The turnover of hexosamine and sialic acid in glycoproteins and mucopolysaccharides of brain

Biochimica et Biophysica Acta, 304 (1973) 413-420

Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands BBA 27094 T H E T U R N O V E R OF H E X O S A M I N E A N D SIALIC ACID IN GLYCOPROTEINS A N D M U C O P O L Y S A C C H A R I D E S OF BRAIN

RENI~E K. MARGOLIS and RICHARD U. MARGOLIS Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, N.Y. 11203 and Department of Pharmacology, New York University School of Medicine, New York, N.Y. 10016 (U.S.A.)

(Received October 17th, 1972)

SUMMARY Two major metabolic pools of glycoproteins were found in rat brain. One of these has a rapid turnover ofhexosamine, sialic acid and sulfate, with half-times of 4.6, 6, and 2.5 days, respectively, while the corresponding half-times for the glycoproteins with a slower turnover rate are 15, 30 and 14 days. One day after the administration of labeled glucosamine or sulfate, the specific activity of sialic acid and sulfate in the glycoproteins extracted by water (containing approximately 15 % of the glycoprotein carbohydrate) was greater than three times that of the remaining material, indicating that the glycoproteins with a rapid turnover are present in relatively soluble cell structures. There were also rapid and slow components for the turnover of hexosamine in hyaluronic acid (t½ ----9 and 45 days) and heparan sulfate (t~ : 3.7 and 10 days), while chondroitin sulfate gave evidence of but a single metabolic pool, with a halftime for hexosamine turnover of 21 days. From a comparison of these results with earlier data obtained for the turnover of sulfate, it would appear that the sulfate groups on chondroitin sulfate and in one fraction of heparan sulfate turn over an average of three times as rapidly as hexosamine in the polysaccharide backbone. Based on the specific activities of the mucopolysaccharides one day after the administration of labeled glucosamine, it can be concluded that the hyaluronic acid with a slow turnover is present in a structurally stable location in the cell, from which it can not be extracted with water or Triton X-100.

INTRODUCTION We have previously reported on the turnover in vivo of sulfate groups in chondroitin sulfate, heparan sulfate, and the sulfated glycoproteins of adult rat brain t. In order to allow comparison of these results with similar data on the turnover of the polysaccharide backbone of the mucopolysaccharides and the oligosaccharide chains of the glycoproteins, we have examined the turnover of hexosamine in the

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R. K. MARGOLIS, R. U. MARGOLIS

glycoproteins and sulfated mucopolysaccharides of rat brain. We have also investigated the turnover of hyaluronic acid, and of sialic acid residues in brain glycoproteins. MATERIALS AND METHODS Adult male Sprague-Dawley rats were injected intraperitoneally with 2.5/~Ci/g of D-[6-3H]glucosamine • HCI (3.6 Ci/mmole, New England Nuclear, Boston, Mass.) dissolved in water at a concentration of 2.5 mCi/ml. The rats were sacrificed by decapitation 1-33 days after injection and the brains were quickly removed and frozen at - 2 0 °C. For each time point 3 brains were pooled and analyzed for glycoprotein and mucopolysaccharide specific radioactivity. Lipids were extracted with chloroform-methanol and the lipid-free protein residue was digested with pronase as described previously 2, 3. After dialysis, the mucopolysaccharides were precipitated with cetylpyridinium chloride from 0.04 M NaCI, and the glycopeptides derived from brain glycoproteins were recovered from the supernatant aftel precipitation of excess cetylpyridinium chloride with KSCN. The sulfated mucopolysaccharides were separated from hyaluronic acid by differential precipitation with cetylpyridinium chloride from 0.3 M NaCI (refs 3, 4), and hyaluronic acid was obtained from the supernatant fluid after removal of excess cetylpyridinium chloride and dialysis 3. Heparan sulfate was separated from the chondroitin sulfate disaccharides (90 ~,, chondroitin 4-sulfate) by digestion with chondroitinase ABC and gel filtration on Sephadex G-25 (ref. 1). For calculation of hexosamine specific radioactivity, the hyaluronic acid, heparan sulfate and chondroitin sulfate disaccharide fractions were lyophilized, redissolved in water, and aliquots were removed for determination of radioactivity and hexosamine content. Glucosamine and galactosamine were determined after acid hydrolysis using the amino acid analyzer. as described previously ~. To calculate the turnover rate of hexosamine and sialic acid residues in brain glycoproteins, glycopeptides were desialylated by mild acid hydrolysis (0.05 M HzSO 4, 1 h, 80 °C) and neutralized with NaOH. Free sialic acid was separated from the desialylated glycopeptides by gel filtration on a I cm × 50 cm column of Sephadex G-25 eluted with distilled water. The desialylated glycopeptides emerged as a sharp peak which was only slightly retarded on the column, while the free sialic acid was well separated from the desialylated glycopeptides and eluted as a single retarded peak. Hexosamine in the desialylated glycopeptides was determined afte~ acid hydrolysis, and sialic acid was measured by the thiobarbituric acid method 5. Sulfate was determined by the barium chloranilate method of Spencer 6 after hydrolysis of glycopeptides for 4 h in I M HCI. Paper chlomatography was performed on Whatman No. I strips using the following solvent systems: propanol-ethyl acetate-water (7: I :2, by vol.); ethyl acetate-acetic acid-water (6 : 3 : 2, by vol.); and butanol-acetic acid-water (50 : 12 : 25, by vol.). Radioactivity was determined using a radiochromatogram scanner, and hexosamine and sialic acid were detected by staining with alkaline AgNO3. For paper chromatography, the fraction of desialylated glycopeptides was hydrolyzed in 2 M trifluoroacetic acid for 2 h at 120 °C, evaporated to dryness in a rotary evaporator and redissolved in 2 M HCI for application to the chromatography strips. We have previously reported the rapid biological exchange of 3H between

