Anticoagulant Heparan Sulfate Precursor Structures in F9 Embryonal Carcinoma Cells*

Lijuan ZhangDagger §, Keiichi Yoshida, Jian LiuDagger , and Robert D. RosenbergDagger parallel **

From the Dagger  Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, the parallel  Department of Medicine, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215, and  Tokyo Research Institute of Seikagaku Corp., Higashiyamato-shi, Tokyo 207, Japan

    ABSTRACT
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Abstract
Introduction
References

To understand the mechanisms that control anticoagulant heparan sulfate (HSact) biosynthesis, we previously showed that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. In this study, HSact precursor structures have been studied by characterizing [6-3H]GlcN metabolically labeled F9 HS tagged with 3-O-sulfates in vitro by 3'-phosphoadenosine 5'-phospho-35S and purified 3-O-sulfotransferase-1. This later in vitro labeling allows the regions of HS destined to become the antithrombin (AT)-binding sites to be tagged for subsequent structural studies. It was shown that six 3-O-sulfation sites exist per HSact precursor chain. At least five out of six 3-O-sulfate-tagged oligosaccharides in HSact precursors bind AT, whereas none of 3-O-sulfate-tagged oligosaccharides from HSinact precursors bind AT. When treated with low pH nitrous or heparitinase, 3-O-sulfate-tagged HSact and HSinact precursors exhibit clearly different structural features. 3-O-Sulfate-tagged HSact hexasaccharides were AT affinity purified and sequenced by chemical and enzymatic degradations. The 3-O-sulfate-tagged HSact hexasaccharides exhibited the following structures, Delta UA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS335S±6S-IdceA2S-[6-3H]GlcNS6S. The underlined 6- and 3-O-sulfates constitute the most critical groups for AT binding in view of the fact that the precursor hexasaccharides possess all the elements for AT binding except for the 3-O-sulfate moiety. The presence of five potential AT-binding precursor hexasaccharides in all HSact precursor chains demonstrates for the first time the processive assembly of specific sequence in HS. The difference in structures around potential 3-O-sulfate acceptor sites in HSact and HSinact precursors suggests that these precursors might be generated by different concerted assembly mechanisms in the same cell. This study permits us to understand better the nature of the HS biosynthetic pathway that leads to the generation of specific saccharide sequences.

    INTRODUCTION
Top
Abstract
Introduction
References

Different heparin/heparan sulfate (HS)1 sequences bind to a large number of growth factors and cytokines (1-6), enzymes (7), protease inhibitors (8-12), virus proteins (13), and selectins (14). Such sequences are usually synthesized in the right place (15) and at the right time (16, 17). These HS species are involved in development, angiogenesis, lipid metabolism, coagulation, virus infection, and inflammation (18-22). To synthesize HS oligosaccharide sequences that bind to these specific protein ligands probably requires the synthesis of specific HS precursor structures. HS precursor structures are then acted upon by a unique sulfotransferase, which is positioned at the end of the biosynthetic pathways and whose levels control the concentration of the specific HS component that is the product of the biosynthetic pathway.

The pentasaccharide sequence, GlcNAc/NS6S-GlcUA-GlcNS3S6S-IdceA2S-GlcNS6S, represents the minimum sequence for antithrombin (AT) binding, where the boldface 3S and 6S constitute the most critical elements involved in the interaction (8, 9). The first sugar residue is either N-acetylated or N-sulfated. The third sugar residue is either with or without a 6-O-sulfate, depending on the source of heparin from which these sequences were originally characterized (23). The AT-binding sequence also exists in HS, but such sequences have never been fully characterized due to limited materials. HSact produced by endothelial cells (1% total HS) is responsible in part for the nonthrombogenic properties of blood vessels (24). Even though it constitutes a small portion of HStotal, the relative abundance of the AT-binding sequence is at least 10-fold greater than would be predicted by completely random assembly of disaccharide constituents (25). These observations suggest that production of the AT-binding sequence requires the coordinated action of several biosynthetic enzymes, i.e. the enzymes that catalyze chain polymerization, GlcNAc N-deacetylation and N-sulfation, GlcUA epimerization, 2-O-sulfation of uronic acid residues, and 3-O- and 6-O-sulfation of glucosaminyl residues. Multiple forms of N-sulfotransferases (26-30) and 3-O-sulfotransferases (31)2 have been reported recently. Due to the substrate specificity of the enzymes involved, the initial distribution of N-sulfate groups strongly influences the subsequent epimerization and O-sulfations (32, 33). The downstream O-sulfation may also be able to influence N-sulfation as well (34). However, the mechanisms that control the coordinated action of these enzymes to generate AT-binding sequences or other sequences in HS and heparin are unknown.

