glucosamine into Fractions I and II (Text-figures 3 and

4).

Having made these observations, we studied additional control specimens. A small quantity of material synthesized by normal corneas eluted from DEAE-Sephacel in about the same position as the aforementioned Fractions I and II. Although Fractions I and II were present in normal corneas, the profile varied in different cases (Text-figures 5-7). At a salt concentration of 0.1-0.2 M lithium chloride (Fraction I) normal corneas eluted material that was readily labeled by prior incubation in radioactive fucose, glucose, glucosamine, or amino acids (Text-figures 8 and 9). The mixed exoglycosidases degraded about 9% of an isolated sample of Fraction II from MH, while heparitinase, nitrous acid, chondroitin ABC lyase, endoglycosidase D, endoglycosidase H, mixed exoglycosidases, and keratan sulfate endo-,B-galactosidase did not degrade more than 1-4% of the material. Fraction I from MN was partially susceptible to chondroitin ABC lyase, suggesting that a portion of it contains /1hexaminidic bonds as in other substrates of this enzyme. Discussion

The corneal tissue in macular corneal dystrophy contains accumulations within the corneal fibroblasts (keratocytes) and frequently also in the corneal endothelium." 2'5 The accumulations stain positively with periodic NORMAL CORNEA

0 .8

~.6

UE

__j

.4

.2

FRACTION NUMBER

TEXT-FIGURE 5-DEAE-Sephacel chromatograph. Normal cornea from an enucleated eye in a 41-year-old man. The position of elution of unincorporated 3Hglucosamine and 3SO4= is also shown.



NORMAL CORNEA

ULCL

u

FRACTION NUMBER TEXT-FIGURF 6-DEAE-Sephacel chromatograph. Normal cornea from an enucleated eye with a retinoblastoma in a 20-month-old child.

NORMAL CORNEA

FRACTION NUMBER TEXT-FiGURE 7-DEAE-Sephacel chromatograph. Normal cornea from an 18-year-old woman obtained 3½ hours after death caused by an automobile accident.

151

152


14C

3H 9000


NORMAL CORNEA

8000 7000

I

6000 ,- 5000

u

4000 3000

2000 1000

L FRACTION NUMBER TEXT-FIGURE 8-DEAE-Sephacel chromatograph. 3H-glucosamine and '4C-mixedamino-acid-labeled material from the normal cornea of a 34-year-old man obtained from an enucleated eye with a massive retinal detachment.

NORMAL CORNEA

u-

u

-

FRACTION NUMBER TEXT-FIGURE 9-DEAE-Sephacel chromatograph. Elution profile of 3H-fucoseand '4C-glucose-labeled material from the normal cornea of an 18-month-old infant.



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acid-Schiff, alcian blue, and metachromatic dyes and possess an affinity for colloidal iron.' Lakes of similar material also occur extracellularly between the collagen fibers of the corneal stroma and within Descemet's membrane.5 By transmission electron microscopy delicate fibrillogranular material is discernible within intracytoplasmic vacuoles of the corneal fibroblasts. These accumulations stain with the periodic acid-Schiff-thiocarbohydrazide-silver proteinate and periodic acid-silver methenamine techniques.5 In contrast to the systemic mucopolysaccharidoses abnormal material also deposits between the collagen fibers in the corneal stroma and within Descemet's membrane. More than a decade ago Klintworth and Vogel 1 proposed that macular corneal dystrophy is a metabolic storage disease restricted to the cornea and characterized by an intra- and extracellular accumulation of excessive quantities of glycosaminoglycans (mucopolysaccharides). This hypothesis is consistent with the disease's autosomal recessive mode of inheritance, as well as with its histochemical and morphologic characteristics. The concept is also compatible with the known propensity of the stromal and endothelial cells of the cornea to synthesize glycosaminoglycans.8'2' Analogous aspects of macular corneal dystrophy and the systemic mucopolysaccharidoses leads one to suspect an inherited enzymatic defect in the degradation of corneal glycosaminoglycans. The cornea synthesizes several glycosaminoglycans under normal or pathologic conditions: keratan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan-4-sulfate, dermatan-6-sulfate, low-sulfated glycosaminoglycans (chondroitin), and hyaluronic acid; but some of these polysaccharides are unlikely to be major storage substances in macular corneal dystrophy because they are susceptible to digestion by enzymes that fail to degrade the naturally occurring accumulations of macular corneal dystrophy." 4 5 By exclusion corneal keratan sulfate, the major corneal sulfated glycosaminoglycan, is the most likely storage substance; and the affinity of the accumulations for alcian blue at low pH with magnesium chloride concentrations with a molarity of up to 0.8 supports this hypothesis.4'22 A storage disorder of corneal keratan sulfate also provides an explanation for the restriction of the clinical disease to the cornea and for the apparent lack of related abnormalities in tissues other than the cornea. Although keratan sulfate is a constituent of both the cornea and cartilage, the structure of the keratan sulfate in these tissues differs in several respects.4 Corneal keratan sulfate (keratan sulfate Type I) is linked by alkali-stable N-glycoside bonds to asparagine residues in the protein core, while the carbohydrate-peptide bond of cartilaginous keratan sulfate (keratan sulfate Type II) involves N-acetylgalactosamine linked in serine

