into calf and beef corneal glycosaminoglycans Claes H. Dohlman, Bernard Wortman,* and Siw Hultman

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1 Incorporation of sulfate- 35 S, N-acetylglucosamine-l- 14 C, glucose-l- 14 C, and galactose-l- 14 C into calf and beef corneal glycosaminoglycans Claes H. Dohlman, Bernard Wortman,* and Siw Hultman Beef corneas were incubated in vitro with N-acetylglucosamine-l-'^C, glucose-l- lj >C, and galactose-l-' J 'C. Calf and beef corneas were incubated in vitro with inorganic sulfate- sr 'S. Corneal glycosaminoglycans were isolated and the mixture resolved by use of ECTEOLA anionexchanger and further fractionation with ethanol. After incubation ivith glucose-l-'tc, a higher specific activity was found in keratan sulfate than in chondroitin-chondroitin sulfate, whereas the opposite toas found after incubation with galactose-l-'*c and sulfate- S5 S. Incubation of corneas with N-acetylglucosamine-l- Ji C resulted in approximately the same specific activity in keratan sulfate and chondroitin-chondroitin sulfate. The results are discussed in terms of the normal biosynthesis of corneal glycosaminoglycans. G,lycosaminoglycans in cornea have been isolated and identified as keratan sulfate, chondroitin, and chondroitin sulfate. 1 The development of methods for the isolation of small amounts of comeal glycosamino- From the Corneal Research Unit, Institute of Biological and Medical Sciences, Retina Foundation, Boston, Mass., and Department of Ophthalmology, Washington UnivWsity School of Medicine, St. Louis, Mo. This investigation was supported in part by Public Health Service Research Grants NB and NB from the National Institute of Neurological Diseases and Blindness, United States Public Health Service, by the Massachusetts Lions Eye Research Fund, and by the St. Louis Heart Association. "This work was done during tenure as an Established Investigator of the American Heart Association. Present address: Department of Pathology, Albany Medical College, Albany, N. Y. 867 glycans by use of the anion-exchanger Ecteola combined with ethanol fractionation on cellulose columns have enabled the extension of studies of cornea. 2-3 These methods, as well as the incorporation of inorganic sulfate- 35 S, have been used to study the cornea in normal and pathological conditions, e.g., wound healing. 4 ' G In the normal cornea, most of the incorporated sulfate- 35 S can be recovered as ester sulfate and is presumed to be in the glycosaminoglycans. However, a small amount of the radioactive label is found to be in a reduced form in methionine and cystine. 7 There is further evidence that cornea can reduce sulfate and incorporate it into an organic compound. s The previous attempt to isolate and identify sulfate- 35 S-labeled glycosaminoglycans disclosed that beef and rabbit corneas incorporated sulfate- 35 S into a glycosaminoglycan which had the same elec-

2 868 Dohlman, Wortman, and Hultman Investigative Ophthalmology October 1965 trophoretic mobility as authentic keratan sulfate. No sulfate- a5 S-labeled chondroitin sulfate could be demonstrated readily by the methods used in that study. 9 Glucose-"C has been found to be incorporated into the hexosamines of corneal glycosaminoglycans; glucosamine had a higher specific activity than did galactosamine. 10 Cornea contains UDPglcA and UDPglc as well as other niicleotides and has the capacity to synthesize glucosamine 6-phosphate from glucose 6-phosphate or fructose 6-phosphate and glutamine. 10 ' X1 Keratan and chondroitin sulfates may be synthesized at different rates in cornea. The biosynthesis of chondroitin and keratan sulfates can be independently influenced by steroids in structures such as nucleus pulposus and cartilage. 