To delineate the biosynthetic pathway that regulates HSact synthesis, our laboratory has purified as well as molecularly cloned 3-O-ST-1 (EC 2.8.2.23). It was demonstrated that 3-O-ST-1, existing in limited amount, acts upon HSact precursor to produce HSact and HSinact precursor to produce 3-O-sulfated HSinact (31, 35). When 3-O-ST-1 is no longer limiting in the F9 cell system, the capacity for HSact generation is determined by the abundance of HSact precursors (36). We have reported that overall HSact and HSinact structures are different at the disaccharide level in the F9 cell system. In vitro 3-O-sulfation with purified 3-O-ST-1 can tag the regions of the HSact precursors destined to become AT-binding sites and allow the HSact precursors to be captured. The tagged regions can then be structurally examined (36). Based on the above observations, we have studied the HSact and HSinact precursor structures by characterizing [6-3H]GlcN metabolically labeled F9 HS tagged with 3-O-sulfates in vitro by PAP35S and purified 3-O-ST-1. We have found that HSact and HSinact precursors in F9 cells have different epimerization and O-sulfation patterns around the 3-O-sulfate acceptor sites in the intact glycosaminoglycan (GAG) chain. We have sequenced the 3-O-sulfate-tagged HSact precursor hexasaccharides that represent the 3-O-sulfate acceptor sites in the HSact precursors, which are destined to become AT-binding sites. This region contains five hexasaccharides, i.e. (Delta UA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS3356S-IdceA2S-[6-3H]GlcNS6S)5, which possess the correct positioning of all critical groups except for the absence of the 3-O-sulfate residue required for AT binding in all HSact precursor chains. This finding demonstrates for the first time the overall structure of the HSact precursors. This information permits us to speculate about the nature of the HS biosynthetic pathway that leads to the generation of specific oligosaccharide sequences.

    EXPERIMENTAL PROCEDURES

Cell Culture-- F9 cells were grown on gelatin (Sigma) coated (0.1%) tissue culture dishes in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated calf serum (Irvine Scientific), penicillin G (100 units/ml), and streptomycin sulfate (100 µg/ml) under an atmosphere of 5% CO2, 95% air and 100% relative humidity. All tissue culture media and reagents were purchased from Life Technologies, Inc., unless otherwise indicated.

HS Preparation-- The methods for HS preparation have been described previously (36). In brief, cell monolayers were labeled with 100-1000 µCi/ml sodium [35S]sulfate (carrier-free, ICN) overnight by incubation in sulfate-deficient DMEM, penicillin G (100 units/ml), and 10% (v/v) fetal bovine serum that had been exhaustively dialyzed against phosphate-buffered saline and supplemented with 200 µM Na2SO4. [6-3H]Glucosamine labeling were conducted in 100 µCi/ml D-[6-3H]glucosamine (40 Ci/mmol, ICN) overnight in 1 mM glucose DMEM with 10% (v/v) dialyzed fetal bovine serum. GAGs were isolated from both monolayer and media. Purified GAGs were resuspended in 0.5 M NaBH4 in 0.4 M NaOH and incubated at 4 °C for 24 h to release the chains from the core protein. beta -Elimination was stopped by adding 5-µl aliquots of 5 M acetic acid until bubble formation ceased. The released GAG chains were purified by DEAE-Sepharose (Sigma) chromatography followed by ethanol precipitation and then resuspended in water. GAGs were digested with 20 milliunits of chondroitinase ABC (Seikagaku) in 50 mM Tris-HCl and 50 mM sodium acetate buffer (pH 8.0). Complete digestion of chondroitin sulfate by chondroitinase ABC was ensured by monitoring the extent of conversion of the carrier to disaccharides (100 µg = 1.14 absorbance units at 232 nm). HS was purified from chondroitinase-degraded products by phenol/chloroform extraction and ethanol precipitation. After washing the pellets with 0.5 ml of 75% ethanol, HS was suspended in H2O for further analysis.

The quality of HS chains prepared was analyzed by anion exchange HPLC (TSK DEAESW, 8 cm × 7.5 mm inner diameter, Tosohaas Inc.). Samples were eluted with a linear gradient of 0.2 to 1 M NaCl in 10 mM KH2PO4, pH 6.0, containing 0.2% CHAPS at a flow rate of 1 ml/min, and radioactivity in the effluent was determined by on-line liquid scintillation spectrometry (Packard).

The absolute amounts of the unlabeled HS were determined by hydrolyzing HS for 3 h with 6 N HCl and 0.1% phenol (v/v) at 100 °C and determining the levels of glucosamine on an Applied Biosystems model 420 Amino Acid Derivatizer with on-line model 130A PTC amino acid analyzer.