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and threonine residues by an 0-glycosidic, alkali-labile bond. Corneal and cartilaginous keratan sulfate also differ with regard to the average length of the polymer chains, the degree of branching, their susceptibility to keratan sulfate endo-,B-galactosidase degradation, and the relative amounts of their amino acids, glucosamine, galactosamine, and sialic acid.4 Corneal keratan sulfate is bound covalently to a protein as a constituent of one of the corneal proteoglycans (keratan sulfate proteoglycan),23'24 but protein does not seem to be a significant component of the stored material in macular corneal dystrophy, because the storage material does not react positively with histochemical reactions for proteins. This investigation indicates that explants of corneal tissue with macular corneal dystrophy incorporate less 35S-sulfate and usually less 3H-glucosamine into corneal keratan sulfate than control specimens and that they synthesize excessive amounts of one or two fractions, which elute from DEAE-Sephacel in characteristic positions. Although the small amounts of these fractions precluded their characterization, normal corneas produce small quantities of material that elutes in a similar position on ion exchange chromatography after preparation and analysis under identical conditions. These pronase-resistant products of normal synthesis contain incorporated glucose, glucosamine, fucose, and amino acids and are probably oligosaccharides or glycopeptides. The observation that the corneal explants with macular corneal dystrophy incorporated less 3S-sulfate, and usually less 3H-glucosamine into keratan sulfate than controls is surprising in view of the original premise that macular corneal dystrophy is a storage disease of corneal keratan sulfate. Especially because other studies employing tissue culture methodology have failed to demonstrate a storage disease of corneal fibroblasts,6'7 one is faced with possibility that the original hypothesis may be wrong. That all 4 corneal explants with macular corneal dystrophy that were chromatographed on DEAE-Sephacel after pronase digestion revealed excessive amounts of unusual fractions raises the possibility that macular corneal dystrophy is a storage disease of some carbohydrate-containing macromolecule other than keratan sulfate and that these abnormal products of the pathologic corneal tissue are related to the naturally occurring storage product. Be this as it may, the present observations, as well as those in previously reported cell culture studies,6'7 remain consistent with the original hypothesis. The diminished incorporation of 3S-sulfate and 3H-glucosamine into keratan sulfate by the corneal explants with macular corneal dystrophy and the unusual products of the corneal explants with macular corneal dystrophy may reflect the abnormal status of the fibroblasts in severely pathologic tissue and not mirror the fundamental meta-