HEXOSAMINE AND SIALIC ACID TURNOVER 1N BRAIN

415

free (but not glycosidically-bound) [aH]tucose and body water in the rat 7. This disappearance of 3H radioactivity from fucose in vivo led us to determine the validity of using D-[6-aH]glueosamine as a label for the hexosamine-containing oligosaccharide and polysaccharide chains of brain glycoproteins and mucopolysa¢charides. Adult rats were injected intraperitoneally with 3 pCi/g of a mixture of [1-14C]glucosamine and [6-3H]glucosamine, in which 25 ~o of the radioactivity was 14C. The rats were sacrificed after 1 and 7 days, and the mucopolysaccharide and glycopeptide fractions were prepared as described above. Since at both time periods the ratio of 14C/3 H radioactivity was the same in the mucopolysaccharide and glycopeptide fractions as that present in the solution used for injection, it can be concluded that [6-3H]glucos amine is a satisfactory label for the carbon backbone in glycoproteins and mucopolysaccharides of rat brain. For studying the specific radioactivity of glycoprotein and mucopolysaccharide fractions extractable by various solvents, adult rats were injected with [3H]glucosamine (1.5 pCi/g) or Na235SO4 (5/~Ci/g) and sacrificed after 24 h. 12-20 pooled brains were homogenized in 10 vol. of cold distilled water and centrifuged for 1 h at 100 000 × #. The pellet was then re-extracted with 1 ~ Triton X-100 (6 ml/g brain) and again centrifuged as described above. The water and Triton extracts and the pellet remaining after Triton extraction were dialyzed, lyophilized, extracted with chlorof0rm-methanol and used for the isolation of glycopeptide and mucopolysaccharide tractions. The pellet remaining after Triton X-100 extraction of the brains of rats which received labeled sulfate was dissolved in 0.3 M lithium diiodosalicylate before dialysis, leaving less than 2 ~o of the glycoproteins and mucopolysaccharides in an insoluble form 8. The specific activities of the mucopolysaccharides and of hexosamine and sialic acid in the glycopeptides were determined in the water, Triton X-100 and residual fractions as described above. RESULTS The disappearance with time of radioactivity in hyaluronic acid, chondroitin sulfate and heparan sultate is shown in Fig. 1. The graph representing the turnover of chondroitin sulfate was linear for a period of over 1 month, and it could be calculated that the hexosamine in this mucopolysaccharide had a half-time of 21 days in adult rat brain. In contrast to the data obtained for chondroitin sulfate, the graphs representing the disappearance of hexosamine radioactivity from hyaluronic acid and heparan sulfate could each be divided into a rapid and a slow component. For hyaluronic acid these had half-times of 9 and 45 days, while the half-times for the rapid and slow components in heparan sulfate were 3.7 and 10 days. The turnover of sialic acid and hexosamine in brain glycoproteins was also examined. Sialic acid was removed by mild acid hydrolysis and separated from the desialylated glycopeptides by gel filtration as described above. Paper chromatography of the sialic acid fraction in three solvent systems demonstrated in each case only a single radioactive component which migrated at the same rate as standard sialic acid. After hydrolysis of the desialylated glycopeptides and paper chromatography there was likewise found to be but a single radioactive component which cochromatographed with a glucosamine standard. (The solvent systems used in this study do not separate glucosamine and galactosamine, but as expected we have found in other