12 " 14 A greater comprehension of the biochemical events and mechanisms involved in corneal wound healing and pathology will be obtained as the biosynthesis and interactions of keratan and chondroitin sulfates are more fully elucidated. For this purpose, calf and beef corneas were studied. Corneas were incubated with inorganic sulfate- 35 S, N- acetylglucosamine-l-^c, glucose-l-^c, and galactose-l- 14 C; glycosaminoglycans were isolated and the specific activities of keratan and chondroitin sulfates determined. Materials and methods Inorganic sulfate- S5 S incorporation into keratan and chondroitin sulfates by calf cornea. Fresh calf corneas were incubated in Simms Z tissue culture solution which contained sodium sulfate- 35 S in a concentration of 20 fie per milliliter. The corneas were incubated for 6 hours at 38 in a shakerwater-bath, rinsed twice in tap water, shaken in saturated Na 2 SO 4 at 4 overnight, rinsed in tap water, and then washed in distilled water for an additional 5 hours. The corneas were homogenized, digested with collagenase and trypsin, lyophilized, and stored according to published methods. 2 The digest was redissolved, dialyzed against water, centrifuged, and the supernatant fluid applied to a 1 by 7 cm. Ecteola column. The initial elution with 0.02M HC1 was followed by stepwise elution with 0.15, 0.25, and 1.95M NaCl in 0.05M HCl." The eluates were dialyzed against water and lyophilized. Portions of these lyophilized fractions were hydrolyzed in 6N HCl for approximately 18 hours at 100 and the hexosamine content determined by a Gardell 16 modification of the Elson and Morgan 17 method. The corneal glycosaminoglycans isolated by the method described above were further fractionated by stepwise elution from 2 by 30 cm. cellulose columns with 80, 50, 35, 25, and 0 per cent ethanol in 0.3 per cent barium acetate. 1S Hexuronic acid and hexose were measured in the eluates by the carbazole 19 method and the anthrone 20 reaction, respectively. The eluates were then dialyzed against water and lyophilized. Total hexosamine as well as glucosamine and galactosamine contents were determined after acid hydrolysis. 16 The sulfate- 35 S was precipitated and counted as barium sulfate. 4 > 7 > 9 Incorporation of various precursors into keratan and chondroitin sulfates by beef cornea. Corneas were obtained from fresh beef eyes and incubated in TC 199 tissue culture medium (Difco Laboratories, Detroit, Mich.) with streptomycin (6 fig per milliliter) and penicillin G (54 units per milliliter). 9 Inorganic sulfate- 35 S (Oak Ridge National Laboratory, Oak Ridge, Tenn.), N-acetylglucosamine-l- 14 C, glucose-l- 14 C or galactose-1-14 C (New England Nuclear Corp., Boston, Mass.) were added in concentrations 1 of 1 fie per milliliter. The corneas were incubated for approximately 18 hours at 38 in a shaker-water-bath. Glycosaminoglycans were isolated and further resolved into fractions which contained keratan sulfate and chondroitin-chondroitin sulfate. 1 ' 3 > Results Incorporation of sulfate- S5 S into chondroitin and keratan sulfates in calf cornea. Ecteola fraction A contained small amounts of hexosamine and low radioactivity (see Fig. 1). Most of the radioactivity was eluted in Ecteola fraction B. A partial separation of keratan sulfate from chondroitin sulfate can be achieved with further fractionation based on ethanol solubility. The glycosaminoglycan which was eluted from the cellulose column in the absence of ethanol contained approximately 93 per cent chondroitin sulfate. The glycosaminoglycan which was eluted from the cellulose column with 35 per cent ethanol contained approximately 90 per cent keratan sulfate. Ecteola fraction C contained glycosaminoglycans which were further resolved into keratan and chondroitin sulfates with ethanol. The total amount of glycosamino-

3 Volume 4 Number 5 Activities of corneal glycosaminoglycans 869 I CHONDROITIN SULFATE II KERATAN SULFATE 0.02 A 0.30 C B 2.00 D I I I I I I I 1 I I I I % ETHANOL Fig. 1. Distribution of corneal glycosaminoglycans after elution from Ecteola column 2 with chloride followed by farther fractionation with ethanol on a cellulose column. 