Oligosaccharide Library-based Assessment of Heparan Sulfate 6-O-Sulfotransferase Substrate Specificity*

Per Jemth {ddagger} §, Emanuel Smeds {ddagger}, Anh-Tri Do {ddagger}, Hiroko Habuchi ¶, Koji Kimata ¶, Ulf Lindahl {ddagger} and Marion Kusche-Gullberg {ddagger} ||

From the {ddagger}Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Center, Box 582, SE-751 23 Uppsala, Sweden and the Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 448-8542, Japan

Received for publication, December 1, 2002 , and in revised form, April 16, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heparan sulfate mediates numerous complex biological processes. Its action critically depends on the amount and the positions of O-sulfate groups (iduronyl 2-O-sulfates, glucosaminyl 6-O- and 3-O-sulfates) that form binding sites for proteins. The structures and distribution of these protein-binding domains are influenced by the expression and substrate specificity of heparan sulfate biosynthetic enzymes. We describe a general approach to assess substrate specificities of enzymes involved in glycosaminoglycan metabolism, here applied to 6-O-sulfotransferases involved in heparan sulfate biosynthesis. To understand how 2-O-sulfation affects subsequent 6-O-sulfation reactions, the substrate specificity of 6-O-sulfotransferase 3 was probed using substrates from a heparin-based octasaccharide library. Purified 3H-labeled N-sulfated octasaccharides from a library designed to sample 2-O-sulfated motifs were used as sulfate acceptors, 3'-phosphoadenosine 5'-phosphosulfate as sulfate donor, and cell extract from 6-O-sulfotransferase 3-overexpressing 293 cells as enzyme source in the 6-O-sulfotransferase-catalyzed reactions. The first 6-O-sulfate group was preferentially incorporated at the internal glucosamine unit of the octasaccharide substrate. As the reaction proceeded, the octasaccharides acquired three 6-O-sulfate groups. The specificities toward competing octasaccharide substrates, for 6-O-sulfotransferase 2 and 6-O-sulfotransferase 3, were determined using overexpressing 293 cell extracts and purified octasaccharides. Both 6-O-sulfotransferases showed a preference for 2-O-sulfated substrates. The specificity toward substrates with two to three 2-O-sulfate groups was three to five times higher as compared with octasaccharides with no or one 2-O-sulfate group.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heparan sulfate (HS)1 is a linear polysaccharide present on virtually all cells and in the extracellular matrix (1). HS chains are heterogeneous, with a large number of complex sequences based on variable patterns of N-acetyl, N-sulfate, and O-sulfate groups (24). The biosynthesis of the polysaccharide chains begins with the synthesis of an oligosaccharide primer co-valently attached to a serine residue in a proteoglycan protein core. HS chains are then generated by the sequential addition of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronate (GlcUA). Along with polymerization the chains undergo a series of modification steps that involve five distinct enzyme families; (i) N-deacetylase/N-sulfotransferase removes the N-acetyl group from GlcNAc units and replaces it with an N-sulfate group (GlcNS); (ii) GlcUA C5 epimerase converts GlcUA to its C5 epimer L-iduronate (IdoUA); (iii) 2-O-sulfotransferase adds 2-O-sulfate groups to IdoUA and GlcUA residues; (iv) 6-O-sulfotransferase (6-OST), and (v) 3-O-sulfotransferase (3-OST) transfer O-sulfate groups to C6 and C3, respectively, of GlcNAc or GlcNS units (24). The N-deacetylase/N-sulfotransferase (58), the 6-OST (9), and the 3-OST (1012) families each contain several members, whereas only one GlcUA C5 epimerase (13, 14) and one hexuronate 2-O-sulfotransferase (15, 16) have been described. During enzymatic HS modification, only parts of the available target units are modified. As a result the final product shows high structural diversity. The factors that regulate HS biosynthesis remain unclear.

Different models for HS biosynthesis have been proposed. One model features an association of all or several of the biosynthetic enzymes in a complex ("gagosome") that subjects the nascent polysaccharide to consecutive modifications in assembly line fashion (4, 17). Other proposals predict that the modifications of the HS chain occur by random encounters between the enzymes and the polysaccharide (see Refs. 2 and 3 for recent reviews). The overall process thus may depend on spatial arrangement (former model) as well as on individual concentrations of the biosynthetic enzymes (latter model). Importantly, in either model the activities of the enzymes would depend on the respective values for all different substrate motifs encountered.