416

R . K . MARGOLIS, R. U. MARGOLIS

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Fig. 1. Time-course of disappearance of hexosamine radioactivity from hyaluronic acid ( ~ - - A ) , chondroitin sulfate ( 0 - - 0 ) , and heparan sulfate ( 0 - - 0 ) in adult rat brain.

experiments that both amino sugars are labeled in brain glycoproteins after administration of [3H]glucosamine.) The sensitivity of the chromatographic procedure used for assaying the radiochemical purity of the sialic acid and hexosamine fractions was such that contaminants representing 3 ~o or more of the total radioactivity would be detectable. Rapid and slow components were also apparent from the graphs representing the turnover of hexosamine and sialic acid in rat brain glycoproteins (Fig. 2). The disappearance of hexosamine radioactivity had half-times of 4.6 and 15 days, while the half-times for the turnover of sialic acid were 6 and 30 days. Our values for the half-time of hexosamine turnover in brain glycoproteins are similar to the average half-times previously reported (1.7 and 12.5 days) for rat brain glycoproteins containing both hexosamine and sialic acid radioactivity9. Approximately 10 ~ of the sialic acid in glycopeptides prepared from brain I0 8

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Fig. 2. Time-course of disappearance of radioactivity from hexosamine ( 0 - - 0 ) ( O - - O ) in glycoproteins of adult rat brain.

and sialic acid

HEXOSAMINE AND SIALIC ACID T U R N O V E R IN I~I~AIN

417

glycoproteins is present in an alkali-labile pentasaccharide linked O-glyeosidically to serine and threonine residues, and having the partial structure: di-N-acetylneuraminyl-D-galactopyranosyl-(1 ~ 3)- [N-acetylneuraminyl-(2 ~ 6)]- O-2-aeetamido-2-deoxy-D-galactose' o. To determine whether these sialic acid residues turn over at the same rate as sialic acid in oligosaccharides linked to protein by alkali-stable N-acetylglucosaminylasparagine linkages, the specific activity of sialic acid was measured in the alkali-labile oligosaccharides obtained by gel filtration and preparative paper chromatography, and compared to that in the alkali-stable glyeopeptides. TABLE I HALF-TIMES FOR THE T U R N O V E R OF SULFATE, HEXOSAMINE, AND SIALIC ACID IN THE GLYCOPROTEINS AND MUCOPOLYSACCHARIDES OF RAT BRAIN Half-times are given in days. Compound

Sulfate*

Hexosamine

Sialic acid

Glycoproteins Hyaluronic acid Chondroitin sulfate Heparan sulfate

2.5, 14 -7 3

4.6, 15 9, 45 21 3.7, 10

6, 30 ----

* Data from ref. 1. TABLE II SPECIFIC ACTIVITY OF GLYCOPROTEIN AND MUCOPOLYSACCHARIDE FRACTIONS ONE DAY AFTER THE ADMINISTRATION OF LABELED G L U C O S A M I N E Compound

Glycoprotein sialic acid Glyeoprotein hexosamine Hyaluronic acid Chondroitin sulfate Heparan sulfate

cpm/l~mole hexosamine or sialic acid Water extract

Triton X-IO0 extract

Residue

42 200 58 300 16 800 17 700 34 400

12 200 46 200 18 200 23 000 44 600

13 000 37 400 7 700 16 600 66 400

TABLE IlI

SPECIFIC ACTIVITY OF GLYCOPROTEIN AND MUCOPOLYSACCHARIDE FRACTIONS ONE DAY A F T E R THE ADMINISTRATION OF LABELED SULFATE Compound

cpm/l~mole hexosamine or glycoprotein sulfate Water extract

Glycoproteins Chondroitin sulfate Heparan sulfate

107 400 28 600 91 500

Triton X-IO0 extract

Residue

36 200 38 200 98 800

30 000 41 800 66 800

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R.K. MARGOLIS, R. U. MARGOLIS

