From the Institut für Laboratoriums- und Transfusionsmedizin,
Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik
der Ruhr-Universität Bochum, Georgstraße 11, 32545 Bad
Oeynhausen, Germany and the Zentrale Proteinanalytik
R0800, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Received for publication, June 13, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Human UDP-D-xylose:proteoglycan
core protein Proteoglycans are the major components of the extracellular
matrices and are present in a variety of cell and basement membranes. Proteoglycans have different functions. They are essential for maintaining the structural integrity of connective tissue, are involved
in cell adhesion and motility, in cell differentiation and
morphogenesis, and may also be responsible, in part, for the nonthrombogenic properties of the vascular endothelium. Both heparan sulfate proteoglycans, present in the membrane of endothelial cells,
and thrombomodulin, a chondroitin sulfate-containing proteoglycan, have
been implicated in procoagulant and anticoagulant mechanisms (1,
2).
UDP-D-xylose:proteoglycan core protein
In contrast to almost all other glycosyltransferases the XT is secreted
into the extracellular space (6). The addition of the
glycosaminoglycans to the core protein in proteoglycans occurs in
vivo in the endoplasmic reticulum or Golgi apparatus (7-9).
We found highly increased XT activities in synovial fluids of patients
with chronic joint diseases (10). The elevation of enzyme activity in
synovial fluids indicates the increased synthesis of proteoglycans in
inflammatory processes. Increased XT activities were also determined in
the serum of patients with systemic sclerosis, closely related to an
elevated proteoglycan biosynthesis. These results demonstrate the
validity of XT as a diagnostic marker for the determination of
sclerotic activity in systemic sclerosis (11, 12).
Isolation of XT has been hampered by difficulties in obtaining a
sufficient amount of the source materials. Therefore, we developed a
method to produce a protein solution containing high XT activity but
low protein concentration by serum-free cultivation of JAR
choriocarcinoma cells using hollow fiber bioreactor technology. Using
this material we isolated an unknown protein in trace amounts and
sequenced 11 peptides from the enzymatically cleaved protein. The amino
acid sequences from the 11 peptides are a basic requirement for
molecular cloning and expression of XT.
Materials
Human JAR choriocarcinoma cells were purchased from ATCC
(Rockville, MD). Dried UltraDOMA-PF medium was obtained from
BioWhittaker (Vervier, Belgium) and aqua ad injecta from
Braun (Melsungen, Germany). Heat-inactivated fetal calf serum,
Dulbecco's phosphate-buffered saline, antibiotic/antimycotic solution,
trypsin-EDTA solution, trypan blue, protamine chloride, and the
bicinchoninic acid protein assay kit were purchased from Sigma
(Deisenhofen, Germany). Cell culture flasks, serological pipettes, and
sterile tubes were purchased from Becton Dickinson (Heidelberg,
Germany). The hybrid hollow-fiber bioreactor TECNOMOUSE was supplied by
Integra Biosciences (Fernwald, Germany), the ACA analyzer by Dade
Diagnostica (München, Germany), and the Super G analyzer by RLT
(Möhnesee, Germany).
UDP-[14C]xylose (9.88 kBq/nmol) came from DuPont (Bad
Homburg, Germany), 25-mm diameter nitrocellulose discs from Sartorius (Göttingen, Germany), scintillation mixture and the liquid
scintillation counter LS500TD was obtained from Beckman Coulter
(Fullerton, CA). Ultrafiltration cells, YM1 membranes, and
polyvinylidene difluoride membranes (Immobilon P) were purchased
from Millipore (Eschborn, Germany). The chromatography media POROS 20 HQ, POROS 20 HE2, POROS 20 AL, POROS 20 EP, and the HPLC work station
Biocad Sprint were supplied by Perseptive Biosystems (Framingham, MA). The gel filtration column TSK G3000 SW (30 cm × 7.5 mm, 10-µm particle size) was obtained from TosoHaas (Montgomeryville, PA). The
MALDI mass spectrometer Reflex II was from Bruker Daltonik GmbH
(Bremen, Germany) and protein sequencer Procise 494 cLC was purchased
from PE Biosystems (Framingham, MA). Precast polyacrylamide gels,
buffers, and NuPAGE electrophoresis system XCell II Mini-Cell and Blot
Module were from Novex (San Diego, CA). The synthetic peptide
CSRQKELLKRKLEQQEK and the rabbit antiserum were purchased from
BioScience (Göttingen, Germany). Peroxidase-conjugated affinipure F(ab')2 fragment goat anti-rabbit IgG (H+L) was purchased
from Dianova (Hamburg, Germany). N-Glycosidase F was
obtained from Roche Molecular Biochemicals (Mannheim, Germany). All
other chemicals were of analytical grade and obtained from Merck
(Darmstadt, Germany).