The determination of substrate specificities for enzymes involved in HS biosynthesis is complicated by the lack of well defined substrates. Most kinetic parameters determined so far are based on more or less heterogeneous saccharide mixtures. Nevertheless, such analyses have provided information regarding the preferred disaccharide target units of 6-OSTs in heterogeneous substrate populations (9), but little data on the influence of flanking disaccharide units. Addressing this question requires access to well defined oligosaccharides of the appropriate structures. In a previous paper (18) we described chemically and enzymatically generated 3H-labeled HS-related oligosaccharide libraries, which were used to assess structure-affinity relationships in fibroblast growth factor-HS interactions. We now explore this concept further, by using purified oligosaccharides isolated from a 3H-end labeled octasaccharide library to study the substrate specificity of 6-OST. The library was designed to sample the 2-O-/N-sulfated segment of the HS sequence space, thus assessing the impact of IdoUA 2-O-sulfate groups on the catalytic activity of 6-OST. The technique used in this study provides a general approach to defining the substrate preferences of glycosaminoglycan-metabolizing enzymes.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of 6-O-Desulfated Octasaccharide Substrates—All reagents were commercial products of high purity unless otherwise indicated. Bovine lung heparin (Upjohn) was N- and partially O-desulfated by treatment with dimethyl sulfoxide/methanol (9:1, v/v) for 2 h (19, 20). The polysaccharide was then re-N-sulfated by incubation with trimethylamine sulfur trioxide (21). The completely 6-O-desulfated partially 2-O-desulfated heparin was subjected to partial deaminative cleavage using nitrous acid at pH 1.5 (22). Approximately 1 mg of O-desulfated heparin was dissolved in 250 µl of 2.5 mM NaNO2, 70 mM H2SO4 and incubated on ice. After 70 min, 80 µl of the reaction mixture, and after 3 h the remaining 170 µl were adjusted to pH ~ 8 by addition of 4 M NaOH. The oligosaccharide fragments formed were reduced with NaB3H4 (5 mCi, 64 Ci/mmol, Amersham Biosciences), to yield reducing terminal 3H-labeled 2,5-anhydromannitol (aManR) residues, followed by reaction with excess unlabeled NaBH4. The labeled O-desulfated heparin oligosaccharides were desalted and lyophilized and were then size fractionated on a Bio-Gel P-10 (Bio-Rad) column (10 x 1500 mm) in 0.5 M NH4HCO3 at a flow rate of 1.8 ml/h. Octasaccharides were recovered, lyophilized, and further separated on a ProPac PA1 anion exchange HPLC column (Dionex, Surrey, UK) using a gradient of 0–1 M NaCl, pH 3.0, at a flow rate of 1.0 ml/min. Elution profiles were monitored by scintillation counting and the central fractions from distinct peaks were pooled and desalted on PD-10 gel filtration columns (Sephadex G-25) (Amersham Biosciences). The resulting purified octasaccharide preparations were subjected to sequence analysis using a combination of chemical (HNO2) and enzymatic (iduronate 2-sulfatase) cleavage as described previously (18, 2325).

Expression of 6-OST2 and 6-OST3 in Human Embryonic Kidney (HEK) 293 Cells—A full-length mouse cDNA clone coding for 6-OST2 was amplified from an adult mouse brain QUICK-Clone cDNA library (Clontech) using the sense primer 5'-CATGGATGAGAAATCTAACAA-3' and the antisense primer 5'-AGCGCCATGTCTCTACG-3'. The amplified product was cloned into pGEM-T (Promega). Insertion of the entire coding region for 6-OST2 was confirmed by sequence analysis. The insert was excised with ApaI/SalI and subcloned into the corresponding site of the Bluescript plasmid vector. The mouse 6-OST2 cDNA was then released from the plasmid vector as a KpnI/EcoRI fragment and cloned into pcDNA3 (Invitrogen), thus creating a stop codon at the 3'-end of the construct. To generate an expression plasmid for mouse 6-OST3 in pcDNA3, pFLAG-CMV2-mHS6ST3 (9) was cleaved with HindIII/XbaI and the produced 6-OST3 cDNA was ligated into corresponding sites in pcDNA3. The expression plasmids were transfected into HEK 293 cells using LipofectAMINE (Invitrogen). Stably transfected cell clones expressing the 6-OST2 or -3, and control clones transfected with vector alone, were selected as described (26). Cells were cultured under an atmosphere of 5% CO2 in air and 100% relative humidity in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum (Invitrogen), 100 µg of streptomycin sulfate/ml, 100 units of penicillin G/ml, and 400 µg of Geneticin/ml (G418 sulfate, Invitrogen).

