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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Key words: affinity chromatography/glycosaminoglycan/glycosyltransferase/proteoglycan/xylosyltransferase
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Introduction |
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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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Chromatography on heparinagarose.
The enzyme solution was dialyzed against buffer B for 12 h and applied to a column (2.5 x 10 cm) of heparinagarose. The column was washed with buffer B until no more protein emerged. Xylosyltransferase was eluted by a linear NaCl gradient (01 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 (01 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 heparinagarose (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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Discussion |
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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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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 sulfatePAGE
The purity of xylosyltransferase was verified by SDS-PAGE using 515% 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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Abbreviations |
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Footnotes |
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References |
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