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Determination of xylosyltransferase activity in serum with recombinant human bikunin as acceptor

Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstr. 11, 32545 Bad Oeynhausen, Germany.
^a Author for correspondence. Fax +49 5731 972307; e-mail HDZ.ILTM{at}post.uni-bielefeld.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Xylosyltransferase (XT) is the chain-initiating enzyme of the biosynthesis of chondroitin sulfate. So far, XT activity has been detected by incorporation of [¹⁴C]xylose in chemically deglycosylated cartilage proteoglycan or silk fibroin. However, these acceptors allow no reliable determination in blood. We found that recombinant bikunin is an excellent acceptor for XT. The Michaelis–Menten constants for the xylosylation of silk, deglycosylated cartilage proteoglycans, and bikunin were 545, 155, and 0.9 µmol/L, respectively. With recombinant bikunin as acceptor, we developed a sensitive assay that allows a precise determination of XT activity in serum. We measured the serum XT activities of 500 blood donors and observed a considerable sex and age dependence. XT activities in men (0.77–1.50 mU/L) were ~30% higher than in women (0.58–1.20 mU/L) and reached a maximum in donors of ~40 years of age. During the menstrual cycle, serum XT activity showed a significant coincidence with the ß-estradiol concentration, and in the first trimester of pregnancy we observed a strong increase in serum XT activity.

Key Words: indexing terms: glycosyltransferases • proteochondroitin sulfates • glycoproteins • xylose • estradiol

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Major components of the extracellular matrix in connective tissue are chondroitin sulfate proteoglycans, in which chondroitin sulfate chains are glycosidically linked by xylose–serine bonds to appointed sites of core proteins. In the biosynthesis of chondroitin sulfate proteoglycans, protein xylosyltransferase (EC 2.4.2.26; XT) catalyzes as initial and rate-limiting step the transfer of xylose from UDP-xylose to the protein (1)(2).¹ Earlier investigations on chondroitin sulfate proteoglycans showed that glycosylated serine residues are mostly followed by a glycine residue (3). Comparison of amino acid sequences of chondroitin sulfate attachment sites in different proteoglycans (Table 1 ) led to a consensus sequence -a-a-a-x-S-G-x-G- with at least one acidic amino acid "a" and variable amino acids "x".

For determination of XT activity according to Stoolmiller et al. (4), the samples were incubated with UDP-[¹⁴C]xylose and an appropriate acceptor. The incorporated radioactivity indicated the amount of XT activity. Acceptors used so far were deglycosylated core proteins from cartilage proteoglycans (4), silk fibroin (5), and several peptides (6).

In previous investigations we found highly increased XT activities in pathological synovial fluids of patients with chronic joint diseases (7). The activities indicated the degree of cartilage destruction, independent of inflammation. However, the test did not allow a precise determination of the lower XT activities in serum.

A natural substrate for XT seems to be bikunin, the inhibitory component of human inter--trypsin inhibitor (Fig. 1 ). Bikunin carries a single chondroitin sulfate chain, which is essential for the structure of the inhibitor. The chondroitin sulfate attachment site of bikunin contains all elements of the consensus sequence, which seem to be responsible for recognition by XT. We expressed bikunin in Escherichia coli and found the nonglycosylated recombinant protein to be a potent acceptor for xylose in the XT assay.