Enzymatic 6-O-Sulfation of Octasaccharides—Crude enzyme preparations from 6-OST2- or 6-OST3-transfected cells were extracted from stably transfected cell clones as described (16). Lysates (corresponding to approximately 20 µg of protein) from 6-OST2- or 6-OST3-transfected cells were incubated with 0.1–2 µM octasaccharide, 5 mM PAPS, 10 mM MnCl2, 5 mM CaCl2, 10 mM MgCl2, 3.5 µM NaF, 0.3% (v/v) Triton X-100, and 50 mM Hepes, pH 7.4, in a total volume of 25 µl. The reactions were incubated at 37 °C for various periods of time (1 min to overnight), depending on the desired degree of 6-O-sulfation and the enzymatic activity. Incubations with microsomal OSTs from a mouse mastocytoma were performed as described (18). The reactions were heat inactivated (90 °C for 1 min) and centrifuged, and the supernatants analyzed by anion exchange chromatography using the ProPac PA1 column. The column was eluted with a linear NaCl gradient, pH 3.0, at a flow rate of 1 ml/min. The gradient was 10 mM NaCl/ml and the initial concentration of NaCl was adjusted according to the number of O-sulfate groups present in the octasaccharides (see legends to Figs. 1 and 3).



View larger version (28K):
[in this window]
[in a new window]
 
FIG. 1.
6-OST3-catalyzed sulfation of a mixture of partially 2-O-desulfated, completely 6-O-desulfated heparin-derived octasaccharides. The 3H-end labeled octamer library was analyzed before (A) and after enzymatic 6-O-sulfation catalyzed by 6-OST3 (B)orby mouse mastocytoma microsomal proteins (C). In panels A and B the column was eluted with a gradient extending from 0 to 1.5 M NaCl, whereas in panel C the gradient extended from 0.5 to 1.5 M NaCl. Indicated in panel A are the number of 2-O-sulfate groups present in the octasaccharide fragments. The arrows show the peaks corresponding to the octasaccharides identified in Fig. 2 (A to H from left to right). The peaks emerging at the beginning of the gradients are 3H-containing impurities (panels A and B). In panel C, this peak also contains 8-mers that would have appeared at ≤0.5 M NaCl.

 


View larger version (25K):
[in this window]
[in a new window]
 
FIG. 3.
Products of 6-OST3-catalyzed O-sulfation. 3H-End labeled octasaccharide substrates (see Fig. 2 for symbols) were incubated with PAPS and lysates of 6-OST3-transfected cells as described under "Experimental Procedures." The resulting 6-O-sulfated octasaccharide products were analyzed by ProPac PA1 anion exchange chromatography. The arrows indicate the elution positions of substrates before incubation with 6-OST3. Up to three O-sulfate groups were transferred to the oligosaccharide substrates. The NaCl gradients were started at 0.25 M (octasaccharide A), 0.35 M (octasaccharide B), 0.50 M (octasaccharides E), 0.60 M (octasaccharides G), and 0.70 M (octasaccharide H). See "Experimental Procedures" for further details.

 



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 2.
Structures of recovered 3H-end labeled heparin-derived octasaccharide substrates. The 3H-end labeled octasaccharide library shown in Fig. 1A was fractionated on the ProPac PA1 anion exchange column and individual peaks were pooled and desalted. The structures of isolated octasaccharides were deduced by HNO2/exoenzyme sequencing (see "Experimental Procedures" and Refs. 18, 24, and 25).

 

Determination of Substrate Specificity of 6-OST3 Using Competing Octasaccharide Substrates—The substrate specificity of 6-OST3 for one octasaccharide substrate over another was assessed by incubating two different characterized octasaccharide preparations with 6-OST and PAPS. The reaction conditions were set to allow O-sulfation of ~2–10% of each of the two octasaccharides (4% (v/v) HEK cell lysate, 5 mM PAPS; reaction time from 1 min up to 30 min at room temperature). Under these conditions (product:substrate ≤ 1:9), product formation was shown to be linear with time, hence giving the respective initial rate, v0. The ratio of these rates (in this case initial rates) gives the substrate specificity according to Equation 1 (27) for substrate Y relative to substrate X.