Cell Culture
JAR choriocarcinoma cells releasing XT in the cell culture
supernatant were cultured in Ultradoma-PF medium containing 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 250 ng/ml amphotericin B in a humidified atmosphere
of 5% CO2 and 95% air at 37 °C. After incubation for 24 h in the serum-containing medium, the cell cultivation was adapted to serum-free conditions as described previously (6). Scaling
up of XT production was carried out in three hybrid hollow-fiber bioreactors TECNOMOUSE. During the exponential growth the cells from
three 175 cm2 T-flasks (>3 × 107 cells)
were detached with 0.5% trypsin and 0.2% EDTA in Dulbecco's phosphate-buffered saline by incubation at 37 °C for 10 min. After centrifugation (5 min, 1000 × g) of the cell
suspension the cell pellet was resuspended in 10 ml of 37 °C warm
serum-free and protein-free Ultradoma-PF medium and washed three times
with 20 ml of the same medium. The cell suspension was drawn into a
10-ml syringe and then inoculated into the extracapillary space (EC
space) of the reactor. The bioreactor was connected with a 2-liter
medium bottle and set to 150 ml/h in the recirculation mode, and the
oxygenation pump was set as described in the operating manual.
Five days after inoculation a 10-ml syringe was connected to the left
hand EC port and 10 ml of cell culture supernatant was harvested from
the EC space. The harvesting was continued every 2 days over a period
of 3 months. Glucose and lactate concentration of the cell culture
supernatant were controlled using the Super G analyzer and the ACA
analyzer, respectively. The 2-liter medium bottle was replaced every 3 days. The viability of the cells was determined by trypan blue exclusion.
Synthesis of the Protamine Affinity Matrix
Protamine chloride was immobilized as ligand on POROS 20 AL. 30 mg of ligand was dissolved in 10 ml of 10 mM
phosphate, 0.15 M NaCl, pH 7.4. After the protein
had been dissolved, 5 ml of 100 mM phosphate, 1.50 M NaCl, pH 7.4, was added. NaCNBH3 was dissolved in the ligand/buffer solution to a final concentration of 5 mg/ml and 1.0 g of POROS 20 AL was suspended in the same solution.
The suspension was mixed gently on a shaker for 1 min at room
temperature. An additional 2 ml of 100 mM phosphate, 1.50 M NaCl, pH 7.4, was added to the suspension and the mixture
was shaken continuously. This step was repeated every 5 min until the
mixture volume was 25 ml. After additional shaking for 2 h, the
medium was filtered on a sintered glass funnel. The matrix was
suspended in 20 ml of 0.2 M Tris-HCl, 5 g/liter
NaCNBH3, pH 7.2, and mixed gently on a shaker for 30 min at
room temperature. After the media had been washed in a sintered glass
funnel using 100 ml of 10 mM phosphate, pH 7.4, 100 ml of
1.0 M NaCl, and another 100 ml of 10 mM
phosphate, pH 7.4, the matrix was packed in a PEEK column (4.6 × 50 mm).
Synthesis of the Aprotinin Affinity Matrix
Aprotinin affinity matrix was synthesized according to the
synthesis of the protamine affinity matrix, but using aprotinin as
ligand. After immobilization of the ligand the matrix was packed in a
PEEK column (4.6 × 50 mm).
Purification of Xylosyltransferase from JAR Cell Culture
Supernatant
Fractionated ammonium sulfate precipitation and chromatography
steps were performed at room temperature, ultrafiltration and diafiltration were carried out at 4 °C. 18.5 liters of JAR cell culture supernatant collected from three hybrid hollow-fiber
bioreactors, TECNOMOUSE, each containing 5 culture cassettes, was
concentrated to 800 ml with ultrafiltration cells using YM1 cellulose
membranes. The retentate was centrifuged at 4,000 × g
for 1 h. The supernatant was decanted, and the pellet was discarded.
Step 1: Fractionated Ammonium Sulfate Precipitation--
Solid
ammonium sulfate was added to the supernatant to 28% saturation. After
1 h at room temperature the suspension was centrifuged at
4,000 × g for 2 h, the supernatant was decanted,
and the precipitate was removed. Additional ammonium sulfate was added
to the solution to the point of 40% saturation, and the suspension was
allowed to stand for 1 h. To recover the precipitate the
supernatant was decanted after the suspension was centrifuged at
4,000 × g for 2 h. Before chromatography on
immobilized heparin the precipitate was dissolved in 460 ml of buffer A
(20 mM sodium acetate, pH 6.0).