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bolic defect. The investigated corneal tissue in all cases of macular corneal dystrophy was obtained late in the course of the disease, when excessive amounts of the abnormal intra- and extracellular accumulations caused sufficient visual impairment to necessitate a corneal graft. As a result, the characteristic accumulations may have influenced the synthetic activity of the corneal fibroblasts. If macular corneal dystrophy is indeed a disorder of corneal keratan sulfate catabolism, it may be that keratan sulfate synthesis by the corneal explants was impaired because of some inhibitory feedback mechanism caused by excessive amounts of keratan sulfate within the pathologic corneal fibroblasts. Perhaps under such circumstances the cells produce excessive amounts of different metabolic products such as glycoproteins or glycopeptides. However, if the impaired synthesis of keratan sulfate by corneal tissue with macular corneal dystrophy results from such a mechanism, one would expect it to operative in vivo; and if so, the disease should progress to a certain point and then reach a steady state. But if this is the case, it does not seem to occur until the entire cornea becomes opaque. Future studies on corneal tissue from normal individuals and from persons with macular corneal dystrophy will, one hopes, permit the relationship between these abnormal products of corneal explants and the innate storage product of corneal fibroblasts in macular corneal dystrophy to be resolved. It is unfortunately not possible to extrapolate from knowledge about factors modulating the biosynthesis of normal corneal glycosaminoglycans and proteoglycans, because such information is virtually nonexistent, especially from the standpoint of corneal keratan sulfate (keratan sulfate I).810162125-27 The synthetic rate of corneal glycosaminoglycans is influenced by the corneal epithelium 8,12,28 and the incorporation of S04= into sulfated corneal glycosaminoglycans is dependent upon Na+ K+-activated ATPase.28 UDP-xylose decreases the synthetic rate of chondroitin and chondroitin sulfate by the cornea but increases the production of keratan sulfate by corneal explants, possibly because of an inhibition of UDP-glucose dehydrogenase by UDP-xylose. Keratan sulfate I is only synthesized in the cornea and as shown in the present study and by others,8,0,',' corneal explants produce it. For reasons that remain unknown this ability is markedly impaired under cultural conditions.8'-2 The enzyme galactosyl transferase, which catalyzes the transfer of galactose from UDP-galactose to N-acetyl-D-glucosamine moieties in the elongating keratan sulfate molecule, is present in cultured corneal fibroblasts, albeit in low concentrations, and the impaired keratan sulfate synthesis is probably not entirely attributable to a loss of this enzyme.27 As shown by MB in the present study, some human corneas obtained within a few hours of death also

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synthesize less keratan sulfate than normally, despite the fact that they are still capable of incorporating considerable amounts of 3S-sulfate and 3Hglucosamine in other glycosaminoglycans. Although the precise structure of corneal keratan sulfate proteoglycan remains to be determined, it appears to be composed of monomers consisting of about 45% protein, 30% keratan sulfate, and 10-12% oligosaccharides and possessing a molecular weight of about 40,000-72,000.3' Puromycin inhibits the incorporation of several labeled moieties into the corneal proteoglycans, and this inhibition affects keratan sulfate proteoglycan less than galactosaminoglycan proteoglycan, suggesting that the protein cores of the proteoglycans are synthesized separately.3 References 1. Klintworth GK, Vogel FS: Macular corneal dystrophy: An inherited acid mucopolysaccharide storage disease of the comeal fibroblast. Am J Pathol 1964, 45:565586 2. Klintworth GK: Current concepts on the ultrastructural pathogenesis of macular and lattice corneal dystrophies. Proceedings of the Second Conference on the Clinical Delineation of Birth Defects. Part 8. Eye. Edited by D Bergsma, VA McKusick, IE Hussels, HE Cross, C Jackson, MW Lorber. Baltimore, Williams & Wilkins, 1971, pp 27-31 3. Klintworth GK: Tissue culture in the inherited corneal dystrophies: Possible applications and problems, The Eye and Inborn Errors of Metabolism (Birth DefectsOriginal Article Series, Vol. 12, No. 3), Edited by D Bergsma, AJ Bron, E Cotlier. New York, Alan R. Liss, 1976, pp 115-132 4. Klintworth GK: The cornea: Structure and macromolecules in health and disease: A review. Am J Pathol 1977, 89:719-808 5. Klintworth GK: Comeal dystrophies, Ophthalmic Pathology Update. Edited by D Nicholson. New York, Masson Publishing, 1980, pp 23-54 6. Klintworth GK, Hawkins HK, Smith CF: Acridine orange particles in cultured fibroblasts: A comparative study of macular corneal dystrophy, systemic mucopolysaccharidoses types I-H and II and normal controls. Arch Pathol Lab Med 1979, 103:297-299 7. Klintworth GK, Smith CF: Macular comeal dystrophy: Studies of sulfated glycosaminoglycans in corneal explant and confluent stromal cell cultures. Am J Pathol 1977, 89:167-182 8. Klintworth GK, Smith CF: A comparative study of extracellular sulfated glycosaminoglycans synthesized by rabbit corneal fibroblasts in organ and confluent cultures. Lab Invest 1976, 35:258-263 9. Gnadinger MC, Schwager-Hubner ME: Biosynthesis of glycosaminoglycans by mammalian comeal epithelium and fibroblasts in vitro II: Approach to specify the GAG from the two cell types. Albrecht von Graefes Arch Klin Exp Ophthalmol 1975, 196:21-30 10. Conrad GW, Dorfman A: Synthesis of sulfated mucopolysaccharides by chick corneal fibroblasts in vitro. Exp Eye Res 1974, 18:421-433 11. Yue BYJT, Baum JL, Silbert JE: The synthesis of glycosaminoglycans by cultures of rabbit comeal endothelial and stromal cells. Biochem J 1976, 158:567-573 12. Bleckmann H, Kresse H: Beeinflussung der Glycosaminoglykansynthese von kultivierten Stromazellen aus Rindercorneae durch Variation der Kulturbedingungen. Albrecht von Graefes Arch Klin Exp Ophthalmol 1979, 210:291-300