(Eq. 1)
[X] and [Y] are the concentrations of the two substrates.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of Octasaccharide Substrate Library—A 3H-labeled, N-sulfated heparin octasaccharide library containing species devoid of 6-O-sulfate groups and with varying degrees of 2-O-sulfation was obtained as described under "Experimental Procedures." These octasaccharides have the general structure [IdoUA ± 2S-GlcNS]3-IdoUA ± 2S-[3H]aManR. Thus, differential 2-O-sulfation of the IdoUA residues can give rise to 16 distinct octasaccharides; one without any 2-O-sulfates, four with one, six with two, four with three, and one with four 2-O-sulfate groups. Accordingly, anion exchange HPLC revealed five oligosaccharide families recognized through sequence analysis and previous analyses of HS and modified heparin octasaccharides (18, 24, 25) (Fig. 1A). Octasaccharide without O-sulfates was eluted at 0.32 M NaCl and a fragment with four 2-O-sulfate groups at ~0.78 M, families carrying one, two, or three 2-O-sulfate groups occurring in between. The latter three groups separated into distinct peaks indicating subspecies with distinct 2-O-sulfation motifs. Purified oligosaccharides (see Fig. 1A) were subjected to radiosequencing involving partial nitrous acid depolymerization and iduronate 2-sulfatase digestion (see "Experimental Procedures"). Deduced structures of recovered 8-mers are shown in Fig. 2. The di- and tri-O-sulfated 8-mers could not be completely separated (Fig. 1A), such that each corresponding final substrate preparation was composed of two distinct octasaccharides (that were sequenced in mixture, cf. Refs. 18 and 25).

Enzymatic 6-O-Sulfation of 3H-Labeled Octasaccharides— The 3H-labeled octasaccharide library was incubated with unlabeled PAPS and, as enzyme source, extracts of either 293 cells expressing 6-OST3 or a microsomal fraction from a heparin-producing mouse mastocytoma. Following incubation with extracts from 6-OST3-transfected cells the octasaccharides assumed overall more retarded elution positions on anion exchange HPLC (Fig. 1B), indicative of 6-O-sulfation. This effect was even more pronounced when the mouse mastocytoma microsomal fraction was used as catalyst2 (Fig. 1C). The retarded elution patterns shown in Fig. 1, B and C, suggest that most fragments present in the 2-O-sulfated octasaccharide library were targets for 6-O-sulfation.

To further evaluate how 2-O-sulfate groups influence 6-O-sulfation, purified and sequenced octasaccharides with 0 to 4 IdoUA2S units (Fig. 2) were used as substrate for heterologously expressed 6-OST3. Analysis of the products by anion exchange chromatography showed changes in elution positions of the fragments corresponding to the addition of one, two, or three 6-O-sulfate groups (Fig. 3). Thus, all octasaccharides tested, including the tetra-2-O-sulfated fragment, were substrates for the recombinant 6-OST3 and all octasaccharides accepted up to three 6-O-sulfate groups.

Reverse transcriptase-PCR analysis using wild type 293 mRNA as template amplified 6-OST1 and 6-OST2S (short form in Ref. 28) cDNAs. No PCR product was observed using primers for 6-OST3, indicating that the 293 cells express 6-OST1 and 6-OST2S mRNA endogenously, as verified by sequencing of the purified PCR fragments (data not shown). The endogenous 6-O-sulfotransferase activity was 10–15-fold lower than that of the overexpressors such that no significant sulfation of the tetra-2-O-sulfated fragment was observed in control incubations performed with cell lysate from vector-transfected cells (data not shown).

Positions of Incorporated 6-O-Sulfate Groups—The data in Fig. 3 show that all three N-sulfated glucosamine residues in an octasaccharide are potential acceptors for 6-OST-catalyzed O-sulfation. To determine the preferred site of 6-O-sulfation, purified octasaccharides (see Fig. 2) were incubated with 6-OST3 and PAPS until ~30–70% of the substrates were consumed. The reaction products were separated by anion exchange HPLC as shown in Fig. 4A for the tri-2-O-sulfated substrate F. The unmodified substrate and the mono-6-O-sulfated product (F and F', respectively, in Fig. 4A) were purified and subjected to partial deaminative cleavage and the resultant di-, tetra-, hexa-, and octasaccharides were then again analyzed on the ProPac PA1 column. Note that only fragments containing the terminal [3H]aManR residue will be detected in the cleavage products; i.e. a disaccharide IdoUA ± 2S-[3H]aManR, a tetrasaccharide IdoUA ± 2S-GlcNS-IdoUA ± 2S-[3H]aManR, etc. The elution positions for both disaccharides and tetrasaccharides were identical for substrate F (Fig. 4B) and product F' (Fig. 4C) indicating that no 6-O-sulfate group had been incorporated on the GlcNS residue closest to the reducing end (in which case the product tetramer would have been retarded, see F' in Fig. 4D). The elution position of the 8-mer was changed, but also that of the 6-mer, thus excluding 6-O-sulfation of the GlcNS unit closest to the non-reducing end. Instead the internal (middle) GlcNS residue of fragment F, recovered in both the hexa- and the octasaccharide, was the prime acceptor for the first 6-O-sulfate group. Similar results were obtained with all substrates except octasaccharide B, where the first 6-O-sulfate group was found also on the GlcNS unit toward the reducing end (see B' in Fig. 4D).