1S The ethanoleluted fractions were analyzed for glucosamine-galactosamine ratio. The specific activity is given under each ethanol fraction and expressed as sulfate- 3s S counts per minute per microgram of hexosamine. Open columns represent ethanol-eluted fractions with insufficient amounts of hexosamine for ratio determination. Table I. Beef corneal glycosaminoglycans Sulfate- 3B S N-acetylglucosamine-l-^'C Clucose-l- 14 C Calactose-1-1 -''C Glycosaminoglycan fraction 60% ethanol insoluble (chondroitinchondroitin sulfate) (counts/'min./ fimole of hexuronic acid) 1,784 5,070 2,635 9,750 70% ethanol insoluble (keratan sulfate) (counts/min./ fimole of hexose) 1,090 5,020 4,200 6,800 Composition of fraction verified by passage through Ecteola"; specific activities include contribution of hexosamine. glycans was less in this fraction than in the previous one. A small amount of hexosamine-containing material with a veiy high specific activity was found in the 50 per cent ethanol eluate. Glycosaminoglycans in Ecteola fraction D consisted mostly of keratan sulfate. The glycosaminoglycan which was eluted from the cellulose column with 35 per cent ethanol contained approximately 95 per cent keratan sulfate of a high specific activity. Approximately 93 per cent of the glycosaminoglycan which was eluted from the cellulose column in the absence of ethanol was found to be chondroitin sulfate. Incorporation of various precursors into chondroitin and keratan sulfates in beef corneas. Chondroitin sulfate incorporated more sulfate- 35 S than did keratan sulfate (see Table I). N-acetylglucosamine-l-^'C was incorporated into the two glycosaminoglycans with a resulting equal specific activity in chondroitin and keratan sulfates. Incubation of cornea with glucose-l- TI C led to a higher specific activity in keratan sulfate than in the chondroitins, whereas the reverse situation was found after incubation of corneas with galactose-l-^'c. Discussion It is evident that calf and beef cornea will incorporate sulfate- 35 S not only into keratan sulfate but also into chondroitin sulfate, in vitro. Calf and beef cornea incorporate sulfate- 35 S into glycosaminoglycans to the extent that chondroitin sulfate demonstrated a higher activity than did keratan sulfate (Fig. 1, Table I). Corneal chondroitin sulfate is known to have a variable sulfate content and, more recently, the variability of the sulfate content of

4 870 Dohlman, Wortman, and Hultman Investigative Ophthalmology October 1965 keratan sulfate has been demonstrated However, the average sulfate: hexosamine ratio of chondroitin sulfate has been found to be much lower than that of keratan sulfate. 1 ' - These data suggest that the turnover of sulfate groups in chondroitin sulfate may be higher than that of keratan sulfate of cornea. Keratan sulfate in cartilage has a much longer half-life than does chondroitin sulfate. 12 ' 13 When earlier results 7 are reviewed in terms of new information, it is suggested that the longer half-life of sulfate- 35 S in cornea compared with that in sclera may be related to the absence of keratan sulfate in sclera rather than the absence of a blood supply in cornea. It is not known at which stage of polymerization of corneal glycosaminoglycans sulfation occurs. While the present study does not offer conclusive data, it was suggested in a previous study that the shorter chain-length keratan sulfate may be a better sulfate acceptor. 21 Published data suggest that in other connective tissues sulfation occurs after synthesis of the polymer. 22 ' 24 Resolution of glycosaminoglycans on Ecteola is in part related to the number of anionic groups and to the molecular size. The specific activity of keratan sulfate was higher in Ecteola fraction D than in fraction B (see Fig. 1). Resolution of keratan sulfate on Ecteola results in fractionation on the basis of molecular weight and keratan sulfate in fraction D would be expected to be the higher molecular weight glycosaminoglycan. 2 The keratan sulfate which binds more strongly to Ecteola may represent the glycosaminoglycan in a more complete or in a more active stage of biosynthesis. Furthermore, the larger weight glycosaminoglycans are replaced by smaller molecular weight glycosaminoglycans in regenerating corneal wounds 0 and much greater concentration of radioactivity can be observed by radioautography at a corneal wound after sodium sulfate- 3S S administration. 4 In addition, the smaller chain-length keratan sulfate shows greater sulfate-acceptor properties than does the larger chain-length keratan sulfate. 