It was found that both sialic acid fractions had the same specific activity 1 day after administration of labeled glucosamine, indicating that sialic acid in both alkali-stable and alkali-labile oligosaccharides turns over at the same (average) rate. For purposes of comparison, the half-times for the turnover of hexosamine in glycoproteins and mucopolysaccharides of rat brain are summarized in Table I, together with the corresponding figures for the turnover of sulfate obtained in a previous study ~. One day after the administration of labeled glucosamine or sulfate, the specific activity of sialic acid and sulfate in the water-soluble glycoproteins was greater than three times that found in the remaining brain glycoproteins (Tables II and III). This would indicate that the pool of brain glycoproteins with a rapid turnover of sialic acid and sulfate is probably located in the soluble fraction of brain, while the pool with a less rapid turnover rate is located primarily in the particulate fraction. The difference in apparent turnover rates for the two glycoprotein hexosamine components (t½ = 5 and 15 days) is not as great as that observed for sialic acid, yet here also there was a decrease in specific activity from the more to the less readily extractable glycoproteins (Table II). The specific activity of hyaluronic acid in the residue remaining after successive extraction of brain with water and Triton X-100 was less than one-half of that found in the water and Triton X-100 extracts, indicating that the pool of hyaluronic acid with a slow turnover rate (t½ = 45 days) is located in structural components which are relatively insoluble in addition to being stable metabolically. In contrast to hyaluronic acid, and hexosamine, sialic acid and sulfate in the brain glycoproteins, all of which were found to have a higher specific activity in the water and/or Triton X-100 extracts, the specific activity of hexosamine in heparan sulfate was lowest in the water extract, and greatest in the lesidue remaining after successive extractions with water and Triton X-100 (Table II). The specific activity of chondroitin sulfate was similar in all three fractions, a finding which is consistent with the linear rate of disappearance of sulfate and hexosamine radioactivity over a period of 3-4 weeks, and which indicates the presence of only one major metabolic pool of chondroitin sulfate in brain. DISCUSSION We have previously reported the presence of two pools of brain glycoproteins, with half-times of 2.5 and 14 days for the turnover of sulfate ~. The results presented here demonstrate two components for the turnover of sialic acid in brain glycoproteins (t~. = 6 and 30 days) which also differ by 5-fold, and support our earlier suggestion that at least two major metabolic pools of glycoproteins are present in brain. Since the specific activity of both sialic acid and sulfate in the glycoproteins extracted with water was much greater than that of the remaining glycoproteins, it appears that the water-soluble glycoproteins contain one or more pools with a relatively rapid turnover of anionic groups. It has been found that fucose has a relatively slow turnover in brain glycoproteins (t~ = 2 weeks in young (2-week-old) rats 7, and approximately 1 month in adult mice ~1). Based on the available information concerning the turnover of fucose and hexosamine in brain glycoproteins, we suggested 7 that the presence of fucose

HEXOSAMINE AND SIAL1CACID TURNOVER IN BRAIN

419

might characterize a population of brain glycoproteins with a slower than average turnover rate, since fucose is always located at a terminal position on the oligosaccharide chains in which it occurs, and cannot turn over more slowly than hexosarnine residues located in more internal positions. Sialic acid is similarly located at a terminal position on the oligosaccharide chains of glycoproteins, and we have found that the Slow component of sialic acid turnover (t½ --~ 1 month) is also much less rapid than that of hexosamine (t~ ---- 2 weeks) in brain glycoproteins. While hexosamine branch groups may turn over independently of and more rapidly than other portions of the oligosaccharide, it would appear that the addition of a terminal fucose or sialic acid residue to an oligosaccharide chain confers a certain measure of metabolic stability on these oligosaccharides. A somewhat analogous phenomenon has been reported in studies demonstrating the role of sialic acid in determining the survival of glycoproteins in the circulation. In these investigations, covering ten plasma proteins and gonadotrophic hormones, it was shown that upon injection into rats, all of the desialylated plasma glycoproteins tested (with the exception of transferrin) were promptly removed from the circulation and taken up by cells of the liver, under conditions where the intact, fully sialylated proteins survived normally throughout the period of obseI vation ~2. In more detailed studies on desialylated ceruloplasmin, this plasma glycoprotein was found to be rapidly catabolizzd by lysosomes in the hepatocytes ~3. Although similar studies have not yet been reported for fucose, these results suggest that the presence or absence of characteristic terminal sugars on oligosaccharides of glycoproteins may be an important factor in determining their rate of turnover. Our results also indicate the presence of two pools of hyaluronic acid and heparan sulfate with different rates of hexosamine turnover (t½ ---- 9 and 45 days for hyalutonic acid, and 3.7 and 10 days for heparan sulfate). In an attempt to obtain preliminary information on the possible differences in localization of these pools, the specific activities of hyaluronic acid, chondroitin sulfate and heparan sulfate were determined in water and Triton X-100 extracts of brain, and in the unextractable residue, 24 h after labeling with [3H]glucosamine or Na235SO4. These experiments were based on the principle that two or more products having different turnover rates but labeled from a common precursor pool will have specific activities which are proportional to their relative metabolic activities after a period of labeling which is short in relation to their turnover times. From the results of these studies it can be seen that the specific activity of hyaluronic acid is approximately equal in the water and Triton X-100 extracts, but only 42-46 ~o as high in the unextractable residue (Table II). Therefore this unextractable material, which comprises more than one-third of the brain hyaluronic acid s, appears to be highly stable metabolically. The specific activity of hexosamine in heparan sulfate increased progressively from the water extract to the Triton X-100 insoluble residue, indicating that the least soluble fractions of brain are enriched in the pool(s) of heparan sulfate with the most rapid turnover of hexosamine (t½ • 3.7 days). Since there was evidence for only one component in the turnover of sulfate in heparan sulfate (t~ ---- 3 days), the sulfate groups in the water-extractable material would appear to turn over approximately three times as rapidly as hexosamine in the polysaccharide backbone (t~ = 10 days for the slow component). The validity of these differences in specific activity is supported by the absence

