Incorporation of sulfate-35S, N-acetylglucosa- mine-l-14C, glucose-l-14C, and galactose-l-14C 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 anion- exchanger 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-S5S. Incubation of corneas with N-acetylglucosamine-l-JiC 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 glycans by use of the anion-exchanger isolated and identified as keratan sulfate, Ecteola combined with ethanol frac- chondroitin, and chondroitin sulfate.1 The tionation on cellulose columns have en- development of methods for the isolation abled the extension of studies of cornea.2-3 of small amounts of comeal glycosamino- These methods, as well as the incorporation of inorganic sulfate-35S, have been used to study the cornea in normal and patholog- 4 G From the Corneal Research Unit, Institute of ical conditions, e.g., wound healing. ' In Biological and Medical Sciences, Retina Founda- the normal cornea, most of the incorporated 35 tion, Boston, Mass., and Department of Ophthal- sulfate- S can be recovered as ester sulfate mology, Washington UnivWsity School of and is presumed to be in the glycosamino- Medicine, St. Louis, Mo. glycans. However, a small amount of the This investigation was supported in part by Public radioactive label is found to be in a re- Health Service Research Grants NB-02220 and duced form in methionine and cystine.7 NB-01911 from the National Institute of Neuro- logical Diseases and Blindness, United States There is further evidence that cornea can Public Health Service, by the Massachusetts reduce sulfate and incorporate it into an s Lions Eye Research Fund, and by the St. Louis organic compound. Heart Association. The previous attempt to isolate and "This work was done during tenure as an Estab- identify sulfate-35S-labeled glycosamino- lished Investigator of the American Heart glycans disclosed that beef and rabbit Association. Present address: Department of 35 Pathology, Albany Medical College, Albany, corneas incorporated sulfate- S into a gly- N. Y. cosaminoglycan which had the same elec- 867

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trophoretic mobility as authentic keratan hours at 100° and the hexosamine content de- a5 termined by a Gardell16 modification of the Elson sulfate. No sulfate- S-labeled chondroitin 17 sulfate could be demonstrated readily by and Morgan method. 9 The corneal glycosaminoglycans isolated by the the methods used in that study. method described above were further fractionated Glucose-"C has been found to be in- by stepwise elution from 2 by 30 cm. cellulose corporated into the hexosamines of corneal columns with 80, 50, 35, 25, and 0 per cent glycosaminoglycans; glucosamine had a ethanol in 0.3 per cent barium acetate.1S Hexuronic acid and hexose were measured in the eluates by higher specific activity than did galactosa- 19 20 10 the carbazole method and the anthrone reac- mine. Cornea contains UDPglcA and tion, respectively. The eluates were then dialyzed UDPglc as well as other niicleotides and against water and lyophilized. Total hexosamine has the capacity to synthesize glucosamine as well as glucosamine and galactosamine con- tents were determined after acid hydrolysis.16 The 6-phosphate from glucose 6-phosphate or 35 fructose 6-phosphate and glutamine.10'X1 sulfate- S was precipitated and counted as barium sulfate.4> 7> 9 Keratan and chondroitin sulfates may be Incorporation of various precursors into keratan synthesized at different rates in cornea. The and chondroitin sulfates by beef cornea. Corneas biosynthesis of chondroitin and keratan sul- were obtained from fresh beef eyes and incubated fates can be independently influenced by in TC 199 tissue culture medium (Difco Labora- steroids in structures such as nucleus pul- tories, Detroit, Mich.) with streptomycin (6 fig 12 14 per milliliter) and penicillin G (54 units per posus and cartilage. " A greater compre- milliliter).9 Inorganic sulfate-35S (Oak Ridge hension of the biochemical events and National Laboratory, Oak Ridge, Tenn.), N-acetyl- mechanisms involved in corneal wound glucosamine-l-14C, glucose-l-14C or galactose-1- healing and pathology will be obtained as 14C (New England Nuclear Corp., Boston, Mass.) 1 the biosynthesis and interactions of keratan were added in concentrations of 1 fie per milli- and chondroitin sulfates are more fully liter. The corneas were incubated for approxi- mately 18 hours at 38° in a shaker-water-bath. elucidated. For this purpose, calf and beef Glycosaminoglycans were isolated and further corneas were studied. Corneas were in- resolved into fractions which contained keratan cubated with inorganic sulfate-35S, N- sulfate and chondroitin-chondroitin sulfate.1' 3> ° acetylglucosamine-l-^C, glucose-l-^C, and 14 galactose-l- C; glycosaminoglycans were Results isolated and the specific activities of keratan S5 and chondroitin sulfates determined. Incorporation of sulfate- S into chon- droitin and keratan sulfates in calf cornea. Materials and methods Ecteola fraction A contained small Inorganic sulfate-S5S incorporation into keratan amounts of hexosamine and low radioac- and chondroitin sulfates by calf cornea. Fresh calf tivity (see Fig. 1). Most of the radio- corneas were incubated in Simms Z tissue culture activity was eluted in Ecteola fraction B. solution which contained sodium sulfate-35S in a A partial separation of keratan sulfate concentration of 20 fie per milliliter. The corneas from chondroitin sulfate can be achieved were incubated for 6 hours at 38° in a shaker- water-bath, rinsed twice in tap water, shaken in with further fractionation based on ethanol saturated Na2SO4 at 4° overnight, rinsed in tap solubility. The glycosaminoglycan which water, and then washed in distilled water for an was eluted from the cellulose column in the additional 5 hours. absence of ethanol contained approximately The corneas were homogenized, digested with 93 per cent chondroitin sulfate. The glyco- collagenase and trypsin, lyophilized, and stored saminoglycan which was eluted from the according to published methods.2 The digest was redissolved, dialyzed against water, centrifuged, cellulose column with 35 per cent ethanol and the supernatant fluid applied to a 1 by 7 cm. contained approximately 90 per cent ker- Ecteola column. The initial elution with 0.02M atan sulfate. HC1 was followed by stepwise elution with 0.15, Ecteola fraction C contained glyco- 0.25, and 1.95M NaCl in 0.05M HCl." The eluates were dialyzed against water and lyo- saminoglycans which were further resolved philized. Portions of these lyophilized fractions into keratan and chondroitin sulfates with were hydrolyzed in 6N HCl for approximately 18 ethanol. The total amount of glycosamino-

