Glycobiology, 2000, Vol. 10, No. 8 803-807
© 2000 Oxford University Press
Purification and some properties of UDP-xylosyltransferase of rat ear cartilage
Institute of Physiological Chemistry, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Karl-Marx-Strasse 3, D-01109 Dresden, Germany
Received on December 9, 1999; revised on February 16, 2000; accepted on February 17, 2000.
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Abstract |
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UDP-xylosyltransferase (UDP-D-xylose:proteoglycan core protein ß-D-xylosyltransferase EC 2.4.2.26) initiates the formation of chondroitin sulfate in the course of proteoglycan biosynthesis. The enzyme catalyzes the transfer of D-xylose from UDP-D-xylose to specific serine residues in the core protein. A procedure for purification of xylosyltransferase from rat ear cartilage was developed which includes ammonium sulfate fractionation, chromatography on heparin–agarose, on Sephacryl S300 and finally a substrate affinity chromatography applying the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G. The specific activity of the purified enzyme was about 420 mU per mg protein. The purification factor was about 26.000 with 27% yield. In SDS-polyacrylamide gel electrophoresis, the highly purified enzyme is homogeneous and yields only a single distinct band of 78 kDa. An apparent molecular mass of 71 kDa was determined for the native enzyme. These data suggest a monomeric structure for the enzyme. Xylosyltransferase activity was found to depend essentially on the presence of divalent metal ions. The Km value for UDP-D-xylose was determined to 6.5 µmol/l and for the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G as xylose acceptor to 8 µmol/l.
Key words: affinity chromatography/glycosaminoglycan/glycosyltransferase/proteoglycan/xylosyltransferase
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Proteoglycans are composed of a central core protein to which a number of highly negatively charged polysaccharide chains are covalently attached. Chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin are conjugated to the core protein through a xylose-galactose-galactose linkage region (Hardingham, 1981
). In the course of glycosaminoglycan biosynthesis, UDP-D-xylose:proteoglycan core protein ß-D-xylosyltransferase (EC 2.4.2.26) catalyzes by transfer of D-xylose from UDP-D-xylose to the hydroxyl groups of some specific serine residues in the core protein the first, rate-limiting step in a sequence of glycosyltransferase reactions (Schwartz, 1976; Roden, 1980).
Acceptors for determination of xylosyltransferase activity used so far were deglycosylated core proteins from cartilage proteoglycans (Sandy, 1979; Coudron et al., 1980; Edge et al., 1981; Olson et al., 1985), silk fibroin (Campbell et al., 1984) and several peptides (Bourdon et al., 1987; Campbell et al., 1990; Kearns et al., 1991). Comparison of amino acid sequences of chondroitin sulfate attachment sites in different proteoglycans led to a consensus sequence for the recognition signal of xylosyltransferase (Esko and Zhang, 1996; Brinkman et al., 1997). Peptides possessing the consensus sequence reveal to be potent acceptor substrates for xylosyltransferase (Weilke et al., 1997). In addition, purification of xylosyltransferase may be accomplished by chromatography on such immobilized peptides. This paper describes a procedure for getting a highly purified, stable, and homogeneous rat ear cartilage xylosyltransferase preparation with a specific activity of about 420 mU per mg protein. The purification involves a specific substrate affinity chromatographic step on a dodeca-peptide (Q-E-E-E-G-S-G-G-G-Q-G-G) with the consensus sequence for recognition of xylosyltransferase. Some molecular and kinetic properties of the enzyme are also presented.
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Results |
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Purification of UDP-xylosyltransferase
Crude homogenate and ammonium sulfate fractionation.
Frozen ears (about 140 g total, 100g after dissecting of surrounding tissue) from Wistar rats were thawed, washed in deionized water containing 0.02% NaN3 and minced in five volumes of buffer A. After homogenization with an ultra turrax and subsequently with a motor driven Teflon pistil, the crude homogenate was centrifuged at 15,000 x g for 15 min. The supernatant was centrifuged again at 100,000 x g for 60 min. The resulting supernatant was fractionated by precipitation with ammonium sulfate between 20% and 55% of saturation.
Chromatography on heparin–agarose.
The enzyme solution was dialyzed against buffer B for 12 h and applied to a column (2.5 x 10 cm) of heparin–agarose. The column was washed with buffer B until no more protein emerged. Xylosyltransferase was eluted by a linear NaCl gradient (0–1 M NaCl in buffer B). Fractions containing xylosyltransferase activity were pooled, the protein was concentrated by ammonium sulfate precipitation.
Gel filtration on Sephacryl S 300.
The precipitated protein was dissolved in 5 ml of buffer C and applied to a column (70 x 2.5 cm) of Sephacryl S 300. Fractions containing xylosyltransferase activity were pooled and dialyzed against buffer D for 12 h.
Affinity chromatography on peptide-Sepharose.
The enzyme solution was applied to a column (0.8 x 9 cm) of the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G coupled to Sepharose 6MB (Figure 1). The column was washed with buffer D until no more protein emerged. Protein unspecifically bound was removed by a linear NaCl gradient (0–1 M in buffer D). Xylosyltransferase was eluted specifically with a solution of the peptide used as affinity ligand (0.1 mM in buffer E).