420

R. K. MARGOLIS, R. U. MA.RGOLIS

of such large differences in the specific activity of sulfate in the various fractions ol heparan sulfate, or of sulfate and hexosamine in chondroitin sulfate (Tables II and III), since in each of these cases only a single metabolic pool was evident from the turnover graphs. It should however be recognized that the calculated half-times are most likely average values which include several pools of glycoproteins or mucopolysaccharides with similar but not identical turnover rates, and that very small pools with turnover rates much faster or slower than average would not be apparent from our data. We are planning to investigate these possibilities in more detail by studies of the metabolism of mucopolysaccharides and glycoproteins in cellular and subcellular fractions of brain. It is of interest that the turnover of sulfate in chondroitin sulfate is three times as rapid as that of hexosamine in the polysaccharide backbone. As mentioned above, there would also appear to be a pool of heparan sulfate in which the turnover of sulfate is three times as rapid as that of hexosamine, in addition to a pool in which the half-times for hexosamine and sulfate are approximately equal. These data suggest that the independent turnover of sulfate on an intact carbon backbone may have some functional significance by providing a mechanism for the addition and removal of anionic groups on cell membranes or in other neural structures. NOTE A D D E D IN PROOF (Received January 16th, 1973)

We have recently found that the small pool of fucose (8 ~o of the total) in the water-soluble glycoproteins o f rat brain has a turnover rate greater than ten times, that in the remaining glycoproteins. ACKNOWLEDGEMENTS

We thank Regina Santella and Donna Atherton for their skillful technical assistance. This work was supported by research grants from the U. S. Public Health Service (NS-09348 and MH-17018).

REFERENCES 1 Margolis, R. U. and Margolis, R. K. (1972) Biochim. Biophys. Acta 264, 426-431 2 Margolis, R. K. and Margolis, R. U. (1970) Biochemistry 9, 4389-4396 3 Margolis, R. U. and Margolis, R. K. (1972) in Research Methods in Neurochemistry (Marks, N. and Rodnight, R., eds), Vol. 1, pp. 249-284, Plenum Press, New York 4 Margolis, R. U. (1967) Biochim. Biophys. Acta 141, 91-102 5 Warren, L. (1959) J. BioL Chem. 234, 1971-1975 6 Spencer, B. (1960) Biochem. J. 75, 435-440 7 Margotis, R. K. and Margolis, R. U. (1972) J. Neurochem. 19, 1023-1030 8 Margolis, R. K. and Margolis, R. U. (1973) J. Neurochem., in the press 9 Holian, O., Dill, D. and Brunngraber, E. G. (1971) Arch. Biochem. Biophys. 142, 111-121 10 Margolis, R. K. and Margolis, R. U. (1973) Biochim. Biophys. Acta 304, 421-429 11 Zatz, M. and Barondes, S. H. (1970) J. Neurochem. 17, 157-163 12 Morell, A. G., Gregoriadis, G., Scheinberg, I. I-L, Hickman, J. and Ashwell, G. (1971) J. Biol. Chem. 246, 1461-1467 13 Gregoriadis, G., Morell, A. G., Sternlieb, I. and Scheinberg, I. H. (1970) J. Biol. Chem. 245, 5833-5837