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I CHONDROITIN II KERATAN SULFATE SULFATE

0.02 A 0.30 C

75 136 272 428 2879 311 998 902

0.20 B 2.00 D

450 135 336 567 135 629 262 631 I I I I I I I 1 I I I I 80 50 35 25 0 80 50 35 25 0 % ETHANOL Fig. 1. Distribution of corneal glycosaminoglycans after elution from Ecteola column2 with chloride followed by farther fractionation with ethanol on a cellulose column.1S The ethanol- eluted fractions were analyzed for glucosamine-galactosamine ratio. The specific activity is given under each ethanol fraction and expressed as sulfate-3sS counts per minute per micro- gram of hexosamine. Open columns represent ethanol-eluted fractions with insufficient amounts of hexosamine for ratio determination.

Table I. Beef corneal glycosaminoglycans glycosaminoglycan which was eluted from the cellulose column in the absence of Glycosaminoglycan fraction • ethanol was found to be chondroitin sul- 60% ethanol fate. insoluble (chondroitin- 70% ethanol Incorporation of various precursors into chondroitin insoluble chondroitin and keratan sulfates in beef sulfate) (keratan corneas. Chondroitin sulfate incorporated (counts/'min./ sulfate) 35 fimole of (counts/min./ more sulfate- S than did keratan sulfate hexuronic fimole of (see Table I). N-acetylglucosamine-l-^'C acid) hexose) was incorporated into the two glycosamino- Sulfate-3BS 1,784 1,090 glycans with a resulting equal specific ac- N-acetylglucosa- 5,070 5,020 tivity in chondroitin and keratan sulfates. mine-l-^'C TI Clucose-l-14C 2,635 4,200 Incubation of cornea with glucose-l- C Calactose-1-1-''C 9,750 6,800 led to a higher specific activity in keratan •Composition of fraction verified by passage through sulfate than in the chondroitins, whereas Ecteola"; specific activities include contribution of hexosamine. the reverse situation was found after in- cubation of corneas with galactose-l-^'C.