Step 2: Heparin Affinity Chromatography on POROS 20 HE2--
The
step 1 product was passed through a 0.2-µm filter. 4.0 ml of the
filtrate was applied to a POROS 20 HE2 column (16 × 100 mm)
equilibrated with buffer A at a flow rate of 40 ml/min. After washing
the column with 100 ml of buffer A the XT activity was eluted with the
same buffer containing NaCl. The NaCl concentration was increased
stepwise: 20 ml of buffer A, 0.09 M NaCl; 20 ml of buffer
A, 0.15 M NaCl; 30 ml of buffer A, 0.24 M NaCl;
24 ml of buffer A, 0.30 M NaCl; 24 ml of buffer A, 0.60 M NaCl; 24 ml of buffer A, 1.00 M NaCl; and 24 ml of buffer A, 1.89 M NaCl. Fractions of 38 ml each were
collected and the XT activity was measured. The procedure was repeated
115 times by cyclic chromatography and the fractions containing XT
activity (115 × 38 ml) were collected.
Step 3: Ion Exchange Chromatography on POROS 20 HQ--
Collected fractions from step 2 were desalted using
diafiltration with YM1 cellulose membranes and ultrafiltration cells. After concentration of the desalted protein solution to 0.05 liter using analogous techniques the XT-enriched solution was subjected to
ion exchange chromatography. 4.0 ml of the XT solution was applied onto
the POROS 20 HQ column (16 × 100 mm) previously equilibrated with
buffer A at a flow rate of 40 ml/min. The column was washed with 80 ml
of buffer A, and the adsorbed protein was eluted stepwise using the
same buffer containing 0.07 M NaCl (88 ml), 0.18 M NaCl (120 ml), and 0.36 M NaCl (120 ml)
followed by a linear gradient of 0.36-1.00 M NaCl (200 ml)
and another step of buffer A, 2.0 M NaCl (120 ml). 50-ml
fractions were collected and assayed for activity and evaluated by
SDS-PAGE. Chromatography was repeated 13 times, and the fractions
exhibiting XT activity (13 × 50 ml) were collected for affinity chromatography.
Step 4: Affinity Chromatography on Protamine
Chloride--
XT-containing solution from step 3 was desalted as
described above and concentrated to 5 ml by ultrafiltration with YM1
cellulose membranes. The ultrafiltration product was passed through a
0.2-µm filter. 100 µl of the filtrate was loaded onto a protamine
chloride-POROS column (4.6 × 50 mm) equilibrated with buffer A. The flow rate was 10 ml/min. The column was washed with 10.0 ml of
buffer A, and the adsorbed fraction was eluted with the same buffer
containing NaCl by a stepwise increase of the NaCl concentration: 6.6 ml of buffer A, 0.04 M NaCl; 6.6 ml of buffer A, 0,06 M NaCl; 6.6 ml of buffer A, 0.23 M NaCl
followed by a linear gradient of 0.23-1.20 M NaCl (4.2 ml)
in buffer A. Fractions of 6.0 ml were collected, assayed for XT
activity, and evaluated by SDS-PAGE. Cyclic chromatography was repeated
50 times. The purified enzyme was collected, concentrated to 1.0 ml
using ultrafiltration techniques and stored at Step 5: SDS-PAGE--
The protein composition of various
fractions was estimated by SDS-PAGE. Briefly, 12.1 µl of sample was
added to 4.7 µl of sample buffer (1.00 M Tris-HCl, 1.17 M sucrose, 0.28 M SDS, 2.08 mM
EDTA, 0.88 mM Serva Blue G250, 0.70 mM phenol
red, 0.10 M dithiothreitol, pH 8.5) and heated for 10 min
at 99 °C. After the sample had been loaded, SDS-polyacrylamide gel
electrophoresis was carried out on a 4-12% bis-tris polyacrylamide
gel with MOPS running buffer (1.00 M MOPS, Tris, 69.3 mM SDS, 20.5 mM EDTA, pH 7.7). Protein bands
were detected by Coomassie Brilliant Blue or silver staining. The
Coomassie bands were excised and characterized by MALDI mass spectrometry and amino acid sequence analysis.
MALDI Mass Spectrometry
Coomassie-stained proteins were excised from the gel, repeatedly
washed with H2O and H2O/acetonitrile, and
digested overnight with trypsin and endoproteinase Lys-C at 37 °C.
The peptides generated in the supernatant were analyzed by MALDI mass
spectrometry. Sample preparation was achieved following the thin film
preparation techniques (13).
Briefly, aliquots of 0.3 µl of a nitrocellulose containing saturated
solution of MALDI mass spectra were recorded in the positive ion mode with delayed
extraction on a Reflex II time-of-flight instrument equipped with a
SCOUT multiprobe inlet and a 337-nm nitrogen laser. Ion acceleration
voltage was set to 20.0 kV, the reflector voltage was set to 21.5 kV
and the first extraction plate was set to 15.4 kV. Mass spectra were
obtained by averaging 50-200 individual laser shots. Calibration of
the spectra was performed internally by a two-point linear fit using
the autolysis products of trypsin at m/z 842.50 and 2211.10.
