Neose Technologies, Inc., 102 Witmer Road, Horsham, PA 19044
Received on February 4, 2004; revised on July 6, 2004; accepted on July 7, 2004
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Abstract |
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Key words: heparan sulfate / K5 polysaccharide / NDST-1 / PAPS cycle / yeast
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Introduction |
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N-deacetylase/N-sulfotransferase (NDST), a bifunctional enzyme in heparin/heparan sulfate biosynthesis, removes acetyl groups from GlcNAc residues, then sulfates the newly formed free amines on the GlcN residues using PAPS as a sulfate donor. N-sulfated glucosamine is required for the subsequent C-5 epimerization of the GlcA to IdoA by C5-epimerase, 2-O-sulfation of IdoA by 2-O-sulfotransferase, and 6-O- or 3-O-sulfation of glucosamine residues by 6-O-sulfotransferase and 3-O-sulfotransferase, respectively (Bame and Esko, 1989; Esko and Lindahl, 2001
; Grobe et al., 2002
; Perrimon and Bernfield, 2000
).
The genes for NDST isozymes 14 have been cloned and expressed from different mammalian sources (Aikawa and Esko, 1999; Aikawa et al., 2001
; Cheung et al., 1996
; Hashimoto et al., 1992
; Kushe-Gullberg et al., 1998
; Orellana et al., 1994
). The NDST isozymes share 6580% sequence identity (Esko and Selleck, 2002
) but differ in their N-deacetylase and N-sulfotransferase activities (Aikawa et al., 2001
). Caenorhabtidis elegans and Drosophila melanogaster possess single orthologs of NDST that are involved in the development of these organisms (Grobe et al., 2002
; Perrimon and Bernfield, 2000
; Selleck, 2000
). NDST-1 and NDST-2 are found in all tissues, whereas localization of NDST-3 and NDST-4 is more restricted, existing only in fetal tissues and adult brain (Aikawa et al., 2001
; Esko and Selleck 2002
; Grobe et al., 2003). NDST-1 is an essential enzyme for development, as its knockout in mice results in neonatal lethality (Grobe et al., 2002
; Ringvall et al., 2000
). NDST-2 knockout mice showed a deficiency in heparin biosynthesis (Grobe et al., 2002
). Esko and Selleck (2002)
have suggested that different isoforms may work on the same heparan sulfate (HS) chain.
NDST-1 has been demonstrated to be a single protein having both N-deacetylase and N-sulfotransferase activities (Pettersson et al., 1991; Wei et al., 1993
). Berninsone and Hirschberg (1998)
demonstrated that in COS-expressed NDST-1, the sulfotransferase activity was located in the carboxyl-terminal half of the protein, containing a PAPS-binding domain that is common in all sulfotransferases (Fukuda et al., 2001
).
All NDSTs containing both activities have been expressed in mammalian cells such as COS (Wei et al., 1993), human kidney 293 cells (Pikas et al., 2000
), and Chinese hamster ovary cells (Aikawa and Esko, 1999
). Sueyoshi et al. (1998)
have expressed the sulfotransferase domain of the human NDST-1 as a glutathione-S-transferase (GST) fusion protein in Escherichia coli, and expression of NDST-2 was recently reported in insect cells (Kuberan et al., 2003
). To date the expression of NDST carrying both N-deacetylase and N-sulfotransferase activities has not been achieved in non-glycosaminoglycan (GAG)containing expression systems, such as yeast or bacteria.
In this work, soluble rat liver (r) NDST-1 was cloned and expressed in Saccharomyces cerevisiae. The lack of background enzymatic activity and endogenous proteoglycans in S. cerevisiae simplifies purification and analysis of the enzyme. The yeast-expressed recombinant rNDST-1 successfully achieved coupled N-deacetylation and N-sulfation on a variety of polysaccharides containing GlcNAc-GlcA repeating units.
