G1cua-Gal-Gal-Xyl-Serine/Poypp

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BIOSYNTHESIS OF THE CHONDROITIN SULFA TE-PROTEIN LINKAGE REGION: PURIFICATION AND PROPERTIES OF A GLUCURONOSYLTRANSFERASE FROM EMl1BRYONIC CHICK BRAIN* BY ALAN E. BRANDT,t JACK DISTLER,t AND GEORGE W. JOURDIAN RACKHAM ARTHRITIS RESEARCH UNIT AND DEPARTMENT OF BIOLOGICAL CHEMISTRY, THE UNIVERSITY OF MICHIGAN, ANN ARBOR Communicated by John L. Oncley, July 11, 1969 Abstract.-The present paper describes the purification and properties of a glucuronosyltransferase isolated from 13-day embryonic chick brain. The enzyme catalyzes transfer of glucuronic acid from UDP-glucuronic acid to a series of low and high molecular weight compounds which contain terminal nonreducing f-D-galactose residues. Studies utilizing enzymatically degraded chondromucoprotein as acceptor suggest that the glucuronosyltransferase ter- minates biosynthesis of the linkage region between protein and polysaccharide of chondromucoprotein.

A number of investigators have shown that naturally occurring mucopoly- saccharides are covalently bound to protein.1-3 A common linkage region between protein and polysaccharide was demonstrated in several mucopolysac- charides including chondroitin sulfate,4-6 heparin,7' 8 and dermatan sulfate.9' 10 The following structure was proposed: /polypeptide G1cUA-Gal-Gal-Xyl-serine/poypppolypeptide Transfer of xylosel-113 and galactose"3 14 from the corresponding uridine di- phosphate sugars to endogenous protein acceptors has been reported to occur with cell-free preparations derived from hen oviduct, mouse mastocytoma, and chick epiphyseal cartilage. Partial characterization of fragments of the proteinaceous products obtained by enzymatic and/or mild acid treatment suggested that synthesis of a trisaccharide sequence similar to that found in the chondroitin sulfate- protein complex linkage region could be initiated. The presence of chondroitin sulfate in vertebrate brain tissue has been demonstrated,15-18 and glycopeptides derived from brain chondromucoprotein were shown to contain monosaccharide units characteristic of the linkage region.19 These latter findings have served to motivate the present investigation on the use of embryonic chick brain extracts for studies of the biosynthesis of the chondromucoprotein linkage region. Materials and Methods.-UDP-GlcUA-U-14C was prepared from UDP-Glc-U-14C by the method of Strominger et al.20 The enzymes employed were obtained from the indicated sources: bovine liver UDP-glucose dehydrogenase and ,3-glucuronidase, Sigma Chemical Co.; testicular hyaluronidase, Worthington Biochemical Corp.; galactose dehydrogenase, Boehringer; and galactose oxidase, K. A. B. I., Sweden. Sialidase was prepared from Clostridium perfringens.21 The following compounds were prepared by described procedures:22 O-j-D-GalNAc (1 -* 4)#-D-GlcUA(1 -- 3)D-GalNAcOCH3 (CS-Trisaccharide),23' 24 O-p-D-Gal(l -i 4)- GlcNAc,25 O-f3-D-Gal-, and O-,3-D-Xyl-serine.20 374 Downloaded by guest on September 29, 2021 VO)L. 642 1969 BIOCHEMISTRY: BRANDT ET AL. 37.5