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Since the activity of purified xylosyltransferase cannot be readily assayed in the presence of the peptide, the samples were rechromatographed on a column of heparin–agarose (0.8 x 2.5 cm). Bound xylosyltransferase was eluted with 0.5 M NaCl in buffer B. The final specific activity was about 420 mU per mg protein. The purification factor was about 26,000 with 27% yield. The purification procedure is summarized in Table I. Figure 2 shows the protein profiles at various steps of the purification procedure as determined by SDS-PAGE under reducing conditions. At the final step of purification, only a single band of 78 kDa was detected. An apparent molecular mass of 71 kDa for the native enzyme was determined by applying analytical HPLC gel filtration (Figure 3). From this, it may be concluded that xylosyltransferase represents a monomeric protein.
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Properties of purified xylosyltransferase
The purified enzyme if stored in a medium containing 50 mM Tris-HCl, pH 7.0 and 50 mM NaCl is stable at –80°C or at –20°C for at least 15 weeks (Figure 4). Storage of the enzyme at either 4°C, 25°C, or 37°C resulted in rapid loss of enzymic activity. The optimum of xylosyltransferase activity in 50 mM Tris-HCl containing 50 mM NaCl was found at pH 7.0. However, as shown in Figure 5, the pH-optimum of xylosyltransferase activity depends on buffer system used. The temperature optimum of the reaction was determined to 37°C. For xylosyltransferase activity, divalent metal cations were found to be essentially required. Ca2+, Mg2+, and Mn2+ show quantitatively similar effects, whereas Zn2+ acts strongly inhibitory (Figure 6).
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The Km value of the enzyme for UDP-D-xylose was determined to be 6.5 µmol/l. To characterize the substrate specificity of xylosyltransferase, a variety of structurally defined peptides were examined. The structures of the peptides and their ability to serve as xylosyl acceptors are presented in Table II. The data demonstrate that the most suitable acceptors are peptides carrying three acidic amino acids located N-terminally of the serine residue, i.e., the peptides 1, 2, and 3. Peptide 2 (Q-E-E-E-G-S-G-G-G-Q-G-G) showed the highest acceptor activity: reduction of the number of glutamate residues (peptides 4, 5, and 6), reduction of the length (peptide 3) or replacement of the C-terminal glycine residues by lysine (peptide 1) resulted in decreasing acceptor activities. No xylosylation was observed when serine was replaced by tyrosine (peptide 8). Peptide 7 possessing a threonine residue instead of serine serves only as a poor xylose acceptor.
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Discussion |
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Although mammalian tissues contain a large number of different glycosyltransferases, only very few of them have been purified to homogeneity. This is largely due to their firm attachment to membrane structures of the cells and their tendency to aggregate (Roden et al., 1972
; Stoolmiller et al., 1972). This paper describes for the first time the purification of UDP-xylosyltransferase from rat ear cartilage. The enzyme belongs together with the xylosyltransferases described by Schwartz and Roden (1974), Stoolmiller et al. (1972), Schwartz and Dorfman (1975), Stoolmiller et al. (1975) and Roden et al. (1994) to a more readily solubilized group of glycosyltransferases. No detergents are necessary for their solubilization. The most successful step in the purification procedure described here represents the specific affinity chromatography on peptide-Sepharose. The affinity ligand is a synthetic dodeca peptide described by Weilke et al. (1997) modified by replacement of two C-terminal lysine residues by glycine (Table II, peptide 2). Compared with the initially described peptide, no specific interaction with proteins other than xylosyltransferase was observed. The peptide Q-E-E-E-G-S-G-G-G-Q-G-G was not only the substrate of choice in selecting a suitable ligand for affinity chromatography but also a useful substrate for sensitive determination of enzyme activity.
Some properties of the enzyme like pH and temperature optimum as well as dependence of enzymic activity on divalent metal ions are similar to that of xylosyltransferases of rat kidney (Roden et al., 1994), rat chondrosarcoma (Schwartz and Dorfman, 1975, Stoolmiller et al., 1975), and embryonic chick cartilage (Stoolmiller et al., 1972; Schwartz and Roden, 1974). On the other hand, there are remarkable differences between them in the molecular mass. Xylosyltransferases from rat ear cartilage and from rat kidney are monomeric enzymes of about 71 kDa and 32 kDa, respectively, whereas the xylosyltransferases from rat chondrosarcoma and from embryonic chick cartilage seem to be tetrameric structures composed of two pairs of nonidentical subunits of 23 kDa and 27 kDa, respectively. Beside different origin of the enzymes, the preparation procedure itself could be a reason for getting xylosyltransferases of different molecular masses. The final step in enzyme purification is always a specific, but in the individual case distinct affinity chromatography. In the case of rat chondrosarcoma and embryonic chick cartilage deglycosylated core protein from cartilage proteoglycans was used as affinity ligand. Xylosyltransferase from rat kidney was prepared by the use of UDP-glucuronic acid-agarose, and xylosyltransferase described in this report was prepared by the use of a dodeca peptide as affinity ligand. From this it may also be assumed that xylosyltransferases of different substrate specificities were isolated.