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Results |
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Acceptor specificity of rNDST-1
rNDST-1 specificity was first tested on different sugar acceptors using yeast lysates, prepared following 24 h induction, under conditions predicted to be optimal for sulfotransferase activity (Pettersson et al., 1991). Using de-N-sulfated heparin (DNSH) as substrate, rNDST-1 had a sulfotransferase activity of 159 pmol/min/ml lysate, whereas desulfated chondroitin, containing repeats of GalNAc instead of GlcNAc, was not a substrate (Figure 1). Using E. coli K5 polysaccharide (K5 or N-acetyl heparosan) as substrate, rNDST-1 had a sulfotransferase activity of 196 pmol/min/ml lysate (Figure 1). Because K5 did not have any free glucosamine residues, the substrate for the N-sulfotransferase, the activity measured indicates coupling of the N-deacetylase and N-sulfotransferase activities of rNDST-1. When a similar acceptor, completely desulfated N-acetylated heparan sulfate (CDSNAcHS), was tested, N-sulfotransferase activity of rNDST-1 was found to be 168 pmol/min/ml lysate (Figure 1). However when N-desulfated N-acetylated heparin (NDSNAcH), containing O-sulfates (2-O, 6-O, and/or 3-O), predicted to inhibit N-deacetylation (Brandan and Hirschberg, 1988
) was used as an acceptor, N-sulfotransferase activity dropped dramatically to 20 pmol/min/ml lysate (Figure 1).
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Optimization of a coupled enzymatic N-deacetylation/N-sulfation reaction
The ability of the rNDST-1 sulfotransferase to successfully add sulfates to a K5 polysaccharide substrate that is completely N-acetylated indicated that the enzyme was functioning in a coupled reaction, with the N-deacetylase activity creating the substrate for the N-sulfotransferase. Because this reaction was carried out under conditions predicted to be optimal for sulfotransferase activity (pH 7.07.4, in the presence of Mn2+, Mg2+, and Ca2+) (Pettersson et al., 1991), we tested the coupled enzyme reaction under reaction conditions predicted to be optimal for the N-deacetylation reaction (pH 6.5, in the presence of Mn2+) (Pettersson et al., 1991
). N-sulfation of K5 polysaccharide by rNDST-1 under conditions optimized for deacetylation was sevenfold higher than N-sulfation in conditions optimized for N-sulfation (Figure 2). This suggests that N-deacetylation is the rate-limiting step in the coupled enzyme reaction. The data showed that Mn2+ was the only divalent cation required for both N-deacetylation and N-sulfation (Figure 2).
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To test the potential incompatibility of the PAPS cycle components with the N-deacetylase activity of rNDST-1, the reaction was uncoupled. N-deacetylation of N-acetyl heparosan was first carried out overnight at 23°C ± 2°C, followed by N-sulfation of the partially N-deacetylated polysaccharides, using the PAPS cycle components, at 23°C ± 2°C. Under these conditions, the N-sulfation yield increased to 24% (Table I), suggesting incompatibility of the N-deacetylase activity of rNDST-1 with the PAPS cycle components. To maximize the uncoupled reaction, the deacetylation reaction was carried out on N-acetyl heparosan at 37°C, followed by N-sulfation of the partially N-deacetylated polysaccharide, using the PAPS cycle components, at 23°C ± 2°C. Under these conditions the N-sulfation yield increased to 38% (Table I). By comparison, coupled reactions carried out with exogenously added PAPS resulted in an N-sulfation yield of 37% at 23°C ± 2°C and 6065% at 37°C (Table I).