View larger version (33K):
[in this window]
[in a new window]
 
FIG. 4.
Purification and analysis of the mono-6-O-sulfated product F' formed by 6-OST3-catalyzed sulfation of fragment F. Octasaccharide F was incubated with 6-OST3 and PAPS, and the products were separated on the ProPac PA1 anion exchange column (panel A). Both the substrate F and the mono-6-O-sulfated product F' were recovered and subjected to partial HNO2/pH 1.5 cleavage. The resulting labeled di-, tetra-, hexa-, and octasaccharides were analyzed on the ProPac PA1 column (panels B and C). The initial 6-O-sulfation of fragment F, and of all other octasaccharides except fragment B involved the middle of the three potential N-sulfoglucosamine units (panel D). See "Results" for further details.

 

Substrate Specificities of 6-OSTs—The effects of 2-O-sulfate groups on the substrate preference of 6-OST3 was studied by incubating two distinct 3H-labeled octasaccharides with lysates from 6-OST3-overexpressing HEK 293 cells in the presence of unlabeled PAPS. The initial rate (v0) of the catalyzed transfer of sulfate from PAPS to the octasaccharide acceptors was measured as the amounts of mono-6-O-sulfated products formed (see "Experimental Procedures"). Product formation was linear with time until ~10% of the substrate was consumed, and then the rate decreased (see Fig. 6). Because all heparin fragments were initially labeled in the same reaction with NaB3H4, the specific 3H activity of the different octasaccharides would be approximately equal (22). Thus, the relative concentrations of substrates and products are reflected by the relative 3H peak areas in anion exchange chromatograms (Fig. 5A). Two distinct substrate preparations (Fig. 2) were mixed and incubated with 6-OST3 and PAPS allowing the substrates to compete for access to the active site of the enzyme. The best substrate of the two was identified as the one showing the largest conversion to mono-6-O-sulfated product, taking into account the substrate concentration (Equation 1). For six different combinations of octasaccharide substrates (B + D, B + F, B + G, D + E, E + G, and F + G), the 6-OST3-catalyzed reaction was quenched at different time points and substrates and products were analyzed. For each reaction, initial rates for 6-OST3 toward the two substrates were estimated by fitting linear or exponential equations to data using the inrate program of the SIMFIT package (29) (Fig. 6). Results were applied to Equation 1 (see "Experimental Procedures") for calculation of relative substrate specificities toward different octamers. Other experiments with two substrates competing for 6-OST3 were conducted for constant periods of time (1–5 independent experiments) under initial rate conditions (i.e. ≤10% substrate consumption, see Fig. 6). Altogether, the results suggest that 2-O-sulfate groups promote the catalytic activity of 6-OST3 (Table I). Di- and tri-2-O-sulfated octamer substrates were favored by 6-OST3 as shown, e.g. by the 3–5-fold increased specificity toward these fragments over the completely O-desulfated octasaccharide A.



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 6.
Formation of mono-6-O-sulfated products as a function of time. A mixture of octasaccharides B and G was incubated with 6-OST3 and PAPS. The reaction was stopped at different time points and the resulting amounts of substrates and products were determined (cf. Fig. 5). The fraction product formed, e.g. B'/(B + B'), is plotted on the y axis, and the slope at the origin thus provides a measure of the initial rate over substrate concentration for the respective octasaccharide. The rate is essentially linear with time up to around 10% product formed. Specificities toward competing substrates for 6-OST3 were calculated according to Equation 1.

 


View larger version (20K):
[in this window]
[in a new window]
 
FIG. 5.
Examples of experiments used to determine the substrate specificities of 6-OST2 and 6-OST3 for octasaccharides with varying 2-O-sulfation. A, two different octasaccharides, A and G (see Fig. 2), were mixed at similar concentrations, as based on 3H contents. The 6-O-sulfation reaction catalyzed by 6-OST3 was allowed to proceed until ~5–10% of the respective substrate was consumed. The reaction products formed were analyzed by anion exchange HPLC and the amounts of mono-6-O-sulfated products A' and G' formed, measured as the areas of their peaks, were used as estimates of their corresponding initial rates. The sum of substrate and product for the respective fragment was taken as substrate concentration. The relative specificity for the two competing substrates was calculated according to Equation 1. B, the octasaccharides, A and D (see Fig. 2), were mixed and incubated with 6-OST2. The amount of di-6-O-sulfated product D'' formed roughly equals the amount of mono-6-O-sulfated A'. Because the concentration of D' (the substrate of the reaction giving D'') is lower than the concentration of A, this result suggests that the di-2-O-sulfated octasaccharide D as well as its mono-6-O-sulfated product D' are both better substrates for 6-OST2 than the O-desulfated octamer A.