Amino Acid Sequence Analysis of XT
The 120-kDa Coomassie-stained protein was excised from the gel,
repeatedly washed with H2O and
H2O/acetonitrile, and digested with trypsin and
endoproteinase Lys-C overnight. For HPLC separation the excised gel
fragment was extracted twice with 0.1% trifluoroacetic acid, 60%
acetonitrile. The extracted enzymatic fragments were separated on a
capillary HPLC system equipped with a 140B solvents delivery system (PE
Biosystems), Acurate splitter (LC-Packings), UV absorbance detector
759A (PE Biosystems), U-Z capillary flow cell (LC-Packings), and
Probot fraction collector (BAI) using a reversed-phase column (Hypersil
C18 BDS, 3 µm, 0.3 × 150 mm) and a linear gradient from 12%
acetonitrile, 0.1% trifluoroacetic acid to 64% acetonitrile, 0.08%
trifluoroacetic acid in 90 min with a flow rate of 4 µl/min at room temperature.
Peptide elution was monitored at 214 nm and individual fractions from
the HPLC separation were analyzed by MALDI mass spectrometry. Sequence
analysis of separated fragments was performed on a Procise Protein
Sequencer 494 cLC using standard programs supplied by PE Biosystems.
Measurement of XT Activity
Determination of XT activity is based on the incorporation of
[14C]D-xylose into the recombinant bikunin
according to a previously described method (14). Briefly, the standard
reaction mixture (100 µl) for the assay contained 50 µl of XT
solution, 25 mM MES, pH 6.5, 25 mM KCl, 5 mM KF, 5 mM MgCl2, 5 mM
MnCl2, 1.0 µM
UDP-[14C]D-xylose, and 1.5 µM
recombinant bikunin. Following incubation at 37 °C for 15 min, the
reaction mixtures were placed on nitrocellulose discs. After drying,
the discs were washed once for 10 min with 10% trichloroacetic acid
and three times with 5% trichloroacetic acid. Incorporated
radioactivity was measured by liquid scintillation counting. The enzyme
activity was expressed in units (1 unit = 1 µmol of incorporated
xylose per min).
Antiserum Preparation
The synthetic peptide CSRQKELLKRKLEQQEK deduced from the
sequenced peptides 2 and 10 of the enzymatically cleaved XT was
synthesized, purified by HPLC, and used for immunization of rabbits
(BioScience, Germany). Polyclonal antiserum was obtained by injection
of the above antigen followed by booster injections at 3-week
intervals, 4 times in total, into Charles rabbits.
Preparation of Solid-phase Antigen
The antigen CSRQKELLKRKLEQQEK was immobilized on POROS 20 EP.
After 1.6 mg of antigen was dissolved in 1.2 ml of 10 mM
phosphate, 0.15 M NaCl, pH 7.4, 0.60 ml of 100 mM phosphate, 1.50 M NaCl, pH 7.4, was added.
400 mg of POROS 20 EP was suspended in the solution and the suspension
was mixed gently on a shaker at room temperature. At 10-min intervals,
five times in total, an additional 0.24 ml of 100 mM
phosphate, 1.50 M NaCl, pH 7.4, was added to the
suspension. After additional shaking for 5 days at room temperature the
suspension was filtered on a sintered glass funnel. The matrix was
suspended in 4 ml of 0.2 M phosphate, 0.1 M
2-mercaptoethanol, pH 7.4, and mixed on a shaker for 2 h at room
temperature. The matrix was washed in a sintered glass funnel using 20 ml of 10 mM phosphate, 0.15 M NaCl, pH 7.4, and
20 ml of 1.0 M NaCl. After additional washing with 20 ml of
10 mM phosphate, pH 7.4, the matrix was packed in a PEEK
column (4.6 × 50 mm).
Antibody Purification
Antiserum was adjusted to 50 mM Tris-HCl, pH 8.0. The solution was clarified by passage through a 0.2-µm filter. 0.4 ml
of filtrate was applied at 10 ml/min to the antigen column previously equilibrated with buffer B (50 mM Tris-HCl, pH 8.0). After
the column was washed with 4.1 ml of buffer B and with 12.5 ml of buffer B, 0.15 M NaCl, the adsorbed antibody was eluted
using 12.4 ml of 50 mM sodium citrate, 0.15 M
NaCl, pH 3.0, followed by 3.4 ml of 100 mM sodium citrate,
1.5 M NaCl, pH 3.0. The eluate was collected as 10-ml
fractions in tubes containing 2 ml of 0.5 M Tris-HCl, pH
8.0, to immediately neutralize the citric acid.