glycans was less in this fraction than in Discussion the previous one. A small amount of hex- It is evident that calf and beef cornea osamine-containing material with a veiy will incorporate sulfate-35S not only into high specific activity was found in the 50 keratan sulfate but also into chondroitin per cent ethanol eluate. sulfate, in vitro. Calf and beef cornea in- Glycosaminoglycans in Ecteola fraction corporate sulfate-35S into glycosaminogly- D consisted mostly of keratan sulfate. cans to the extent that chondroitin sulfate The glycosaminoglycan which was eluted demonstrated a higher activity than did from the cellulose column with 35 per cent keratan sulfate (Fig. 1, Table I). Corneal ethanol contained approximately 95 per chondroitin sulfate is known to have a cent keratan sulfate of a high specific ac- variable sulfate content and, more recently, tivity. Approximately 93 per cent of the the variability of the sulfate content of

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keratan sulfate has been demonstrated.-0- -1 sulfate shows greater sulfate-acceptor prop- However, the average sulfate: hexosamine erties than does the larger chain-length ratio of chondroitin sulfate has been found keratan sulfate.21 Glucose and galactose to be much lower than that of keratan are not incorporated so as to yield the sulfate.1' - same specific activity in both corneal gly- These data suggest that the turnover of cosaminoglycans. It is speculated that both sulfate groups in chondroitin sulfate may corneal glycosaminoglycans may not turn be higher than that of keratan sulfate of over at the same rate, and the glycosamino- cornea. Keratan sulfate in cartilage has a glycans may be sulfated, as acceptor sites much longer half-life than does chon- are made available during biosynthesis. droitin sulfate.12'13 When earlier results7 Although cell-free extracts from beef- are reviewed in terms of new information, cornea epithelium are capable of incorpor- it is suggested that the longer half-life of ating inorganic sulfate-35S into glycosamin- sulfate-35S in cornea compared with that oglycans, it seems unlikely that the stromal in sclera may be related to the absence of glycosaminoglycans are synthesized in the keratan sulfate in sclera rather than the epithelium.25 Corneal stroma can incorpor- absence of a blood supply in cornea. ate inorganic sulfate-35S at a normal rate It is not known at which stage of poly- in some species in vitro26 and in vivo27 merization of corneal glycosaminoglycans when denuded of epithelium and endo- sulfation occurs. While the present study thelium. It is more logical to conclude that does not offer conclusive data, it was sug- epithelial cells have the capacity to syn- gested in a previous study that the shorter thesize 3'-phosphoadenosine 5'-phosphosul- chain-length keratan sulfate may be a fate (active sulfate) and to transfer sulfate better sulfate acceptor.21 Published data groups to suitable acceptor compounds. suggest that in other connective tissues sulfation occurs after synthesis of the REFERENCES polymer.22'24 1. Meyer, K., Linker, A., Davidson, E. A., and Weissman, B.: The mucopolysaccharides of Resolution of glycosaminoglycans on bovine cornea, J. Biol. Chem. 205: 611, 1953. Ecteola is in part related to the number 2. Anseth, A., and Laurent, T. C: Studies on of anionic groups and to the molecular corneal polysaccharides. I. Separation, Exper. size. The specific activity of keratan sulfate Eye Res. 1: 25,1961. was higher in Ecteola fraction D than 3. Wortman, B.: Quantitation of keratan sulfate in the presence of other corneal glycosamino- in fraction B (see Fig. 1). Resolution of glycans, Anal. Biochem. 4: 10, 1962. keratan sulfate on Ecteola results in 4. Dohlman, C. H.: On the metabolism of the fractionation on the basis of molecular corneal graft, Acta ophth. 35: 303, 1957. 5. Dunnington, J. H., and Smelser, G. K.: In- weight and keratan sulfate in fraction D 35 would be expected to be the higher molecu- corporation of S in healing wounds in nor- 2 mal and devitalized corneas, Arch. Ophth. 60: lar weight glycosaminoglycan. The kera- 116, 1958. tan sulfate which binds more strongly to 6. Anseth, A., and Laurent, T. C.: Polysac- Ecteola may represent the glycosamino- charides in normal and pathologic corneas, glycan in a more complete or in a more INVEST. OPHTH. 1: 195, 1962. active stage of biosynthesis. Furthermore, 7. Dohlman, C. H.: Incorporation of radioactive sulfate into the rabbit eye, Acta ophth. 35: the larger weight glycosaminoglycans are 115, 1957. replaced by smaller molecular weight gly- 8. Wortman, B.: Pyridine nucleotide-stimulated cosaminoglycans in regenerating corneal production of 3'-phosphoadenosine 5'-phos- wounds0 and much greater concentration phate by beef-cornea-epithelial extract, of radioactivity can be observed by radio- Biochim. et biophys. acta 77: 65, 1963. 9. Wortman, B., and Strominger, J. L.: Incor- autography at a corneal wound after so- 35 3S 4 poration of inorganic sulphate S into sul- dium sulfate- S administration. In addi- phated mucopolysaccharides of cornea in tion, the smaller chain-length keratan vitro, Am. J. Ophth. 44: 291, 1957.