View larger version (31K): [in this window] [in a new window]   Figure 1. Structure of human inter--trypsin inhibitor and the chondroitin sulfate attachment site of bikunin. Bold amino acids are essential or important for recognition by XT in chondroitin sulfate biosynthesis. Gal, galactose; GlcA, glucuronic acid; GalNAcSO3, N-acetylgalactosamine sulfate.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
materials
UDP-[¹⁴C]xylose (9.88 kBq/nmol) was purchased from DuPont (Bad Homburg, Germany), nitrocellulose discs (25 mm diameter) from Sartorius (Göttingen, Germany), and Immobilon-AV membrane from Millipore (Eschborn, Germany). The liquid scintillation counter LS 500TD and scintillation cocktail were supplied by Beckman (Fullerton, CA). Chang liver cell line was obtained from ICN (Meckenheim, Germany), RNA Insta-Pure^TM from Eurogentec (Seraing, Belgium), the enzymes used for cDNA cloning from Boehringer Mannheim (Mannheim, Germany), the vector pET 15b and the E. coli strain BL21 DE3 from Novagen (Madison, WI), and chelating Sepharose and Q-Sepharose from Pharmacia (Uppsala, Sweden). The oligonucleotides were synthesized by Genosys (Cambridge, UK). Minimum essential medium (MEM), pronase E, collagenase XI, fetal calf serum, and antibiotic/antimycotic solution were obtained from Sigma (Deisenhofen, Germany). The peptide QEEEGSGGGQKK was synthesized by Quality Controlled Biochemicals (Hopkinton, MA). Silk fibroin and HF- and trifluoromethanesulfonic acid (TFMS)-degraded proteoglycan from bovine nasal septum cartilage were prepared as described elsewhere (7). All other chemicals in p.a. quality were purchased from Merck (Darmstadt, Germany).

serum samples
Venous blood samples were collected in serum monovettes from Sarstedt (Nümbrecht, Germany). After clotting and centrifugation the serum was stored at -70 °C.

Samples were collected from (a) male (n = 294) and female (n = 266) blood donors, 18 to 65 years old; (b) healthy men (n = 3) and women not using oral contraceptives (n = 5), three times a week for 6 weeks; (c) a 28-year-old pregnant woman, three times a week during the first 2 months after conception and once a week until birth; and (d) pregnant women in the third trimester (n = 30), every 2 weeks.

preparation of human chondrocyte cultures
Small pieces of sternal cartilage were obtained during open heart surgery. The cartilage was cut aseptically, incubated for 90 min at 37 °C in 10 g/L pronase E in MEM, and for several hours in 2.5 g/L collagenase XI, until the tissue was digested (8). The free cells were cultivated in MEM supplemented with 100 mL/L fetal calf serum and antibiotic/antimycotic solution. After 4–5 days the conditioned medium was separated by ion-exchange column chromatography on Q-Sepharose. The proteins were bound to the resin in 50 mmol/L Tris-HCl buffer (pH 8.0) and eluted by a gradient from 0 to 1.0 mol/L NaCl in Tris buffer. Fractions between 0.40 and 0.45 mol/L NaCl showed high XT activities.

synthesis of recombinant bikunin
cDNA cloning was performed with standard methods (9). The RNA from Chang liver cells was isolated with RNA Insta-Pure. Bikunin cDNA was synthesized by reverse transcription with oligo(dT)₁₅ primer and Moloney murine leukemia virus reverse transcriptase and subsequent PCR with the bikunin-specific primers 5'-TCTCAGCATATGGCTGT-GCTACCCCAAGAA and 5'-GGCCAGGGATCCTCAGGAGAAGCGCAGCAG. The amplified product was cut with the restriction enzymes BamHI and NdeI, ligated into the vector pET 15b, and expressed in the E. coli strain BL 21 DE3 (10). The recombinant protein carried a leader sequence with six histidines and was purified in one step by affinity chromatography with Ni²⁺-chelating resin.

optimization of substrate concentrations, reaction time, and temperature for xylosylation
To investigate the dependence of the xylose transfer rate by XT on the substrate concentrations, 50 µL of a partially purified and enriched XT preparation from chondrocyte cultures was incubated with 25 mmol/L 4-morpholinoethanesulfonic acid (pH 6.5), 25 mmol/L KCl, 5 mmol/L KF, 5 mmol/L MgCl₂, 5 mmol/L MnCl₂, and various amounts of UDP-[¹⁴C]xylose and recombinant bikunin in a total volume of 100 µL. After incubation at 34 °C for 1 h, the mixture was placed on a nitrocellulose disc and allowed to dry. It was washed for 10 min with 10% trifluoroacetic acid and three times with 5% trifluoroacetic acid. Incorporated radioactivity was determined by liquid scintillation counting.