21 Glucose and galactose are not incorporated so as to yield the same specific activity in both corneal glycosaminoglycans. It is speculated that both corneal glycosaminoglycans may not turn over at the same rate, and the glycosaminoglycans may be sulfated, as acceptor sites are made available during biosynthesis. Although cell-free extracts from beefcornea epithelium are capable of incorporating inorganic sulfate- 35 S into glycosaminoglycans, it seems unlikely that the stromal glycosaminoglycans are synthesized in the epithelium. 25 Corneal stroma can incorporate inorganic sulfate- 35 S at a normal rate in some species in vitro 26 and in vivo 27 when denuded of epithelium and endothelium. It is more logical to conclude that epithelial cells have the capacity to synthesize 3'-phosphoadenosine 5'-phosphosulfate (active sulfate) and to transfer sulfate groups to suitable acceptor compounds. REFERENCES 1. Meyer, K., Linker, A., Davidson, E. A., and Weissman, B.: The mucopolysaccharides of bovine cornea, J. Biol. Chem. 205: 611, Anseth, A., and Laurent, T. C: Studies on corneal polysaccharides. I. Separation, Exper. Eye Res. 1: 25, Wortman, B.: Quantitation of keratan sulfate in the presence of other corneal glycosaminoglycans, Anal. Biochem. 4: 10, Dohlman, C. H.: On the metabolism of the corneal graft, Acta ophth. 35: 303, Dunnington, J. H., and Smelser, G. K.: Incorporation of S 35 in healing wounds in normal and devitalized corneas, Arch. Ophth. 60: 116, Anseth, A., and Laurent, T. C.: Polysaccharides in normal and pathologic corneas, INVEST. OPHTH. 1: 195, Dohlman, C. H.: Incorporation of radioactive sulfate into the rabbit eye, Acta ophth. 35: 115, Wortman, B.: Pyridine nucleotide-stimulated production of 3'-phosphoadenosine 5'-phosphate by beef-cornea-epithelial extract, Biochim. et biophys. acta 77: 65, Wortman, B., and Strominger, J. L.: Incorporation of inorganic sulphate S 35 into sulphated mucopolysaccharides of cornea in vitro, Am. J. Ophth. 44: 291, 1957.

5 Volume 4 Number 5 Activities of corneal glycosaminoglycans Pogell, B. M., and Koenig, D. F.: Studies on mucopolysacchari.de biosynthesis in the cornea, J. Biol. Chem. 234: 2504, Duda, H., and Pogell, B. M.: Acid-soluble nucleotides of bovine lens and cornea, Arch. Biochem. & Biophys. 73: 100, Davidson, E. A., and Small, W.: Metabolism in vivo of connective-tissue mucopolysaccharides. I. Chondroitin sulfate C and keratosulfate of nucleus pulposus, Biochim. et biophys. acta 69: 445, Davidson, E. A., and Small, W.: Metabolism in vivo of connective-tissue mucopolysaccharides. III. Chondroitin sulfate and keratosulfate of cartilage, Biochim. et biophys. acta 69: 459, Kaplan, D., and Fisher, B.: The effect of methylprednisolone on mucopolysaccharides of rabbit vitreous humor and costal cartilage, Biochim. et biophys. acta 83: 102, Anseth, A.: Studies on corneal polysaccharides. III. Topographic and comparative biochemistry, Exper. Eye Res. 1: 106, Gardell, S.: Separation on Dowex 50 ion exchange resin of gkicosamine and galactosamine and their quantitative determination, Acta chem. scandinav. 7: 207, Elson, L. A., and Morgan, W. T. J.: A colorimetric method for the determination of glucosamine and chondrosamine, Biochem. f. 27: 1824, Gardell, S.: Separation of mucopolysaccharides on a cellulose column, Acta chem. scandinav. 11: 668, Dische, Z.: A new specific color reaction of hexuronic acids, J. Biol. Chem. 167: 189, Dreywood, R.: Qualitative test for carbohydrate material, Indust. & Engin. Chem. (Anal.) 18: 499, Wortman, B.: Variability of keratan sulfate isolated from beef cornea, Biochim. et biophys. acta 83: 288, Adams, J. B.: The biosynthesis of chondroitin sulfate, Biochem. J. 76: 520, Spolter, L., Rice, L. I., and Marx, W.: Biosynthesis of heparin. Evidence for the transfer of radioactive sulfate to small-molecular-weight acceptors, Biochim. et biophys. acta 74: 188, Perlman, R. L., Tesler, A., and Dorfman, A.: The biosynthesis of chondroitin sulfate bv a cell-free preparation, J. Biol. Chem. 239: 3623, Wortman, B.: Enzymatic sulfation of corneal mucopolysaccharides by beef cornea epithelial extract, J. Biol. Chem. 236: 974, Wortman, B.: Metabolism of sulfate by beef and rabbit cornea, Am. J. Physiol. 198: 779, I Dohlman, C. H., and Gasset, A. R.: Unpublished observations, 1964.