Chondromucoprotein was prepared from freshly excised bovine nasal septa,27 treated with hvaluronidase and the degraded protein complex isolated by gel filtration.21 Termi- nal glucuronic acid residues were removed with ,B-glucuronidase. The degraded complex was adsorbed on DEAE-cellulose and eluted with 0.3 Ml KCl. Colorimetric analyses6 of the glycosidase-free product, hereafter referred to as Degraded CS-Protein, gave the following molar ratios: xylose, 1.00; galactose, 1.95; glucuronic acid, 0.68; galactosamine, 0.80. Identity of the carbohydrate constituents was established by paper chromatographic and paper electrophoresis techniques. The terminal galactose content (0.96,4mole/mg protein) was determined with galactose oxidase. In addition, the product (1) eluted from Sephadex G-200 as a single peak with a molecular weight in excess of 60,000,29 and (2) exhibited a single major protein-staining band and 3 trace components after disk gel electrophoresis at pH 9.5. Preparation and assay of the enzyme: All operations were conducted at 0-4' unless otherwise stated. Approximately 90 gm of 13-day embryonic chick brain were homogenized with an equal volume of Tyrode's buffer. The homogenate was centrifuged at 270 X g for 15 min, the pellet washed first with 60 ml and then with 30 ml of buffer, and the combined supernatants centrifuged at 200,000 X g for 1 hr. The pellet was suspended in 26 ml of 0.05 I1 imidazole buffer, pH 7.0 and recentrifuged at 200,000 X g for 1 hr. The sedimented fraction was gently suspended in 26 ml of water and an acetone powder prepared as described by Morton.30 Acetone powder (1 gm) was homogenized in 30 ml of a buffer containing 0.05 M imidazole, pH 9, 0.15 M KCl, and 1% Triton X-100, and the suspension dialyzed against the same buffer lacking Triton X-100 for 12 hr. After centrifugation of the retent at 200,000 X g for 1 hr, the clear supernatant was dialyzed against distilled water for 8 hr, and chromatographed on DEAE-cellulose (Fig. 1). The specific activity of the final preparation was approximately 65-fold higher than that of the crude homogenate; the yield of enzyme ranged from 4-10%. Typical enzyme incubations contained the following components in final volumes of 0.05 ml: 0.002 311 UDP-GlcUA-U-14C (1 /Ac/Mmole); 0.1 M lactose; 0.1 Al imidazole buffer, pH 7.0; 0.007 211 MnCl2; protein, 40 ,ug. After incubation at 370 for 6 hr, an ali- quot was assayed by paper electrophoresis in 0.05 AI pyridyl acetate buffer, pH 6.5 for 30 min at 80 v/cm. Low molecular weight products migrated toward the cathode while

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00 200 300 400O 50C 600" 7~-r EFFLUENT VCLUME ml) FIG. 1.-Chromatography of acetone powder extract on DEAE-cellulose. The protein fraction (300 mg) was applied to a 2 X 15-cm DEAE-cellulose column and eluted in a batchwise manner with 0.05 M imidazole buffer, pH 7.0 containing the indicated concentrations of potassium chloride. Protein was determined by a modified biuret procedure and plotted as mg protein/10 ml fraction, 0. Enzy- matic activity was determined as described in the text with the exception that incubations were conducted for 6 hr at 370 in the presence of 0.1 mM UDP- GlcUA-U-14C (50 isc/hmole), *. Downloaded by guest on September 29, 2021 376 BIOCHEMISTRY: BRANDT ET AL. PROC. N. A. S.