 

View this table:
[in this window]
[in a new window]
 
TABLE I
Specificities for competing substrates for 6-OST3 and 6-OST2

 

Similar results were obtained for 6-OST2, which likewise displayed a preference for 2-O-sulfated octamers over the fragment devoid of O-sulfates. In these experiments, the reactions were allowed to proceed long enough for di-6-O-sulfated species to emerge from the di-, tri-, and tetra-2-O-sulfated substrates (Fig. 5B). This approach revealed that also the mono-6-O-sulfated di-, tri-, and tetra-2-O-sulfated octamers were better substrates for 6-OST2 than the O-desulfated octasaccharide A. For example, the experiment illustrated in Fig. 5B shows about equal formation of di-6-O-sulfated D'' and mono-6-O-sulfated A' despite a higher concentration of substrate A than of substrate D'. Moreover, the average time for the production of D'' is shorter than that for formation of A', because D' must first be formed from D.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study demonstrates the use of library-type oligosaccharides in determining substrate specificities of enzymes involved in heparin/heparan sulfate biosynthesis. Experiments with competing, well defined, oligosaccharides allow the assessment of relative specificities that define the preferred substrates of the enzymes. We have applied this concept to 6-OSTs that catalyze a late step in biosynthetic polymer modification of HS. Notably, the number and positions of glucosamine 6-O-sulfate groups are important determinants in a variety of biological phenomena (30). The 6-O-sulfation reaction is preceded by C5 epimerization of GlcUA to IdoUA units, followed by 2-O-sulfation of, in particular, IdoUA units (24). In a separate study we have shown that the epimeric configuration of the hexuronate residue directly upstream of the acceptor glucosamine unit influences the substrate preference of the 6-OSTs, an IdoUA residue in this position providing a better acceptor sequence than a GlcUA unit (31). Because the arrangement of preformed 2-O-sulfate groups on the maturing HS chain might also regulate 6-O-sulfation we studied the substrate specificity of 6-OST3 with regard to distinct octasaccharides with varying degrees and patterns of 2-O-sulfation.

The results of these experiments show that 2-O-sulfate groups consistently promote the enzymatic activity of 6-OST3. Octasaccharides with two or more 2-O-sulfate groups were better substrates than octasaccharides with no or only one 2-O-sulfate group. No clear differences in specificity were observed toward substrates of similar charge, suggesting that the mere presence of 2- (and 6-) O-sulfates is more important for substrate discrimination than the exact positions of the sulfates. However, all possible substrates were not tested and we cannot exclude the occurrence of more preferred substrates.

Previous work showed that 2-O-sulfation of IdoUA is precluded by 6-O-sulfation of an adjacent GlcN unit (32). It is therefore of interest to note that 6-O-sulfation will readily take place between two 2-O-sulfated IdoUA residues, as demonstrated by sulfation of the tetra-2-O-sulfated substrate H (Fig. 3).