Preparation of Immunoaffinity Column
Purified antibody was concentrated to a protein concentration of
0.3 mg/ml using ultrafiltration with YM1 cellulose membranes. The
antibody solution was adjusted to 10 mM phosphate, 0.15 M NaCl, pH 7.4. After filtration of the solution through a
0.2-µm filter 100 µl of filtrate was applied at 0.2 ml/min to a
POROS 20 PA column (2.1 × 30 mm). This step was repeated 17 times.
Adsorbed antibody was cross-linked using cross-linking solution (100 mM triethanolamine, pH 8.5). After the column was washed with 5 ml of buffer C (10 mM phosphate, 0.15 M
NaCl, pH 7.4) 2 ml of cross-linking solution was applied at 0.5 ml/min
onto the cartridge. The procedure was repeated 6 more times, using a
total volume of 14 ml of cross-linking solution. To block unreacted functional groups on the cross-linking reagents 2 ml of 100 mM monoethanolamine, pH 9.0 (quenching solution), was
loaded onto the cartridge at 0.5 ml/min. The column was washed using 2 ml of buffer C and the cross-linking step was repeated using another 2-ml quenching solution. The immunoaffinity column was cycled between
buffer C and 12 mM HCl, 0.15 M NaCl 3 times
using a total volume of 12 ml of solution.
Immunoaffinity Column Purification of XT
XT-containing fractions eluted from the heparin affinity matrix
were desalted using diafiltration with YM1-cellulose membranes and
passed through a 0.2-µm filter. 100 µl of this XT sample was applied to the immunoaffinity column equilibrated with buffer D (20 mM Tris-HCl, pH 8.0) at a flow rate of 1 ml/min. The column was washed with 1.4 ml of buffer D and with 8.5 ml of buffer D, 0.15 M NaCl. The XT activity was eluted with 4.2 ml of 12 mM HCl followed by 1.2 ml of 12 mM HCl, 1.5 M NaCl. Alternatively the elution was performed using 100 µl of antigen at 1 mg/ml in buffer D. Fractions (1 ml) were collected
into tubes containing 1 ml of 0.1 M Tris-HCl, pH 8.0. The
XT activities of the fractions were determined.
Aprotinin Affinity Chromatography
200 µl of desalted XT solution from the heparin purification
step was applied at 10 ml/min to the aprotinin column previously equilibrated with buffer A. After washing the column with 6.6 ml of
buffer A the adsorbed protein was eluted stepwise using the same buffer
containing 0.3 M NaCl (10.0 ml), 0.54 M NaCl
(10.0 ml), 1.00 M NaCl (10.0 ml), and 1.50 M
NaCl (2.4 ml). Fractions of 5 ml were collected and assayed for XT activity.
Western Blot Analysis
For Western blot analysis, proteins were transferred to
polyvinylidene difluoride membrane in a semi-dry instrument (Novex). After transfer nonspecific antibody-binding sites were blocked with 2%
bovine serum albumin in 0.1 M Tris-HCl, pH 7.2, for 1 h at room temperature. The membrane was incubated with antiserum in 50 mM phosphate, 0.15 M NaCl, 0.5 ml/liter Tween
20, pH 7.4, at 1:1000 dilution for 1 h. Bound antibody was
detected using a second anti-rabbit goat immunoglobulin coupled to
horseradish peroxidase at a 1:1000 dilution. The blot was developed
using 4-chloro-1-naphthol.
Gel Filtration Chromatography
A sample of 100 µl from the heparin purification step was
applied at 1.0 ml/min to a TSK G3000 SW column (30 cm × 7.5 mm, 10-µm particle size) which had previously been equilibrated with buffer A, 0.15 M NaCl. Proteins were eluted with the same
buffer. Fractions of 200 µl were collected and tested for XT
activity. Column calibration was performed using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa),
ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A
(13.7 kDa).
N-Glycosidase F Digestion
Aliquots (1 µg) of XT were digested with 3, 1 × 10 Measurement of Protein Concentration
Protein concentration was estimated by absorbance at 280 nm
assuming E1 cm1% = 10.0 or with
the bicinchoninic acid protein assay using bovine serum albumin as a standard.
Isolation of Xylosyltransferase from Cell Culture Supernatant
18.5 liters of enriched cell culture supernatant (equivalent to
2,000 liters normal cell culture supernatant) of serum-free cultivated
JAR choriocarcinoma cells was required to isolate the XT in trace
amounts. The so far unknown protein with a molecular mass of 120 kDa
was enzymatically digested and a partial amino acid sequence was
determined by Edman degradation and MALDI-TOF mass spectrometry. A
summary of the purification is shown in Table I and explained in detail below.