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Discussion |
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Of the enzymes involved in the synthesis and modification of HSs, the NDST isozymes have the unique role of defining areas on the polysaccharide backbone where subsequent modifications can occur (reviewed in Grobe et al., 2002). The bifunctional NDST has been studied extensively in natural and cloned forms from mammalian sources, but analysis of this enzyme has been complicated by the existence of endogenous GAGs, as well as the enzymes that synthesize and modify these polysaccharides in all mammalian tissues and cell lines. Here we report for the first time expression of rNDST-1 in S. cerevisae and the demonstration of both N-deacetylase and N-sulfotransferase activities of the expressed enzyme. The use of S. cerevisae offers a clear advantage over mammalian expression systems in that a yeast expression system contains a background free of GAGs and GAG-synthesizing and -modifying enzymes. Although bacterial expression systems provide a similar background to yeast, the intracellular environment of bacteria is not conducive to the recovery of a bifunctionally active NDST-1. The N-sulfotransferase domain of human NDST-1 has been successfully expressed in E. coli (Sueyoshi et al., 1998
), but expression of both N-deacetylase and N-sulfotransferase activities of NDST-1 in bacteria was not successful (Kusche-Gullberg and Kjellen, 2003
). Our attempts to express the rNDST-1 in bacteria resulted in inclusion body formation, and attempts at refolding of the inclusion bodies resulted in a soluble enzyme with N-sulfotransferase activity but lacking N-deacetylase activity (unpublished data).
The N-deacetylation and N-sulfation activities of NDST-1 are coupled in vivo, with N-deacetylation a prerequisite for N-sulfation. We chose to focus on enzymatic studies using unmodified K5 polysaccharide as substrate. This polysaccharide is identical to the heparan backbone and this substrate is sufficient for demonstrating the bifunctional activity of NDST-1. Kusche et al. (1991) showed that incubation of mastocytoma microsomal fractions with K5 polysaccharide resulted in N- and O-sulfated sugars resembling heparin/HS, indicating that NDST-1 would use this polysaccharide substrate. Most reports in the literature have investigated the individual enzyme activities, optimizing reactions for either the N-deacetylase or N-sulfotransferase functions. It was reported that addition of PAPS to microsomal preparations from mast cell tumors (Silbert, 1967
) or rat liver (Riesenfeld et al., 1982
) controlled the extent of N-deacetylation of HS precursors, suggesting that increase in N-deacetylation was caused by concomitant N-sulfation due to a higher affinity of N-deacetylase for partially N-sulfated substrates. Bame et al. (1991)
have found that adding exogenous PAPS did not increase the N-deacetylation in microsomal preparations from wild-type or mutant Chinese hamster ovary cells containing threefold lower N-sulfotransferase activity. The extent of N-deacetylation was found to be equal to the extent of N-sulfation.
Our results using S. cerevisaeexpressed rNDST-1 clearly demonstrated the coupled nature of the two activities as suggested by early in vivo work by Silbert (1967) and Riesenfeld et al. (1982)
. In vitro N-deacetylation of N-acetyl heparosan (K5 polysaccharide or PM PS) in the absence of N-sulfation reached a maximum of 3035%, but addition of exogenous PAPS to this reaction, initiating N-sulfation, allowed the recovery of 6065% N-sulfated products. These results demonstrate that N-sulfation of N-deacetylated residues in the K5 polysaccharide (or PM PS) allowed further N-deacetylation of neighboring residues. Bengtsson et al. (2003)
have recently demonstrated that introducing single mutations in each domain of NDST-1 created mutants having either N-deacetylase or N-sulfotransferase activities. The experiment with these mutants has shown that rate-limiting step of the overall reaction was N-deacetylation, which also determined the degree of N-sulfation, agreeing with our studies.
We developed a coupled reaction incorporating N-deacetylation and N-sulfation of N-acetyl heparosan (K5 or PM PS) in a one-pot reaction. It was discovered that optimal N-sulfation of N-acetyl heparosan in a coupled reaction occurred when the reaction was optimized for the N-deacetylation reaction (Pettersson et al., 1991), and required only the presence of Mn2+ and PAPS. We also attempted to develop a coupled reaction of N-deacetylation/N-sulfation of N-acetyl heparosan with NDST-1 using the PAPS cycle (Burkart et al., 2000
). It was discovered that the N-sulfation yields were extremely low when coupled reactions using the PAPS cycle were performed at 23°C ± 2°C, as required for optimum PAPS cycle enzyme activities. However, if N-deacetylation was performed first at 37°C, followed by N-sulfation at 23°C ± 2°C with the PAPS cycle enzymes, 3738% N-sulfated polysaccharide was obtained. Further optimization of the PAPS cycle coupled reaction will be required to make this a viable large-scale process. Figure 7 summarizes production of N-sulfated heparosan using coupled NDST reactions in a pH 6.5 buffer containing Mn2+ and endogenous PAPS. It is also shown that the PAPS cycle can be used for auxiliary sulfate regeneration. We established a simple one-pot production of N-sulfated polysaccharides that are ready for the subsequent steps in HS and heparin biosynthesis.