For the determination of the temperature optimum of XT, reaction mixtures with 0.5 µmol/L UDP-[¹⁴C]xylose and 1.5 µmol/L bikunin were measured after incubation at 4, 25, 30, 34, 37, 40, and 45 °C. The time dependence of the xylosylation rate was investigated with incubation times of 20, 40, 60, 90, 120, and 180 min. All tests were performed three times.

determination of xt activity
The method for determination of XT activity is based on the binding of [¹⁴C]xylose to recombinant bikunin as acceptor. The reaction mixture for the assay contains, in a total volume of 100 µL, 50 µL of sample, 25 mmol/L 4-morpholinoethanesulfonic acid (pH 6.5), 25 mmol/L KCl, 5 mmol/L KF, 5 mmol/L MgCl₂, 5 mmol/L MnCl₂, 0.5 µmol/L UDP-[¹⁴C]xylose, and 1.5 µmol/L recombinant bikunin. After incubation at 34 °C for 1 h, the mixture was placed on a nitrocellulose disc and allowed to dry. It was washed for 10 min with 10% trifluoracetic acid and three times with 5% trifluoracetic acid. Incorpo- rated radioactivity was determined by liquid scintillation counting. The enzyme activity was: 1 U = 1 µmol of incorporated xylose · min^-1).

To establish the normal range of XT activity, we measured the enzyme activities in blood donors, calculated the mean values, and estimated the 90% ranges graphically.

precision, recovery, and detection limit of the xt assay
The precision of the assay was determined by using three serum samples with different XT activities (mean values 0.60, 0.95, 1.72 mU/L). The intraassay and interassay precision was obtained when each sample was measured in triplicate on 20 different days.

The accuracy was investigated by measuring the recovery of enzyme activity in heat-inactivated serum or PBS, to which different amounts of an XT-enriched solution were added. For definition of the detection limit of the assay, serum was heated 3 h at 65 °C to inactivate XT. The inactivated serum was measured 10 times and the concentration corresponding to the upper 3 SD limit was defined as the detection limit.

determination of michaelis–menten constants (K_M) for xylosylation of different acceptors
For the determination of the K_M values of different acceptors, various concentrations of the acceptor proteins were incubated with partially purified and enriched XT solution from chondrocyte culture supernatant and UDP-[¹⁴C]xylose under assay conditions. After incubation the mixtures were placed on discs of Immobilon-AV membrane, which immobilize small peptides quantitatively by covalent links. The membrane discs were washed four times for 10 min with 1 mL/L Tween 20 in PBS and measured by liquid scintillation counting.

The concentrations of the acceptor proteins were calculated per xylosylation site. The potential xylosylation sites of fibroin are contained in the repetitive hexapeptide GSGAGA. Silk fibroin consists of ~60% of this repetition (11)(12), so the weight per mole of xylosylation sites is ~667 g/mol. The cartilage proteoglycan core protein with 210 kDa has ~100 xylosylation sites (13)(14)(15)(16) (2100 g/mol). Because of the inhomogeneity of silk fibroin and the core protein preparations, the real molecular mass of the acceptor protein may show differences from the theoretical values. However, the calculation of the K_M values on the basis of the molar concentrations enables a comparison of the acceptor affinities of XT. The recombinant bikunin with a short leader sequence and one xylosylation site per molecule has a molecular mass of 17.52 kDa (17) (17 520 g/mol). The K_M of UDP-xylose was determined by using 1.5 µmol/L recombinant bikunin and different UDP-xylose concentrations. K_M and V_max were calculated on the basis of nonlinear regression analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
xt affinities for different acceptors
We compared the affinity of XT for different acceptors by determination of the Michaelis–Menten constants for the xylosylation (Table 2 ). XT showed the highest affinity for recombinant bikunin. The affinity of XT for silk fibroin was considerably lower than for the core protein from cartilage (chemically degraded proteoglycans). The enzyme affinity for recombinant bikunin, however, was ~200-fold higher than that for the degraded proteoglycans and ~600-fold higher than that for silk fibroin. The acceptor activity (V_max/K_M) of recombinant bikunin was 250-fold higher than that for the degraded proteoglycans and 600-fold higher than that for silk.