-D-xylosyltransferase (EC 2.4.2.26, XT)
initiates the biosynthesis of glycosaminoglycan lateral chains in
proteoglycans by transfer of xylose from UDP-xylose to specific serine
residues of the core protein. In this study, we report the first
isolation of the XT and present the first partial amino acid sequence
of this enzyme. We purified XT 4,700-fold with 1% yield from
serum-free JAR choriocarcinoma cell culture supernatant. The
isolation procedure included a combination of ammonium sulfate
precipitation, heparin affinity chromatography, ion exchange
chromatography, and protamine affinity chromatography. Among other
proteins an unknown protein was detected by matrix-assisted laser
desorption ionization mass spectrometry-time of flight analysis in the purified sample. The molecular mass of this protein was determined as 120 kDa by SDS-polyacrylamide gel electrophoresis. The
isolated protein was enzymatically cleaved by trypsin and endoproteinase Lys-C. Eleven peptide fragments were sequenced by Edman
degradation. Searches with the amino acid sequences in protein and EST
data bases showed no homology to known sequences. XT was enriched by
immunoaffinity chromatography with an immobilized antibody against a
synthetic peptide deduced from the sequenced peptide fragments and was
specifically eluted with the antigen. In addition, XT was purified
alternatively with an aprotinin affinity chromatography and was
detected by Western blot analysis in the enzyme-containing fraction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-xylosyltransferase (EC 2.4.2.26,
XT)1 initiates the
biosynthesis of glycosaminoglycan lateral chains in proteoglycans by
the transfer of xylose from UDP-D-xylose to specific serine
residues of the core protein (3, 4). Only selected serine residues of
the core protein were recognized by XT. Several comparisons of amino
acid sequences of known glycosaminoglycan attachment sites in different
proteins resulted in a recognition sequence composed of the amino acids
a-a-a-a-G-S-G-a-b-a, with a = E or D and b = G, E, or D (5).
The highest sensitivity for XT activity in a radiochemical test was
reached using a protein acceptor containing this recognition sequence.
This was confirmed by the determination of Michaelis-Menten
(Km) constants for in vitro xylosylation
of different proteins and synthetic peptides in comparison to silk,
which was formerly used. The use of recombinant bikunin, which contains
the recognition sequence as acceptor, enables an accurate and precise
determination of XT activity even in serum. Bikunin is the inhibitory
component of the human inter-
-trypsin inhibitor and a natural core
protein which is quantitatively modified by one chondroitin sulfate chain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
75 °C.
-cyano-4-hydroxycinnamic acid in acetone were deposited
onto individual spots on the target. Subsequently, 0.8 µl of 10%
formic acid and 0.4 µl of the digest sample was loaded on top of the
thin film spots and allowed to dry slowly at ambient temperature. To
remove salts from the digestion buffer the spots were washed with 10%
formic acid and with H2O.
3 units of N-glycosidase F at 37 °C for 1 and 12 h according to the method recommended by the manufacturer.
The samples were then subjected to SDS-PAGE, and protein bands were
detected by silver staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Purification of XT
Step 1: Fractionated Ammonium Sulfate Precipitation-- XT of the ammonium sulfate precipitable fraction was dissolved in 0.46 liters of buffer A with solubilization of 79.5% of the original activity.
Step 2: Heparin Affinity Chromatography on POROS 20 HE--
4 ml
of XT-enriched solution from step 1 was loaded onto the POROS 20 HE
column. XT activity was completely retained on the column. More than
70% of total protein passed through the column. Contaminating protein
was eluted at a low NaCl concentration. 44% of the XT activity bound
to the heparin matrix emerged at 0.5 M NaCl (Fig.
1A).
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Step 3: Ion Exchange Chromatography on POROS 20 HQ-- 4 ml of the desalted XT-containing fraction from step 2 was loaded onto the POROS 20 HQ column equilibrated in buffer A. More than 98% of the XT activity bound to the resin. The column was then eluted stepwise with NaCl in buffer A (Fig. 1B). XT-containing fractions were collected.
Step 4: Affinity Chromatography on Protamine Chloride--
The
product of step 3 was desalted and concentrated using dia- and
ultrafiltration. 100 µl of the protein solution was applied to the
POROS protamine chloride column previously equilibrated with buffer A. Approximately 95% of the transferase activity bound to the column,
whereas 75% of the contaminating protein did not. Additional proteins
were eluted with buffer A containing low NaCl concentrations. Enzyme
activity was eluted at ~0.15 M NaCl (Fig. 1C).
The enzyme activity was stable for at least 6 months at 75 °C.
Step 5: SDS-PAGE--
XT-containing fractions from steps 1-4 were
subjected to SDS-PAGE on a 4-12% gradient polyacrylamide gel (Fig.