"C-labeled Degraded CS-Protein and other high molecular weight compounds remained at the origin. The appropriate radioactive areas were cut out and counted by liquid scintillation techniques. Paper chromatography was performed on Whatman #1 or 3 MM papers. The papers were irrigated in a descending fashion with ethyl acetate, pyridine, water (8:2:1). Results.-Kinetic studies with the 65-fold purified preparation showed (a) that product formation was proportional to time of incubation and protein concentration at all stages of purification; (b) that maximum activity was obtained with 0.007 M Mn++ [an effect which could be partially replaced by Mg++ or Co++ (65%), while Cu ++ and Ni ++ showed lower but significant activity (51 and 38%) ]; and (c) that the approximate Km values were 1 X 10-4 Jf for UDP-GlcUA, 8 X 10-3 M for lactose, 5.5 X 10-4 M for acetyllactosamine, 2 X 10-2 M for CS- Trisaccharide, 7 X 10-5 M for Degraded CS- Protein (based on teminal galactose content), and 0.18 M for galactose. The 65-fold purified glucuronosyltransferase preparation showed activity with a number of low molecular weight acceptors, particularly those containing terminal nonreducing /3-D-galactose residues. The following sugars and sugar derivatives were active: O-f3-D-Gal(1 4) D-Glc, 100; CS-Trisaccharide, 180; O-fl-D-Gal(1 -- 4)D-GlcNAc, 176; O-f3-D-Gal(1 -> 3)D-Gal, 112; O-f3-D-Gal(1 -I 4)- D-Gal, 103; O-f3-D-Gal(1 -- 6)D-Gal, 47; O-fl-D-Gal-serine, 23; and D-Gal, 5. Inactive compounds (less than 1% as active as lactose) included O-a-Gal(1--*-6)- D-Glc, o-aminophenol, GalNAc, O-fl-D-Xyl-serine, cellobiose, lactosylceramide, galactosylceramide, and monogalactosyldiglyceride. The observation that embryonic chick brain utilized lactose, containing a nonreducing galactose terminus, and CS-Trisaccharide, containing a nonreducing acetylgalactosamine terminus, as acceptors suggested the possible presence of 2 glucuronosyltransferases. While the ratio of activity of CS-Trisaccharide to lactose remained constant (1.8) at each step during purification, different enzyme sources showed different relative activities. For example, the 200,000 X g pellet from epiphyseal cartilage exhibited a ratio of 18. Substrate competition studies (Table 1) have provided additional evidence for the presence of two glucuronosyltransferases. With the assay conditions described, v/vmax is equal to the fraction of enzyme occurring as enzyme-substrate complex.3' Under conditions of substrate saturation, v/vmax approaches but cannot exceed one. If one enzyme (or active site) catalyzes two reactions, the sum of v/vmax should be less than one; with two enzymes (or two independent active sites) the sum of v/vmax should be less than two. A value of 1.6 was obtained for the sum of

TABLE 1. Substrate competition study.* VLactose VCs-Trisaccharide Substrate RatetLactose Ratetc&Trisacmharide VmaxLactose VmaxcS.Trisaccharide Lactose 1.6 0 0.95 0 CS-Trisaccharide 0 3.0 0 0.90 Lactose + CS-Trisaccharide 1.3 2.4 0.80 0.83 * Incubation conditions were those described in the text for the purified enzyme. Lactose and CS-Trisaccharide were present at concentrations 0.1 M in final volumes of 0.025 ml. t mjsmoles of product formed during 3-hr incubation at 37°. Downloaded by guest on September 29, 2021 VOL. 64, 1969 BIOCHEMISTRY: BRANDT ET AL. 377

v/vmax in the presence of high concentrations of lactose and CS-Trisaccharide acceptor. Characterization of the "4C-product with lactose as acceptor: To characterize the product formed from lactose, the stated incubation conditions (described in Materials and Methods) were augmented 16-fold. The 14C product was isolated by paper electrophoresis and after further purification by descending paper chromatography, was obtained in a 40 per cent yield. The following molar ratios were observed when the 14C-product was analyzed: 14C, 1.00; galactose (galactose oxidase), 0.93; glucose (glucose oxidase), 0.97. Partial acid hydroly- sis6 yielded a 14C-disaccharide in 70 per cent yield. Vigorous acid hydrolysis of the disaccharide released galactose, determined by galactose dehydrogenase. Treatment of the "4C-di- and trisaccharides with f-glucuronidase quantitatively released "4C-glucuronic acid. After periodate oxidation of the trisaccharide (0.05 M periodate, pH 4.5, 16 hr at 00), 51 per