Download Purification and some properties of UDP

Glycobiology vol. 10 no. 8 pp. 803–807, 2000
Purification and some properties of UDP-xylosyltransferase of rat ear cartilage
Uwe Pfeil and Klaus-Wolfgang Wenzel1
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
UDP-xylosyltransferase (UDP-D-xylose:proteoglycan core
protein β-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-Dxylose 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
Introduction
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 β-Dxylosyltransferase (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
1To
whom correspondence should be addressed
© 2000 Oxford University Press
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.
Results
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 × g for 15 min. The
supernatant was centrifuged again at 100,000 × 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 × 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
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U.Pfeil and K.-W.Wenzel
Table I. Purification of xylosyltransferase from rat ear cartilage (100 g)
Purification step
Total activity (mU)
Specific activity (mU/mg)
Purification (-fold)
Crude homogenate
29.5
0.016
1
Ammonium sulfate fractionation
26.1
0.031
1.9
Yield (%)
100
88.5
Heparin–agarose
25.0
0.224
14
84.7
Sephacryl S300
19.3
1.192
74.5
65.4
Affinity chromatography
8.1
418.7
26197
27.5
(70 × 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 × 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).
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 × 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.
Fig. 2. SDS-PAGE at various stages of xylosytransferase purification. Proteins
were visualized by silver staining. Lanes 1 and 7, molecular mass standards;
lane 2, crude homogenate; lane 3, ammonium sulfate fractionation; lane 4,
chromatography on heparin–agarose; lane 5, gel filtration on Sephacryl S 300;
lane 6, affinity chromatography on Q-E-E-E-G-S-G-G-G-Q-G-G-Sepharose.
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
Fig. 3. HPLC gel filtration of purified xylosyltransferase. Arrows indicate the
elution of standard proteins: 1, 670 kDa; 2, 158 kDa; 3, 44 kDa; 4, 17 kDa; 5,
1.35 kDa. Protein, circles; xylosyltransferase activity, triangles.
Fig. 1. Affinity chromatography of xylosyltransferase on Q-E-E-E-G-S-G-GG-Q-G-G-Sepharose. Protein, open circles; xylosyltransferase activity, open
triangles; dashed line, NaCl gradient.
804
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.
Purification and some properties of xylosyltransferase
Fig. 4. Stability of xylosyltransferase in dependence on temperature of storage.
The enzyme was stored in 50 mM Tris-HCl, pH 7.0 containing 50 mM NaCl. 80°C, diamonds; –20°C, asterisks; 4°C, triangles; 25°C, squares; 37°C, circles.
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).
The Km value of the enzyme for UDP-D-xylose was determined to be 6.5 µmol/l. To characterize the substrate specifi-
Fig. 6. Activity of xylosyltransferase as a function of divalent metal ions.
Endogenous metal ions were removed from the enzyme solution by exhaustive
dialysis against buffer E containing 5 mM EDTA. Dependence of enzymic
activity on the concentration of either MnCl2 (diamonds), MgCl2 (squares),
CaCl2 (triangles), or ZnCl2 (circles).
city 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.
Table II. Acceptor specificity of xylosyltransferase
Fig. 5. Influence of buffers on pH optimum of xylosyltransferase activity. The
buffers were 50 mM MES-NaOH, pH 5.0–7.5 containing 50 mM NaCl
(squares), 50 mM HEPES-NaOH, pH 5.5–8.5 containing 50 mM NaCl
(circles), and 50 mM Tris-HCl, pH 6.5–9.0 containing 50 mM NaCl
(triangles).
Acceptor
Km [mmol/l]
1. Q-E-E-E-G-S-G-G-G-Q-K-K
0.093
385
2. Q-E-E-E-G-S-G-G-G-Q-G-G
0.008
3050
3. Q-E-E-E-G-S-G-G-G
0.110
164
4. Q-E-E-G-G-S-G-G-G-Q-G-G
0.47
31
5. Q-E-G-G-G-S-G-G-G-Q-G-G
1.01
8.5
6. Q-G-G-G-G-S-G-G-G-Q-G-G
8.60
0.7
7. Q-E-E-E-G-T-G-G-G-Q-G-G
0.82
1.4
8. Q-E-E-E-G-Y-G-G-G-Q-G-G
n.d.
Vmax/Km
Michaelis-Menten constants and maximal reaction rates were calculated by
incubating various concentrations of the respective acceptor substrates with
xylosyltransferase under standard assay conditions. n.d., Not detectable.
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U.Pfeil and K.-W.Wenzel
Discussion
Materials and methods
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-EG-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 UDPglucuronic 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.
Materials
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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 × 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-EE-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.
Purification and some properties of xylosyltransferase
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.
Acknowledgments
We thank A.Hientzsch for excellent technical assistance. This
work was supported by the Bundesministerium für Bildung,
Wissenschaft, Forschung, und Technologie (01ZZ5904).
Abbreviations
HPLC, high-performance liquid chromatography; SDS, sodium
dodecyl sulfate; PAGE, polyacryl amide gel electrophoresis;
Xyl-T, xylosyltransferase.
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