assay optimization
By varying the concentrations of UDP-xylose and bikunin, we determined the dependence of the reaction rate on the respective concentrations (Fig. 2 ). For an efficient utilization of the two substrates, we chose a ratio of 1:3 for the concentrations of UDP-xylose and recombinant bikunin. Concentrations of 0.5 µmol/L UDP-xylose and 1.5 µmol/L recombinant bikunin provided an appropriate sensitivity and precision at low costs.

View larger version (17K): [in this window] [in a new window]   Figure 2. Determination of the xylosyl transfer rate with various bikunin or UDP-xylose concentrations. For • the UDP-xylose concentration was a constant 0.5 µmol/L; for the bikunin concentration was 1.5 µmol/L.

According to the temperature and time dependence of the xylose transfer reaction (Fig. 3 ), a temperature of 34 °C and an incubation time of 1 h were used for the assay.

View larger version (22K): [in this window] [in a new window]   Figure 3. Xylosyl transfer dependence on temperature and time. The xylosyltransferase turnover rate of UDP-xylose and bikunin was determined for different incubation temperatures. The turnover at 34 °C was measured after increasing incubation times.

precision, recovery, and detection limit of xt activity assay
With recombinant bikunin as acceptor, the intra- and interassay CVs for the determination of enzyme activities in the range of 0.60–1.72 mU/L in human serum were 5.4–6.1% and 6.8–7.3%, respectively. When added to heat-inactivated serum or PBS, 94–96% of XT activity was recovered. A linear correlation between enzyme activity and XT concentration was observed in the range 0.02–3.0 mU/L (Fig. 4 ). The lower detection limit was 0.01 mU/L.

View larger version (15K): [in this window] [in a new window]   Figure 4. Linear correlation of measured XT activity and different dilutions of a XT preparation from human chondrocyte cultures.

determination of xt activity in serum of blood donors
We determined the XT activity in serum of male (n = 294) and female (n = 206) blood donors, and observed a considerable sex and age dependence (Fig. 5 ). The activities in serum of men (0.77–1.50 mU/L) were ~30% higher than in those of women (0.58–1.20 mU/L) and reached a maximum in men between 35 and 40 years old, whereas only a moderate increase was observed in 40–50-year-old women.

View larger version (48K): [in this window] [in a new window]   Figure 5. XT activities in blood serum of male and female blood donors. Mean values (bold line) and 90% ranges are shown (n = 560, 25–30 donors per group).

serum xt activity during menstrual cycle and pregnancy
We observed the serum XT activity in women over a period of 6 weeks and found cyclic changes that corresponded to alterations in the menstrual cycle. The enzyme activity was rather constant for 2 weeks, followed by an activity peak 2 days before ovulation (100% increase) and a lower increase in the luteal phase. A conspicuous coincidence of changes in serum XT activity and ß-estradiol concentration was observed (Fig. 6 ). Longitudinal studies in men (n = 3) showed only slight and irregular alterations in the serum XT activity (not shown).

View larger version (66K): [in this window] [in a new window]   Figure 6. XT activity and ß-estradiol concentration in serum of five women during menstrual cycle.

In one woman, XT was determined from the early phase of pregnancy until birth (Fig. 7 ). At 2 weeks after conception the enzyme activity strongly increased and, at 5 weeks after conception, reached a maximum of about three times the activity before pregnancy. While the ß-estradiol concentration continued to increase during pregnancy, the serum XT activity declined in the following weeks and reached a steady state of ~1.5 times the normal activity until birth (Fig. 7 ). In 30 women the serum XT activity was observed during the third trimester of pregnancy and was increased in the same extent.