cent of the galactose was recovered, suggesting the occurrence of af1 -- 3 glycosidic bond between glucuronic acid and galactose. Periodate oxidation of the methyl glycoside of an isolated disaccharide where galactose-14C served as acceptor gave similar results. High molecular weight acceptors: Glycoproteins containing terminal, nonreducing galactose or N-acetylgalactosamine residues were also acceptors for glucuronic acid. In order to study the maximum incorporation into these compounds, incubations were carried out for longer periods of time (12-24 hr) to en- sure complete reaction (Fig. 2). The following results were obtained when glycoproteins served as acceptors (indicated as per cent of 14C-glucuronic acid incorporated per available terminal galactose or acetyigalactosamine residue): De- graded CS-Protein, 100; OSM and orosomucoid pretreated with sialidase, 38 and 27, respectively. Compounds inactive as acceptors were OSM, orosomucoid, keratosulfate, collagen, and Degraded CS-Protein pretreated with galactose oxidase. Characterization of degraded CS-Protein-14C-GlcUA: Degraded CS-Protein

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E 0 4 8 12 16 20 24 28 32 34 36 E HOURS FIG. 2.-Formation of Degraded CS-Protein-'4C-GlcUA catalyzed by chick brain glucuronosyltransferase. An incubation similar to that indicated in the text was in- creased 10-fold, using Degraded CS-Protein (1 mg/ml) as acceptor. Aliquots were removed at the indicated times and assayed as described. At 25 hr (arrow) the same amount of enzyme and UDP-14C-GlcUA were added. Theory indicates the available terminal galactose in the acceptor, as determined by galactose oxidase. Downloaded by guest on September 29, 2021 378 BIOCHEMISTRY: BRANDT ET AL. PROC. N. A.S. was labeled with "4C-glucuronic acid during the course of an incubation similar to that described above. The product (Degraded CS-Protein-14C-GlcUA) when fractionated on DEAE-cellulose and Sephadex G-200, in each instance eluted as a homogeneous 14C peak which cochromatographed with Degraded CS-Protein. Disk gel electrophoresis gave two anodic "4C-bands accounting for 90 and 10 per cent of the 14C activity. In addition, the product was nondialyzable, precipita- ble with phosphotungstic acid or 80 per cent ethanol and yielded alkali-labile 14C- glycopeptides after digestion with papain. ,3-Glucuronidase quantitatively released 14C identified as 14C-GlcUA by electrophoresis in 0.05 M pyridyl acetate, pH 6.5. Partial acid hydrolysis of Degraded CS-Protein-'4C-GlcUA using conditions previously employed to characterize CS-glycopeptides,6 released 66 per cent of the "4C-activity as a disaccharide. The latter was identified as '4C-glucuronosylgalactose as described above. Less than 1 per cent "4C-glucu- ronosylgalactosamine (the compound expected if terminal N-acetylgalactosamine end groups were substituted with 14C-glucuronic acid) was detected after paper electrophoresis. Tissue distribution of UDP-glucuronic acid: galactoside transferase: The results of a survey of vertebrate tissues for glucuronosyltransferase activity are shown in Table 2. While many particulate preparations were active, brain yielded the most active preparations. Some tissues, especially brain, contained considerable amounts of uncharacterized high molecular weight endogeneous acceptors which were removed by further purification. Discussion.-The data presented in this paper describe the purification and properties of a UDP-glucuronic acid: galactoside transferase. With the exception of the CS-Trisaccharide and OS\IM (pretreated with sialidase), both of which contain terminal N-acetylgalactosamine end groups, the purified protein preparation shows strict specificity towards compounds containing nonreducing 3-D-galactopyranosyl end groups. In addition, a number of factors, e.g., the nature of the penultimate sugar, the type of glycosidic linkage to the penultimate sugar, the molecular weight and the type of acceptor (glycoprotein, glycolipid, TABLE 2. Glucuronosyltransferase activity in vertebrate tissues.* 14C in GlcUA-Gal-Glc (cpm/mg protein/6 hr) Tissue Chick Guinea pig Human Brain 8240 3720 ... Gray matter ...... 1180 White matter ...... 7720 Choroid, eye 3640 ...... Skin 1840 ...... Epiphyseal cartilage 1760 ...... Chorioallantoic membrane 750 ... Liver 730 ...... Heart 360 330 ... Lung ... 