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10. Pogell, B. M., and Koenig, D. F.: Studies on 18. Gardell, S.: Separation of mucopolysaccha- mucopolysacchari.de biosynthesis in the cornea, rides on a cellulose column, Acta chem. J. Biol. Chem. 234: 2504, 1959. scandinav. 11: 668, 1957. 11. Duda, H., and Pogell, B. M.: Acid-soluble 19. Dische, Z.: A new specific color reaction of nucleotides of bovine lens and cornea, Arch. hexuronic acids, J. Biol. Chem. 167: 189, Biochem. & Biophys. 73: 100, 1958. 1947. 12. Davidson, E. A., and Small, W.: Metabolism 20. Dreywood, R.: Qualitative test for carbohy- in vivo of connective-tissue mucopolysac- drate material, Indust. & Engin. Chem. charides. I. Chondroitin sulfate C and kerato- (Anal.) 18: 499, 1946. sulfate of nucleus pulposus, Biochim. et 21. Wortman, B.: Variability of keratan sulfate biophys. acta 69: 445, 1963. isolated from beef cornea, Biochim. et biophys. 13. Davidson, E. A., and Small, W.: Metabolism acta 83: 288, 1964. in vivo of connective-tissue mucopolysac- 22. Adams, J. B.: The biosynthesis of chondroitin charides. III. Chondroitin sulfate and kerato- sulfate, Biochem. J. 76: 520, 1960. sulfate of cartilage, Biochim. et biophys. acta 23. Spolter, L., Rice, L. I., and Marx, W.: 69: 459, 1963. Biosynthesis of heparin. Evidence for the 14. Kaplan, D., and Fisher, B.: The effect of transfer of radioactive sulfate to small-molec- methylprednisolone on mucopolysaccharides ular-weight acceptors, Biochim. et biophys. of rabbit vitreous humor and costal cartilage, acta 74: 188, 1963. Biochim. et biophys. acta 83: 102, 1964. 24. Perlman, R. L., Tesler, A., and Dorfman, A.: 15. Anseth, A.: Studies on corneal polysaccha- The biosynthesis of chondroitin sulfate bv a rides. III. Topographic and comparative bio- cell-free preparation, J. Biol. Chem. 239: chemistry, Exper. Eye Res. 1: 106, 1961. 3623, 1964. 16. Gardell, S.: Separation on Dowex 50 ion ex- 25. Wortman, B.: Enzymatic sulfation of corneal change resin of gkicosamine and galactosa- mucopolysaccharides by beef cornea epithelial mine and their quantitative determination, extract, J. Biol. Chem. 236: 974, 1961. Acta chem. scandinav. 7: 207, 1953. 26. Wortman, B.: Metabolism of sulfate by beef 17. Elson, L. A., and Morgan, W. T. J.: A colori- and rabbit cornea, Am. J. Physiol. 198: 779, metric method for the determination of I960. glucosamine and chondrosamine, Biochem. f. 27. Dohlman, C. H., and Gasset, A. R.: Unpub- 27: 1824, 1933. lished observations, 1964.

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