View larger version (31K): [in this window] [in a new window]   Figure 7. XT activity and ß-estradiol concentration in serum of a 28-year-old woman during pregnancy. In addition, mean values and 90% ranges of the serum XT activities of 30 women during the third trimester are shown. The enzyme activity in serum of these women was measured every 2 weeks (~15 values per week of pregnancy). The gray area shows the normal activity range of nonpregnant women.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
XT initiates the formation of chondroitin sulfate chains by the transfer of xylose to appointed serine residues in core proteins. The in vivo substrate for XT is not a single, well-defined compound, but rather a wide spectrum of different proteins. So far, XT activity was determined by the incorporation of radioactively marked xylose into deglycosylated cartilage proteoglycans or solutions of silk fibroin, which have a high number of potential glycosylation sites per molecule (5)(14). As extreme chemical conditions are necessary for the deglycosylation of proteoglycans, different preparations showed considerably varying acceptor activities. Silk fibroin consists of ~60% of the sequence repetition (SGAGAG)_n and was shown to be an appropriate substrate for XT, though no glycosylation is known in vivo. However, the composition of silk varies and silk solutions are very unstable. Therefore, these acceptor substrates made a reliable determination of XT activity difficult.

To find a better acceptor substrate for XT, we compared the amino acid sequences of chondroitin sulfate attachment sites in different proteoglycans and found the consensus sequence a-a-a-x-S-G-x-G with acidic amino acids in the positions "a" and variable amino acids "x". Proteoglycans of the extracellular matrix often show a high number of potential xylosylation sites that are not quantitatively glycosylated. These sites often lack the second glycine residue and contain only one or two acidic amino acids N-terminal to the serine residue. Other proteoglycans with single chondroitin sulfate chains that are functionally important are always glycosylated, for example the invariant chain of human class II MHC molecules (17) or bikunin (18). In these proteins the chondroitin sulfate attachment sites contain the second glycine residue and acidic amino acids in all "a" positions. The sequence of the XT recognition site seems to be a regulatory factor determining the probability of xylosylation.

For a quantification of the affinities of XT for different acceptors, we determined the Michaelis–Menten constants for the xylosylation. Recombinant bikunin and the synthetic peptide QEEEGSGGGQKK with a bikunin analog partial sequence proved to be the best acceptors. The affinity of XT for bikunin was >200-fold higher than for deglycosylated cartilage proteoglycans, and the acceptor activity of bikunin was >250-fold higher. Thus, so far, the sequence of the chondroitin sulfate attachment site in bikunin ⁶EEEGSGGG¹³ seems to be best suited for recognition by XT.

We developed an assay for the determination of XT activity with recombinant bikunin as acceptor for [¹⁴C]xylose. This assay provided a considerably higher sensitivity than other XT assays with deglycosylated cartilage proteoglycan or silk as acceptor, though the V_max values of the different substrates were similar. However, the precision of the assay with bikunin was higher because, in contrast to the degraded proteoglycan and silk, recombinant bikunin is a pure and stable substance with a reproducible acceptor activity. With acceptor concentrations of 1.5 µmol/L the xylose incorporation rate of bikunin was ~100-fold higher than that of deglycosylated proteoglycan and 240-fold higher than that of silk. About 1000-fold higher concentrations of the latter acceptor substrates were required for an appropriate xylose incorporation. Because of such high protein concentrations near the limit of solubility, the imprecision was increased and precipitation sometimes occurred during incubation with XT. The CV of the assay with bikunin was <7.3%, whereas with deglycosylated cartilage proteoglycan or silk it was 10–20% (7) and >30% with different preparations of the degraded proteoglycan. Since the higher precision of the assay with bikunin as substrate enabled a better distinction between samples with low activities and XT-negative samples, the lower detection limit was reduced from ~0.2 mU/L with the former acceptors to 0.01 mU/L with bikunin.