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13. Klintworth GK, Smith CF: Abnormal metabolic product synthesized by corneal explants in macular corneal dystrophy. Fed Proc 1979, 38:1452 14. Klintworth GK, Smith CF: Probable glycopeptide abnormality in corneal explants from patients with macular corneal dystrophy. Invest Ophthalmol Vis Sci 1979, 18 (suppl):36 15. Hart GW: Biosynthesis of glycosaminoglycans during corneal development. J Biol Chem 1976, 251:6513-6521 16. Cifonelli JA, King J: The distribution of 2-acetamido-2-deoxy-D-glucose residues in mammalian heparins. Carbohydr Res 1972, 21:173-186 17. Cifonelli JA, King J: Structural studies on heparins with unusually high N-acetylglucosamine contents. Biochim Biophys Acta 1973, 320:331-340 18. Conrad GW, Hart GW: Heparan sulfate biosynthesis by embryonic tissues and primary fibroblast populations. Dev Biol 1975, 44:253-269 19. Saito H, Yamagata T, Suzuki S: Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J Biol Chem 1968, 243:1536-1542 20. Partridge SM: Aniline hydrogen phthalate as a spraying reagent for chromatography of sugars. Nature 1949, 164:443 21. Yue BYJT, Baum JL, Silbert JE: Synthesis of glycosaminoglycans by cultures of normal human corneal endothelial and stromal cells. Invest Ophthal Vis Sci 1978, 17:523-527 22. Garner A: Histochemistry of corneal macular dystrophy. Invest Ophthal 1969, 8:475-483 23. Axelsson I, Heinegard D: Characterization of the keratan sulphate proteoglycans from bovine corneal stroma. Biochem J 1978, 169:517-530 24. Hassell JR, Newsome DA, Hascall VC: Characterization and biosynthesis of proteoglycans of corneal stroma from rhesus monkey. J Biol Chem 1979, 254:1234612354 25. Bleckmann, H. and Wollensak, J.: Hemmung des Hornhaustoffwechsels durch Glukocorticosteroide: Eine in vitro Studie. Albrecht Von Graefes Arch. Klin Expl Opthalmol 1975, 193:57-65 26. Handley CJ, Phelps CF: The biosynthesis in vitro of keratan sulphate in bovine cornea. Biochem J 1972, 128:205-213 27. Christner JE, Distler JJ, Jourdian GW: Biosynthesis of keratan sulfate: Purification and properties of a galactosyltransferase from bovine cornea. Arch Biochem Biophys 1979, 192:548-558 28. Wortman B: Metabolism of sulfate by beef and rabbit cornea. Am J Physiol 1960, 198:779-783 29. Balduini C, Brovelli A, Castellani AA: Biosynthesis of glycosaminoglycans in bovine cornea. Biochem J 1970, 120:719-723 30. Hart GW: Glycosaminoglycan sulfotransferases of the developing chick cornea. J. Biol Chem 1978, 253:347-353 31. Greiling H, Stuhlsatz HW, Kisters R: Structure and metabolism of proteokeratan sulfate, Chemistry and Molecular Biology of the Intercellular Matrix. Vol 2. Edited by EA Balazs. London, Academic Press, 1970, pp 873-878

Acknowledgment Keratan sulfate endo-,8-galactosidase and heparitinase were gifts from Dr. K. Nakazawa and Dr. A. Linker, respectively.

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