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Materials and methods |
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Yeast and bacterial strains
InvSc1 yeast cells were from Invitrogen. E.coli K5 (ATCC #23506) and P. multocida (ATCC #43019) bacterial strains were from ATCC (Manassas, VA).
Cloning rNDST-1 into the pYES2/NTC yeast expression vector
To make a 132-bp truncation in the 5' end of the rNDST-1 gene, the following polymerase chain reaction (PCR) primers were designed (restriction sites introduced are underscored): sense, NDASTXH1(5'-TTATTCTGGAGCCCTC GGCAGATGCTTCTGAG-3'), antisense, NDASTXB-1 (5'-CGGCAGATCTCTACCTGGTGTTCTGGAGGTCT T-3'). One nanogram of rat liver cDNA (Clontech) was amplified with the primers NDASTXH1 and NDASTXB1 using Stratagene's Herculase high-performance PCR system. The 2.5-kb PCR product, containing the truncated rNDST-1 coding sequence was excised and purified from an agarose gel and cloned into the pCR-Blunt vector (Invitrogen). pCR-Blunt-rNDST-1 was digested with Xba I and Xho I and the resultant insert was cloned into Xba I and Xho Idigested pYES2/NTC, creating pYES2-rNDST-1.
Expression of rNDST-1 in yeast cells
S. cerevisiae InvSc1 cells were made competent, and pYES2-rNDST-1 DNA was transformed into the competent cells using the S.c. EasyComp Transformation kit (Invitrogen) according to the manufacturer's instructions. Transformed colonies, InvScl/pYES2-rNDST-1, were selected on glucose minus uracil agar plates (Teknova). Single colonies were picked and inoculated in 5-ml CM-glucose minus uracil media (Teknova) and grown at 30°C with shaking overnight. Overnight cultures were inoculated in 100 ml CM-glucose minus uracil and grown until the OD620nm was 0.5. The cultures were harvested by centrifugation (3000 x g for 5 min), and the pellets were resuspended in 90 ml induction media (CM-galactose minus uracil, Teknova). Five-milliliter aliquots were taken at 0 h induction, centrifuged (1500 x g at 4°C) and washed with dH2O (1/10 culture volume). The pellet was stored at 80°C until it was processed. The remaining culture was grown for 16 h at 30°C. After 16 h, 45 ml of the culture (OD620nm = 1.12) was withdrawn, centrifuged, and washed as described and stored at 80°C. The remainder of the culture was allowed to grow for 24 h after induction (OD620nm = 1.2). The cultures were then harvested, washed, and stored at 80°C as described.
Preparation of yeast cell extracts
The frozen yeast cell pellets were thawed and treated with yeast-PE LB yeast protein extraction kit (Genotech). An equal volume of yeast suspension buffer supplemented with ß-mercaptoethanol (143 mM) was added to the pellets. The yeast cell suspension was vortexed to obtain a homogenous suspension. Longlife Zymolyase enzyme (Genotech) was added to this suspension. The contents were mixed gently followed by incubation at 37°C for 1 h. At the end of the incubation, the suspension was centrifuged at 10,000 x g for 5 min. Supernatants were discarded and the pellets were treated with 510 volumes of yeast-PE LB containing 1 mM dithiothreitol and 1 mM phenylmethanesulfonyl fluoride. The suspensions were vortexed and incubated on ice 30 min followed by 12-min incubation at 37°C to obtain yeast cell lysates. The lysates were centrifuged at maximum speed in an Eppendorf microcentrifuge for 1 h at 4°C. Clarified lysates were used for assays and purification.