350 ... *Activities are given as 14C incorporated into product (GlcUA-Gal-Glc), and are expressed as counts per min per mg protein per 6 hr of incubation at 37°. Incubation conditions were those described in the text; washed 200,000 X g pellet was used as enzyme source. Product formation was proportional both to time of incubation and to protein concentration. Preparations yielding less than 300 cpm/mg protein were considered inactive; these included chick and guinea pig striated muscle, and preparations from guinea pig liver, kidney, spleen, and small intestine. Downloaded by guest on September 29, 2021 VOL. 64, 1969 BIOCHEMISTRY: BRANDT ET AL. 379

etc.) play a significant role in determining acceptor specificity. For example, the order of decreasing activity of acceptors is as follows: Degraded CS-Protein, orosomucoid pretreated with sialidase, lactose, galactosylserine, galactose, melibiose, and lactosylceramide. The present studies with Degraded CS-Protein as acceptor indicate that the glucuronosyltransferase may terminate biosynthesis of the linkage region. These results, in conjunction with those previously reported,11-14 suggest the possibility that each of the glycosyltransferases involved in the synthesis of the linkage region requires, in addition to its corresponding sugar nucleotide, a glycoprotein acceptor containing a specific terminal monosaccharide residue. If such is the case, the specificity exhibited toward the acceptor would predetermine the or- dered sequential synthesis of the tetrasaccharide comprising the CS-protein linkage region. The fact that the 65-fold purified protein preparations effect transfer of glucuronic acid to compounds containing either galactose or N-acetylgalactosamine end groups suggests that it contains two glucuronosyltransferases. The results of substrate competition studies support this contention. Also, cell-free preparations from embryonic chick tissues exhibited different activities with lactose and CS-Trisaccharide as acceptors. The glucuronosyltransferase that transfers glucuronic acid to N-acetylgalactosamine end groups may be the same or similar to that described by Dorfman and co-workers32' 33 in particulate preparations from chick epiphyseal cartilage. Using a similar particulate preparation, Helt- ing and Rod6n14 noted the presence of glucuronosyltransferase activity that added glucuronic acid to galactosylgalactosylxylose; however, no attempt was made to establish whether this transferase differed from that described by Dorfman and co-workers. The present observation that the glucuronosyltransferases were not resolved from one another at any stage of the 65-fold purification procedure is striking. This is emphasized by the finding that fractionation of extracts of acetone powder preparations on DEAE-cellulose resulted in the separation of several glycosyltransferases from the glucuronosyltransferases; the former include two gluco- syltransferases34 35 and three galactosyltransferases.4' 35. 36 While the purified glucuronosyltransferase system behaves as a soluble protein (i.e., it could not be sedimented at 200,000 X g), the possibility exists that it consists of subunits derived from cellular membranes. Indeed, gel filtration studies reveal that the purified protein(s) may have a molecular size in excess of 200,000. Whether other enzymes that participate in chondromucoprotein biosynthesis are associated with this protein is under investigation. Note added in proof: Since submission of this manuscript, Helting and Rod6n (J. Biol. Chem., 244, 2799 (1969)) reported the presence of a similar glucuronosyltransferase system in particulate preparations from embryonic chick epiphyseal cartilage. Competition studies with small molecular weight acceptors containing terminal nonreducing galactose or N- acetylgalactosamine also suggested the presence of two glucuronosyltransferases. * The Rackham Arthritis Research Unit is supported by a grant from the Horace H. Rack- ham School of Graduate Studies of The University of Michigan. This investigation was supported by a grant from the US Public Health Service (AM 10531). t Recipient of a predoctoral training grant from the NIH (GM-00187-11). Downloaded by guest on September 29, 2021 380 BIOCHEMISTRY: BRANDT ET AL. PROC. N. A. S.