The amino acid sequence of the xylosylation side as a primary signal for the transfer of xylose to serine was investigated by comparison of the acceptor efficiencies (Vmax/Km) of different synthetic peptides. In agreement with the findings of Brinkman et al. (1997) and Esko and Zhang (1996), a minimum length of the peptide and acidic amino acids located N-terminally of the serine residue are required for effective xylose acceptor function.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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Materials
UDP-[14C]-D-xylose (9.9 GBq/mmol) was purchased from NEN Life Science Products GmbH. UDP-D-xylose, CNBr-activated Sepharose 6 MB, heparin–agarose and electrophoresis-grade reagents were obtained from Bio-Rad. Sephacryl S 300 was from Pharmacia and Rotiszint Eco Plus scintillation mixture from Roth. Polyethylene glycol-polystyrene (PEG-PS) supports and amino acids were purchased from PER SEPTIVE Biosystems.
Buffers and solutions
Buffer A: 0.1 M Tris-HCl, pH 7.0 containing 0.25 M NaCl, 1 mM EDTA, 5 mM benzamidine hydrochloride, 2 mM iodoacetic acid, and 1 µM soybean trypsin inhibitor. Buffer B: 0.1 M Tris-HCl, pH 7.0 containing 1 mM EDTA. Buffer C: 0.1 M Tris-HCl, pH 7.0 containing 0.25 M NaCl and 1 mM EDTA. Buffer D: 50 mM Tris-HCl, pH 7.0 containing 50 mM NaCl, and 5 mM MnCl2. Buffer E: 50 mM Tris-HCl, pH 7.0 containing 50 mM NaCl.
Determination of UDP-xylosyltransferase activity
Reaction mixtures for the assay of UDP-xylosyltransferase contained in a final volume of 100 µl: 320 µM acceptor peptide of the sequence Q-E-E-E-G-S-G-G-G-Q-G-G, 0.46 µM UDP-[14C]-D-xylose, 68 µM UDP-D-xylose, 5 mM MnCl2 and varying amounts of enzyme protein in buffer E. After incubation for 20 min at 37°C, 1.5 mg of bovine serum albumin and 0.5 ml of 10% trichloroacetic acid/4% phosphotungstic acid were added. Precipitated protein was collected by centrifugation at 30,000 x g for 15 min, washed twice with 0.5 ml of 5% trichloroacetic acid, and dissolved in 0.2 ml of 1 M NaOH for liquid scintillation counting. Xylosyltransferase activity was calculated from the difference of UDP-D-xylose initially employed and D-xylose bound to the acceptor peptide. One milliunit of enzymic activity represents the incorporation of 1 nmol xylose/min into the acceptor peptide.
Determination of the acceptor activities for xylosylation of different acceptors
Michealis-Menten constants (Km) and maximal reaction rates (Vmax) were determined for xylosylation of different acceptor peptides. The ratio Vmax/Km is according to Kearns et al. (1991) defined as acceptor activity.
Peptide synthesis
Peptides were obtained by solid-phase peptide synthesis (9050 PepSynthesizer, MilliGen/Biosearch) employing Fmoc-amino acid pentafluorophenylesters. Cleaving of the peptides from the support and deprotection of side chains were achieved by incubation in trifluoroacetic acid containing 5% phenol and 5% 4-(methylthio)phenol. The peptides were pecipitated with diethylether and purified by chromatography on Sephadex G 15.
Preparation of peptide-Sepharose resin
Fifteen milligrams of the dodeca peptide of the sequence Q-E-E-E-G-S-G-G-G-Q-G-G were coupled to 1 g of cyanogen bromide-activated Sepharose 6 MB. Any remaining active groups were blocked by reaction with 1 M ethanolamine.
Sodium dodecyl sulfate–PAGE
The purity of xylosyltransferase was verified by SDS-PAGE using 5–15% gradient-separating and 3% stacking gels. After electrophoresis, proteins were visualized by silver staining. The relative molecular mass of xylosyltransferase was estimated using a SDS-PAGE molecular broad range standard from Bio-Rad.
Size-exclusion HPLC
Size-exclusion HPLC was carried out using a Bio-Silect 125-5 column (Bio-Rad) equilibrated with buffer C. For calculating the relative molecular mass of xylosyltransferase, the column was calibrated with a gel filtration standard from Bio-Rad.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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We thank A.Hientzsch for excellent technical assistance. This work was supported by the Bundesministerium für Bildung, Wissenschaft, Forschung, und Technologie (01ZZ5904).
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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HPLC, high-performance liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacryl amide gel electrophoresis; Xyl-T, xylosyltransferase.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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Bourdon,M.A., Kruisus,S., Campbell,S.C., Schwartz,N.B. and Rouslahti,E. (1987) Identification and synthesis of a recogition signal for the attachment of glycosaminoglycans to proteins. Proc. Natl. Acad. Sci. USA, 84, 3194–3198.
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