Purification of rNDST-1 on a heparin-Sepharose CL-6B column
Yeast culture, induced for 24 h, was used for preparation of yeast cell extracts. Approximately 1.4 g yeast pellet was obtained, and 6 ml yeast cell extracts were prepared as described. Heparin-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) was packed into a 1 x 10 cm column, washed extensively with dH2O, and equilibrated with buffer A (10 mM TrisHCl, pH 7.2, 20 mM MgCl2, 2 mM CaCl2, 10 mM ß-mercaptoethanol, 0.1% Triton X-100, and 20% glycerol). Clarified yeast extracts containing the enzyme rNDST-1 were loaded onto the column, and the column was washed with buffer A until the baseline returned to absorbance return to zero at 280 nm. Linear gradient elution was carried out in buffer A from 0.15 to 0.65 M NaCl. The fractions were tested for NDST activity using the radioactive sulfotransferase assay. The fractions exhibiting highest NDST activity were pooled and concentrated using Apollo7 centrifugal concentrators (Orbital Biosciences, Topsfield, MA).
N-deacetylation and N-sulfation reactions
N-deacetylation of K5 or PM PS by rNDST-1 (510 µU) was carried out in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6.5, in the presence of 10 mM MnCl2 at either 23°C ± 2°C or 3537°C. N-sulfation of K5 or PM PS by rNDST-1 was carried out in 10 mM HEPES buffer, pH 7.0, with 10 mM MnCl2, 10 mM MgCl2, and 5 mM CaCl2, except during optimized, coupled reactions, where N-sulfation was carried out in the same buffer as N-deacetylation. In the coupled reactions, N-sulfation was initiated by addition of excess PAPS (200400 µM) and additional rNDST-1 (510 µU). All reactions were stopped either by heating the reaction mixture at 98°C or by storage at 20°C.
The rate of N-sulfation was monitored using a radioactive sulfotransferase assay. In each reaction, 10 µg acceptor sugar, 10 µM PAPS, and 400,000500,000 cpm 35S-PAPS were incubated with 1030 µl of cell lysate or 110 µl (110 µU) purified enzyme in 100 µl final volume at 37°C or 23°C ± 2°C, as indicated. The reactions were stopped by the addition of 10 µl chondroitin sulfate (20 mg/ml) and 480 µl 100% EtOH, followed by overnight incubation at 20°C to allow precipitation of the sugars. The following day, the tubes were centrifuged in a microcentrifuge at maximum speed for 10 min. The supernatants were removed completely and the pellets were dissolved in 50 µl TE buffer (10 mM TrisHCl, pH 8.8, 0.1 M NaCl, 1 mM ethylenediamine tetra-acetic acid). Thirty-five microliters of resuspended reaction products were applied to prespun Quick Spin Columns (G-25 Sephadex, Roche). The columns were centrifuged at 1100 x g for 2 min at 23°C ± 2°C, the flow-through fractions were collected and mixed with liquid scintillation fluid, and radioactivity was determined in a liquid scintillation counter.
The N-deacetylation rate was monitored by HPLC analysis of the formation of GlcNH-containing disaccharides following enzymatic digestion of polysaccharides (see next section).
HPLC analysis of N-deacetylated and N-sulfated polysaccharides
The method was based on the work by Kinoshita and Sugahara (1999). Reaction samples (50200 µl), containing 5100 µg modified/unmodified polysaccharide, were heated to 98°C for 2 min and centrifuged for 5 min at maximum speed (microcentrifuge) to deactivate and precipitate enzymes. The supernatants were dialyzed against 100 ml water on V Series Membranes (0.025 µm, Millipore) for 1 h and then evaporated to dryness. The samples were then digested to disaccharides by incubating first at 30°C for 2 h and then at 37°C overnight in 60 µl 20 mM TrisHCl buffer, pH 7.1, containing heparinase I (5 U), heparinase II (1 U), heparitinase I (0.002 U), 50 mM NaCl, and 4 mM CaCl2. The resulting disaccharides contain UV-absorbing unsaturated UAs at their nonreducing termini.