Postdoctoral fellow, the Arthritis Foundation. 1 Scheinthal, B. M., and M. Schubert, J. Biol. Chem., 238, 1935 (1963). 2 Sandson, J., and D. Hamerman, J. Clin. Invest., 41, 1817 (1962). 3 Muir, H., Biochem. J., 69, 195 (1958). 4Lindahl, U., and L. Rod6n, J. Biol. Chem., 241, 2113 (1966). S Rod6n, L., and R. Smith, J. Bwl. Chem., 241, 5949 (1966). 6 Rod6n, L., and G. Armand, J. Biol. Chem., 241, 65 (1966). 7 Lindahl, U., Biochim. Biophys. Acta, 130, 361 (1966). 8 Ibid., 130, 368 (1966). 9 Stern, E. L., Federation Proc., 27, 596 (1968). 0 Bella, A., Jr., and I. Danishefsky, J. Biol. Chem., 243, 2660 (1968). 11 Grebner, E. E., C. W. Hall, and E. F. Neufeld, Biochem. Biophys. Res. Commun., 22, 672 (1966). 12 Grebner, E. E., C. W. Hall, and E. F. Neufeld, Arch. Biochem. Biophys., 116, 391 (1966). 13 Robinson, H. C.. A. Telser. and A. Dorfman, these PROCEEDINGS, 56, 1859 (1966). 14Helting, T., and iJ. Rod6n, Biochem. Biophys. Res. Commun., 31, 786 (1968). 12 Clausen, J., and P. Rosenkast, J. Neurochem., 9, 393 (1962). 16Margolis, R. U., Biochim. Biophys. Acta, 141, 91 (1967). 17 Szabo, M. M., and E. Roboz-Einstein, Arch. Biochem. Biophys., 98, 406 (1962). 18 VoS, J., K. Kuriyama, and E. Roberts, Brain Res., 12, 172 (1969). 19 Wardi, A. H., W. S. Allen, D. L. Turner, and Z. Stary, Arch. Biochem. Biophys., 117, 44 (1966). 10 Strominger, J. L., H. AI. Kalckar, J. Axelrod, and E. S. Maxwell, J. Am. Chem. Soc., 76, 6411 (1954). 21 Cassidy, J. T., G. W. Jourdian, and S. Roseman, J. Biol. Chem., 240, 3501 (1965). 22 We are most grateful for the following gifts of compounds and tissues: O-O-D-Gal(l- 3)- D-Gal, Dr. D. M. W. Anderson; O-#-D-Gal(l 4)1-Gal, Dr. P. A. Gorin; O-,B-D-Gal(1 - 6)- D-Gal, Dr. J. H. Pazur; O-,j-D-Gal(1 -* 3)D-GlcNAc, Dr. M. C. Glick; keratosulfate, Dr. K. Meyer; orosomucoid, Dr. K. Schmidt; ovine submaxillary mucin (OSM), Dr. J. T. Cassidy; lactosylceramide and galactosylceramide, Dr. N. S. Radin; and human brain, Dr. W. W. Tourtellotte. 23 Fransson, L. A., L. Rod6n, and M. Spach, Anal. Biochem., 21, 317 (1968). 24Kantor, T. G., and M. Schubert, J. Am. Chem. Soc., 79, 152 (1952). 2 Kuhn, R., and W. Kirschenlohr, Liebigs. Ann. Chem., 600, 135 (1956). 2' Kum, K., and S. Roseman, Biochemistry, 5, 3061 (1966). 27 Malawista, I., and M. Schubert, J. Biol. Chem., 230, 535 (1958). 28Gregory, J. D., T. C. Laurent, and L. Rod6n, J. Biol. Chem., 239, 3312 (1964). 29Andrews, P., Biochem. J., 91, 222 (1964). 30 Morton, R. K., in Methods in Enzymology, ed. S. P. Colowick, and N. 0. Kaplan (New York: Academic Press, 1955), vol. 1, p. 25. 31 Dixon, M., and E. C. Webb, Enzymes (New York: Academic Press, 1964). 32 Telser, A., H. C. Robinson, and A. Dorfman, Arch. Biochem. Biophys., 116, 458 (1966). 83 Perlman, R. L., A. Telser, and A. Dorfman, J. Biol. Chem., 239, 3623 (1964). 34 Distler, J., and G. W. Jourdian, Federation Proc., 26, 345 (1967). 15 Spiro, R. G., and M. J. Spiro, Federation Proc., 27, 345 (1968). 36 McGuire, E. J., G. W. Jourdian, D. M. Carlson, and S. Roseman, J. Biol. Chem., 240, PC 4112 (1965). Downloaded by guest on September 29, 2021