The contribution of each 6-OST isoform in generating specific heparan sulfate protein binding sequences is currently not known. Here we have mainly studied the substrate preference of 6-OST3 and to a lesser extent that of 6-OST2. No striking differences were observed in catalytic specificity toward the different octasaccharide substrates. The latter would suggest that distinct complex sequences are unlikely to be formed in vivo by these two enzymes. However, at present it is difficult to assess the significance of the observed differences, because of the paucity of quantitative data for the other biosynthetic enzymes. The most important aspect of the present work is therefore the potential for unbiased comparison of defined saccharide sequences with regard to substrate specificity, which should generate a more detailed understanding of HS biosynthesis as other enzymes and substrates are studied. This development was enabled through the use of the library-based saccharide substrates (pending access to purely synthetic analogs of authentic saccharide structures). In principle, a similar approach should be feasible in studies of a variety of polymer-modifying enzymes. The present work relied on the ability to elucidate the structures of octasaccharide substrates and products. Unfortunately, current sequencing methodology is not readily adapted to physiologically more relevant substrates, because HS oligosaccharides longer than octa/decamers are not easily sequenced. Still, octasaccharide substrates are most probably relevant from an enzymatic point-of-view. There is no crystal structure available for any of the HS O-sulfotransferases, but the structure for the homologous N-sulfotransferase domain of an N-deacetylase/N-sulfotransferase, involved in HS biosynthesis, has been described (33). Whereas the sequence similarity between this domain and soluble sulfotransferases such as estrogen and hydroxysteroid sulfotransferase is low their crystal structures are similar (34), and it is thus unlikely that the structures of the 6-OSTs would differ markedly from that of the N-sulfotransferase domain. A homology model of HS 3-O-sulfotransferase based on this N-sulfotransferase structure (35) underscores the structural similarities of the HS sulfotransferases. The cleft that binds the polysaccharide substrate can contain a hexasaccharide according to the crystal structure of the N-sulfotransferase domain (33), whereas the saccharide pocket in the 3-O-sulfotransferase model can accommodate at least a tetrasaccharide (35). Thus, the polysaccharide moiety interacting with the 6-OSTs probably does not encompass more than three disaccharide units, such that octasaccharides, composed of four hexuronate and three GlcN units, and with a aManR unit at the reducing end, would be a good substitute for the native substrate. Indeed, the finding that the middle GlcNS unit is the preferred position for O-sulfation (Fig. 4) is in line with a binding site that can accommodate a hexasaccharide and utilize the two flanking disaccharide units to increase the affinity for the transition state of the catalyzed reaction.


    FOOTNOTES
 
* This work was supported by Swedish Medical Research Council Grant 13401, The Swedish Foundation for Strategic Research (the program "Glycoconjugates in Biological Systems"), Magnus Bergvalls stiftelse, Polysackaridforskning AB (Uppsala, Sweden), Konung Gustav V 80-års fond, and European Commission Contract QLK3-CT99-00536. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence may be addressed: Present address: Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, United Kingdom. E-mail: pj{at}mrc-lmb.cam.ac.uk.

|| To whom correspondence may be addressed. Tel.: 46-18-471-4242; Fax: 46-18-471-4209; E-mail: Marion.Kusche{at}imbim.uu.se.

1 The abbreviations used are: HS, heparan sulfate; 6-OST2, -3, 6-O-sulfotransferase 2, -3; GlcN, D-glucosamine; GlcUA, D-glucuronate; IdoUA, L-iduronate; GlcNAc, N-acetyl-D-glucosamine; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; aManR, 2,5-anhydro-D-mannitol (formed by reduction of terminal 2,5-anhydromannose residues with NaBH4); NS, N-sulfate group; 2S, 2-O-sulfate group; 6S, 6-O-sulfate group; HPLC, high performance liquid chromatography; HEK, human embryonic kidney. Back