2, panel A). Coomassie-stained
protein bands were excised and characterized by MALDI-TOF mass
spectrometry after tryptic digestion. The molecular mass of an unknown
protein was determined as 120 kDa (Fig. 2, panel B).
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Amino Acid Sequence Analysis of XT
The 120-kDa protein from the excised band was digested with trypsin and endoproteinase Lys-C. The proteolytic fragments were separated by reversed-phase HPLC, and selected peptides were subjected to automatic amino acid sequence analysis. Table II shows the obtained amino acid sequences.
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Immunochemical Detection of XT
Polyclonal antibodies against the synthetic peptide
CSRQKELLKRKLEQQEK deduced from the peptides 2 and 10 of the
enzymatically cleaved 120-kDa protein were covalently bound on POROS 20 PA. About 50% of the XT activity of an applied sample was bound (Fig. 3, panel A) when a partially
purified XT sample obtained by heparin affinity chromatography
(purification step 2) was loaded onto the column. 58% of the adsorbed
XT activity was eluted with 150 mM NaCl, and the rest was
eluted with 12 mM HCl. Furthermore, the adsorbed XT
activity was also eluted from the solid phase when 100 µl (1 mg/ml)
of the synthetic peptide was added to the mobile phase (Fig. 3,
panel C). When immobilized preimmune serum was used as
affinity matrix (negative control) no XT activity was adsorbed to or
eluted from the matrix (Fig. 3, panel B).
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The desalted XT fraction after heparin affinity chromatography
(purification step 2) was loaded on an aprotinin affinity column. The
elution profile shows four major protein peaks (Fig.
4, panel I). 61% of the XT
activity adsorbed to the aprotinin matrix emerged at 0.30 M
NaCl and another 21% at 0.54 M NaCl. A single 120-kDa band
of the XT-containing fractions was detected by Western blot analysis
with the polyclonal antibodies (Fig. 4, panel B).
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Determination of the Molecular Weight of XT
100 µl of heparin affinity purified XT was separated under
nonreducing and nondenaturating conditions using a TSK G3000 SW column.
Two XT activity maxima were detected at 500 and 120 kDa (Fig.
5, panel A). The molecular
mass of the 120-kDa protein was reduced about 3% after
N-glycosidase F digestion as shown by SDS-PAGE (Fig. 5,
panel B).
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DISCUSSION |
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We have purified UDP-D-xylose:proteoglycan core
protein -D-xylosyltransferase to apparent homogeneity
from JAR choriocarcinoma cell culture. The isolated protein was a
single-stranded polypeptide with a molecular mass of 120 kDa. The
protein was enzymatically cleaved and 11 peptide fragments were
sequenced by Edman degradation. Our results do not agree with previous
reports on the isolation of the enzyme from embryonic chicken cartilage
(15) and rat chondrosarcoma (16). No amino acid sequence data of the
protein were reported; however, a complex heteromerous tetramer
structure of a 120-kDa protein was postulated.
Like other glycosyltransferases, XT is present only in very small amounts in animal tissues but unlike other glycosyltransferases, more than 90% of XT is enriched in the medium of cultured cells (11). The highest secretion of XT activity was measured in JAR choriocarcinoma cell culture as shown in our previous study (6), in which sternal cartilage chondrocytes and 21 different human cell lines were examined. To produce a highly enriched XT solution for the isolation of XT, we adapted JAR choriocarcinoma cells to hollow fiber culture conditions using a novel bioreactor (TECNOMOUSE) and Ultradoma-PF medium without serum addition as nutrient.
For purification of XT a combination of classic separation methods and new affinity matrices was employed. Previous studies have shown that heparin reduced XT activity indicating a strong interaction of heparin with the enzyme (10). Therefore, we used a heparin matrix as an affinity ligand for the XT. When applied to immobilized heparin, XT was completely adsorbed at the matrix and the XT activity was eluted only with a high salt concentration after most contaminating proteins were removed from the matrix.
Protamine chloride is well known as cationic activator for several sulfotransferases (17-20), so we investigated the effect of protamine chloride on the XT. An increased XT activity was measured when protamine chloride was added to the XT assay solution,2 indicating an interaction of these arginine-rich proteins with XT. Therefore, we synthesized a protamine chloride affinity matrix using an aldehyde-activated perfusion medium as support. The interaction of XT with this affinity matrix resulted in a 13.6 times enrichment of XT. The protamine chloride affinity chromatography was the most efficient purification step during the isolation of the XT.
Immobilized aprotinin, a Kunitz-type proteinase inhibitor, was found to be another appropriate affinity matrix for enrichment of XT, as it was able to adsorb XT quantitatively. Therefore, it was used for alternative purification of XT.