The new assay enabled the determination of XT in blood serum and other body fluids. It was optimized with minimal amounts of the expensive and radioactive substrates. The lower limit of the normal range of enzyme activity in human serum was ~70 times higher than the lower detection limit of the assay. However, an even higher sensitivity could be achieved by using higher concentrations of UDP-[¹⁴C]xylose and recombinant bikunin.

The XT activities in serum of blood donors showed a considerable sex and age dependence. Hitherto, we were not able to find a provable explanation for that. Investigating the serum XT activity of women during menstrual cycle and pregnancy, we observed that the enzyme activity correlates well with the ß-estradiol concentration. High estradiol concentrations during pregnancy and menstrual cycle lead to an increased water content in the connective tissue. This seems to be caused by an increased synthesis of polyanionic chondroitin sulfate proteoglycans, which are highly hydrated. However, in chondrocyte cultures we could not find a direct stimulation of XT expression by ß-estradiol treatment. We have already shown that XT is secreted into the extracellular matrix simultaneously with the synthesized chondroitin sulfate proteoglycan to which it is attached (19). Therefore, XT is an appropriate marker enzyme for the synthesis and secretion process of chondroitin sulfate proteoglycans in connective tissue cells.

By using deglycosylated cartilage proteoglycans or silk fibroin as acceptors, our previous investigations showed that in synovial fluids of patients with chronic joint diseases, the XT activity increased considerably, depending on the degree of cartilage destruction and the extent of newly synthesized proteoglycans (7). Now there is a new assay available that allows the measurement of XT activity, especially in serum, with much higher sensitivity and precision, thereby offering new chances in the diagnosis of disorders of connective tissue metabolism.

	Acknowledgments

 
We are indebted to Ernst Hartog, Frauenklinik Bielefeld, for providing serum samples of pregnant women. We also thank Grainne Delany for linguistic advice.

	Footnotes

 
¹ Nonstandard abbreviations: XT, xylosyltransferase; TFMS, trifluoromethanesulfonic acid; and MEM, minimum essential medium.

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				N.-S. Seo, A. M. Hocking, M. Hook, and D. J. McQuillan Decorin Core Protein Secretion Is Regulated by N-Linked Oligosaccharide and Glycosaminoglycan Additions J. Biol. Chem., December 30, 2005; 280(52): 42774 - 42784. [Abstract] [Full Text] [PDF]

				C. Gotting, S. Muller, M. Schottler, S. Schon, C. Prante, T. Brinkmann, J. Kuhn, and K. Kleesiek Analysis of the DXD Motifs in Human Xylosyltransferase I Required for Enzyme Activity J. Biol. Chem., October 8, 2004; 279(41): 42566 - 42573. [Abstract] [Full Text] [PDF]

				C. Gotting, J. Kuhn, H.-R. Tinneberg, T. Brinkmann, and K. Kleesiek High xylosyltransferase activities in human follicular fluid and cultured granulosa-lutein cells Mol. Hum. Reprod., December 1, 2002; 8(12): 1079 - 1086. [Abstract] [Full Text] [PDF]

				U. Pfeil and K.-W. Wenzel Purification and some properties of UDP-xylosyltransferase of rat ear cartilage Glycobiology, August 1, 2000; 10(8): 803 - 807. [Abstract] [Full Text] [PDF]

				T. Brinkmann, C. Weilke, and K. Kleesiek Recognition of Acceptor Proteins by UDP-D-xylose Proteoglycan Core Protein beta -D-Xylosyltransferase J. Biol. Chem., April 25, 1997; 272(17): 11171 - 11175. [Abstract] [Full Text] [PDF]

				J. Kuhn, C. Gotting, M. Schnolzer, T. Kempf, T. Brinkmann, and K. Kleesiek First Isolation of Human UDP-D-Xylose: Proteoglycan Core Protein beta -D-Xylosyltransferase Secreted from Cultured JAR Choriocarcinoma Cells J. Biol. Chem., February 9, 2001; 276(7): 4940 - 4947. [Abstract] [Full Text] [PDF]

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