2 Previous experiments showed that, under the experimental conditions used, 6-O-sulfation is more readily achieved than 2-O-sulfation using the microsomal fraction as catalyst (18). Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bernfield, M., Götte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Annu. Rev. Biochem. 68, 729–777[CrossRef][Medline] [Order article via Infotrieve]
  2. Lindahl, U., Kusche-Gullberg, M., and Kjellén, L. (1998) J. Biol. Chem. 273, 24979–24982[Free Full Text]
  3. Esko, J. D., and Lindahl, U. (2001) J. Clin. Invest. 108, 169–173[Free Full Text]
  4. Esko, J. D., and Selleck, S. B. (2002) Annu. Rev. Biochem. 71, 435–471[CrossRef][Medline] [Order article via Infotrieve]
  5. Orellana, A., Hirschberg, C. B., Wei, Z., Swiedler, S. J., and Ishihara, M. (1994) J. Biol. Chem. 269, 2270–2276[Abstract/Free Full Text]
  6. Eriksson, I., Sandbäck, D., Ek, B., Lindahl, U., and Kjellén, L. (1994) J. Biol. Chem. 269, 10438–10443[Abstract/Free Full Text]
  7. Aikawa, J., and Esko, J. D. (1999) J. Biol. Chem. 274, 2690–2695[Abstract/Free Full Text]
  8. Aikawa, J., Grobe, K., Tsujimoto, M., and Esko, J. D. (2001) J. Biol. Chem. 276, 5876–5882[Abstract/Free Full Text]
  9. Habuchi, H., Tanaka, M., Habuchi, O., Yoshida, K., Suzuki, H., Ban, K., and Kimata, K. (2000) J. Biol. Chem. 275, 2859–2868[Abstract/Free Full Text]
  10. Shworak, N. W., Liu, J., Fritze, L. M. S., Schwartz, J. J., Zhang, L., Logeart, D., and Rosenberg, R. D. (1997) J. Biol. Chem. 272, 28008–28019[Abstract/Free Full Text]
  11. Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5170–5184[Abstract/Free Full Text]
  12. Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J-P., Malmström, A., Shukla, D., and Liu, J. (2002) J. Biol. Chem. 277, 37912–37919[Abstract/Free Full Text]
  13. Li, J. P., Gong, F., El Darwish, K., Jalkanen, M., and Lindahl, U. (2001) J. Biol. Chem. 276, 20069–20077[Abstract/Free Full Text]
  14. Crawford, B. E., Olson, S. K., Esko, J. D., and Pinhal, M. A. S. (2001) J. Biol. Chem. 276, 21538–21543[Abstract/Free Full Text]
  15. Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980–13985[Abstract/Free Full Text]
  16. Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2001) Biochemistry 40, 5548–5555[CrossRef][Medline] [Order article via Infotrieve]
  17. Salmivirta, M., Lidholt, K., and Lindahl, U. (1996) FASEB J. 10, 1270–1279[Abstract/Free Full Text]
  18. Jemth, P., Kreuger, J., Kusche-Gullberg, M., Sturiale, L., Giménez-Gallego, G., and Lindahl, U. (2002) J. Biol. Chem. 277, 30567–30573[Abstract/Free Full Text]
  19. Nagasawa, K., Inoue, Y., and Kamata, T. (1977) Carbohydr. Res. 58, 47–55[CrossRef][Medline] [Order article via Infotrieve]
  20. Spillmann, D., Witt, D., and Lindahl, U. (1998) J. Biol. Chem. 273, 15487–15493[Abstract/Free Full Text]
  21. Levy, L., and Petracek, F. J. (1962) Proc. Soc. Exp. Biol. Med. 190, 901–905
  22. Shively, J. E., and Conrad, H. E. (1976) Biochemistry 15, 3932–3942[Medline] [Order article via Infotrieve]
  23. Turnbull, J. E., Hopwood, J. J., and Gallagher, J. T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2698–2703[Abstract/Free Full Text]
  24. Vivès, R. R., Pye, D. A., Salmivirta, M., Hopwood, J. J., Lindahl, U., and Gallagher, J. T. (1999) Biochem. J. 339, 767–773[CrossRef][Medline] [Order article via Infotrieve]
  25. Kreuger, J., Salmivirta, M., Sturiale, L., Giménez-Gallego, G., and Lindahl, U. (2001) J. Biol. Chem. 276, 30744–30752[Abstract/Free Full Text]
  26. Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463–468[CrossRef][Medline] [Order article via Infotrieve]
  27. Fersht, A. (1999) in Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding, W. H. Freeman and Co., New York
  28. Habuchi, H., Miyake, G., Nogami, K., Kuroiwa, A., Matsuda, Y., Kusche-Gullberg, M., Habuchi, O., Tanaka, M., and Kimata, K. (2003) Biochem. J. 371, 131–142[CrossRef][Medline] [Order article via Infotrieve]
  29. Bardsley, W. G., Bukhari, N. A. J., Ferguson, M. W. J., Cachaza, J. A., and Burguillo, F. J. (1995) Comput. Chem. 19, 75–84[CrossRef]
  30. Gallagher, J. T. (2001) J. Clin. Invest. 108, 357–361[Free Full Text]
  31. Smeds, E., Habuchi, H., Hjertson, E., Grundberg, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2003) Biochem. J. 372, 371–380[CrossRef][Medline] [Order article via Infotrieve]
  32. Jacobsson, I., and Lindahl, U. (1980) J. Biol. Chem. 255, 5094–5100[Abstract/Free Full Text]
  33. Kakuta, Y., Sueyoshi, T., Negishi, M., and Pedersen, L. C. (1999) J. Biol. Chem. 274, 10673–10676[Abstract/Free Full Text]
  34. Negishi, M., Pedersen, L. G., Petrotchenko, E., Shevtsov, S., Gorokhov, A., Kakuta, Y., and Pedersen, L. C. (2001) Arch. Biochem. Biophys. 390, 149–157[CrossRef][Medline] [Order article via Infotrieve]
  35. Raman, R., Myette, J., Venkataraman, G., Sasisekharan, V., and Sasisekharan, R. (2002) Biochem. Biophys. Res. Commun. 290, 1214–1219[CrossRef][Medline] [Order article via Infotrieve]