Different lines of evidence showed that the isolated protein corresponds to the XT: (a) XT activity was enriched using immobilized antibodies raised against a synthetic peptide deduced from the 120-kDa protein, and the XT activity could be competitively eluted with this peptide. (b) Immunoblot analysis of aprotinin affinity purified XT corresponds with the 120-kDa protein.
The unknown protein in the sample obtained by the combined purification steps migrated as a broad band on SDS-PAGE with an molecular mass of 120 kDa. Stoolmiller et al. (21) determined the molecular mass of XT from chicken cartilage as 110 to 120 kDa by gel filtration on Sephadex G-200, which is in agreement with our results. The enzyme appears to be a monomer based on the similarity between its molecular weight on SDS-PAGE and gel filtration. However, nonreducing and nondenaturating gel filtration chromatography with heparin affinity-purified XT from JAR cell culture supernatant shows an additional peak of XT activity at a molecular mass of ~500 kDa. Our previous results demonstrated that the enzyme is secreted simultaneously with chondroitin sulfate proteoglycans into the extracellular space, suggesting that XT is associated with proteoglycans (11).
Treatment of XT with N-glycosidase F resulted in a decrease of the molecular mass from 120 to 116 kDa, suggesting that the XT is a glycoprotein. Most glycosyltransferases purified so far are glycoproteins, which contain up to 15% carbohydrate by weight.
In eukaryotes, most glycosyltransferases are located in the endoplasmatic reticulum and Golgi membranes and require the addition of detergents for solubilization. However, glycosyltransferases were also found in body fluids as soluble enzymes, and were purified to homogeneity (22, 23). Glycosyltransferases have virtually no sequence homology to each other, although these Golgi enzymes share a common domain structure. They are all type II membrane proteins, composed of a short NH2-terminal cytoplasmic domain, a transmembrane domain, a stem region of variable length, and a large COOH-terminal globular catalytic domain (24, 25). Several investigations suggest that soluble glycosyltransferases were formed from membrane-bound enzymes by proteolytic cleavage between the catalytic domain and the transmembrane domain (26). Most reactions catalyzed by glycosyltransferases take place in the lumen of the Golgi apparatus, but xylosylation is performed in a pre-Golgi compartment in chicken chondrocytes (4, 9).
A comparison of the molecular mass of XT with other glycosyltransferases involved in biosynthesis of proteoglycans shows that the XT is larger than the other enzymes. Another difference is that nearly all proteoglycan glycosyltransferases are tightly bound to the membrane of the endoplasmic reticulum, whereas XT is secreted into the extracellular space (11).
The size of the XT and its secretion into the extracellular space may suggest that the enzyme is involved in additional molecular processes. Phosphorylation of C-2 of xylose has been discovered in both chondroitin sulfate (27) and heparan sulfate proteoglycans (28). Xylose is generally not phosphorylated in proteoglycans (29, 30), because the addition of the first glucuronic acid residue is followed by a rapid dephosphorylation of the xylose (31). It is possible that the XT is involved in the phosphorylation or dephosphorylation process. The function of the xylose phosphorylation is not clear, but it might provide a signal for secretory transport of proteoglycans (32).
Like other glycosyltransferases, XT requires a divalent metal ion for enzyme activity. Manganese is most effective for XT activity followed by magnesium (33). Many glycosyltransferases contain a DxD motif (34-36), suggesting that this motif is involved in binding the metal-ion cofactor and the donator substrate (37). A DxD sequence was also found in peptide 8 obtained from the enzymatically cleaved XT.
In conclusion, we have determined the first partial amino acid sequence
of human XT, and produced antibodies raised against this enzyme that
initiates the glycosaminoglycan synthesis in proteoglycans. On the
basis of this sequence, it should be possible to clone the cDNA
encoding the XT using degenerated primers and polymerase chain reaction
cloning strategy. Furthermore, the antibodies against the XT can be
used to develop an immunological test system for the a rapid and
sensitive measurement of this protein as a new diagnostic tool in
clinical praxis.
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ACKNOWLEDGEMENTS |
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We thank Anne-Kathrin Vollmer, Anja Reuße-Kaup, and Sabine Fiedler for excellent technical assistance and Grainne Delany for linguistic advice.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 49-5731-971390; Fax: 49-5731-972307; E-mail: kkleesiek@hdz-nrw.de.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M005111200
2 J. Kuhn, C. Götting, M. Schnölzer, T. Kempf, T. Brinkmann, and K. Kleesiek, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: XT, xylosyltransferase; HPLC, high performance liquid chromatography; MOPS, 3-(N-morpholino)propanesulfonic acid; MALDI-TOF mass spectrometry, matrix-assisted laser desorption/ionization time of flight mass spectrometry; EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid.
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