Following digestion the solutions were heated to 98°C for 2 min and centrifuged (microcentrifuge) for 5 min at maximum speed to remove the digestion enzymes. The supernatants were analyzed by RP-HPLC on a Chromolith Performance RP-18e column (10 cm x 4.6 mm) (Merck KGaA, Darmstadt, Germany). The separation was performed in an isocratic mode using 5% acetonitrile (ACN) in 5 mM Tetrabutylammonium dihydrogen phosphate, pH 6.75, as a mobile phase running at 5 ml/min. The analytes were monitored at 232 nm.
Alternatively, the supernatants were removed and evaporated to dryness. The resulting variously sulfated disaccharides were labeled with fluorescent tag 2-AB by adding 5 µl 2-AB (0.7 M) in 30% HOAc/70% dimethyl sulfoxide and 5 µl NaBH3CN (1 M) in tetrahydrofuran and incubating at 60°C for 2 h. The labeled disaccharides were suspended in 1 ml 95% ACN, and passed through a filter (MF Support Pad, 13 mm, Millipore), which was prewetted by passing 1 ml water followed by 3 ml 95% ACN. The samples were then washed with 3 ml ACN and eluted with 1 ml 20% ACN. The solutions were dried, redissolved in 100 µl water, and analyzed by ion exchange HPLC on a YMC-Pack, Polyamine II column (25 cm x 4.6 mm, 5 µm, Waters, Milford, MA). A linear gradient was employed from 0 to 100% B (A: 15 mM NaH2PO4, B: 800 mM NaH2PO4) in 30 min. The flow rate was 1 ml/min.
The analytes were detected by fluorescence with excitation and emission wavelengths of 330 nm and 420 nm, respectively. Retention times of different disaccharides were determined by processing commercially obtained disaccharide standards (UA-GlcNAc,
UA-GlcNAc6S,
UA-GlcNS,
UA-GlcNS6S,
UA2S-GlcNS,
UA2S-GlcNS6S) following the same procedure (Kinoshita and Sugahara, 1999
).
UA-GlcN coelutes with the unincorporated 2-AB fluorescent tag at 3.3 min. There was also high degree of variation in the amount of free 2-AB from sample to sample.
N-sulfation of N-deacetylated heparosan in PAPS cycle
The PAPS cycle buffer Bis-tris-propane, pH 7.0 (Burkart et al., 2000), was substituted with MES buffer, pH 6.5, to accommodate NDST reactions. MnCl2 was also included in the cycle to ensure NDST activity. N-deacetylation of K5 polysaccharide was carried out overnight as described. The N-deacetylated reaction mixture was added to an equal volume of 50 mM MES (pH 6.5) buffer containing 10 mM MnCl2, 186 µM PAP, 100 mM PNPS, 0.5 µl dithiothreitol, and 10 µl refolded PAP-free ß-arylsulfotransferase IV (GST-ß-ASTIV, Sar
ba
, unpublished data). Several minutes after the PAPS cycle was initiated, additional rNDST-1 (510 µU) was added. The reactions were allowed to run overnight and the products were kept frozen at 20°C until analysis. The cycle was monitored by measuring p-nitrophenol formation at 405 nm as described earlier (Burkart et al., 2000
). In parallel, N-deacetylated heparosan was N-sulfated by addition of PAPS and partially purified rNDST-1 at 23°C ± 2°C. This reaction was run along with the PAPS cycle reaction. Analyses of N-deacetylated and N-sulfated products were done as described.
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Acknowledgements |
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Footnotes |
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2 Present address: Department of Chemistry, University of California-Davis, Davis, CA 95616
3 Present address: Department of Plant Sciences, University of Arizona, Tucson, AZ 85721
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Abbreviations |
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References |
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