HSact and HSinact Precursors Were Tagged by 3-O-Sulfation with Purified 3-O-ST-1 and PAPS-- The standard 50-µl reaction contains 400 units of purified 3-O-sulfotransferase-1 (3-O-ST-1) (EC 2.8.2.23), 60 µg of bovine serum albumin, metabolically labeled [3H]HS, [35S]HS, or cold HS chains prepared from F9 cells suspended in 1 mM CaCl2, 5 mM MgCl2, 10 mM MnCl2, 0.375 µg/ml protamine, 0.4 mg/ml chondroitin sulfate, 1% Triton X-100, 50 mM MES (pH 7.0), and 1 mM PAPS or 25 µM (1 × 108 cpm) PAP35S. The reactions were incubated at 37 °C overnight and then terminated by boiling for 1 min. After adding 100 µg of chondroitin sulfate as cold carrier, the radiolabeled HS was purified by phenol/chloroform extraction, DEAE-Sepharose chromatography, and ethanol precipitation. After washing the pellets with 0.5 ml of 75% ethanol, 3-O-sulfate-tagged HSact and HSinact precursors were separated by AT affinity assay as described below.

Separation of 3-O-Sulfate-tagged HSact and HSinact Precursors by AT Affinity Assay-- AT affinity assay was used as described previously (36). In brief, AT complexes were created by mixing 3-O-sulfate-tagged HStotal in 500 µl of HB (150 mM NaCl, 10 mM Tris-Cl (pH 7.4)) with 2.5 mM AT, 100 µg of chondroitin sulfate, 0.002% Triton X-100, 1 mM each of CaCl2, MgCl2, and MnCl2. 60 µl of HB containing ~50% concanavalin A-Sepharose 4B was then added. AT complexes were bound to concanavalin A by mixing the reaction mixtures for 1 h at room temperature. Beads were pelleted by a brief centrifugation at 10,000 × g. The supernatant was collected, and the beads were washed three times with 1.25 ml of HB containing 0.0004% Triton X-100. The supernatant and washing solutions were combined as 3-O-sulfate-tagged HSinact precursors. 3-O-Sulfate-tagged HSact precursors were eluted with three successive 200-µl washes of HB containing 1.0 M NaCl, 0.0004% Triton X-100 and pooled. After adding 100 µg of chondroitin sulfate as cold carrier to 3-O-sulfate-tagged HSact precursors, the pooled 3-O-sulfate-tagged HSact and HSinact precursors were cleaned by phenol/chloroform extraction followed by DEAE-Sepharose chromatography and ethanol precipitation. After washing the pellets with 0.5 ml of 75% ethanol and dried briefly by Speed-Vac, 3-O-sulfate-tagged HSact and HSinact precursors were resuspended in H2O and used for chemical and enzymatic structural studies.

Low pH Nitrous Acid Degradation-- 3-O-Sulfate-tagged HSact and HSinact precursors or 3-O-sulfate-tagged tetra- or hexasaccharide were mixed with 10 µg of bovine kidney HS (ICN), treated at room temperature with low pH nitrous acid (pH 1.5) for 10 min, and then reduced under alkaline conditions with 0.5 M NaBH4 for another 10 min. Bio-Gel P6 or P2 chromatography as described below was used to analyze further the resultant products.

Bio-Gel P2 and P6 Gel Filtration Chromatography of Chemically or Enzymatically Degraded 3-O-Sulfate-tagged HSact and HSinact Precursors-- Bio-Gel P2 (0.75 × 200 cm) and P6 (0.75 × 200 cm) columns were equilibrated with 100 mM ammonium bicarbonate at a flow rate of 4 ml/h. 200 µl of radiolabeled sample mixed with dextran blue (5 µg) and phenol red (5 µg) was loaded on the column. 0.4 ml per fraction was collected. A 10-µl sample from each fraction was counted for 3H and/or 35S radioactivity unless otherwise indicated. The desired fractions were pooled and dried by Speed-Vac to remove ammonium bicarbonate and used for further analysis. A small amount of free sulfate generated during the experimental procedures was quantitated by re-running the collected disaccharide fractions on a polyamine HPLC. The free sulfate is subtracted from disaccharides to get the accurate di-, tetra-, and oligosaccharide quantitations indicated under "Results."

Digestion of 3-O-Sulfate-tagged HSact and HSinact Precursors with Heparitinase I, Heparitinase II, Heparinase, and Heparitinase IV-- Heparitinase I (EC 4.2.2.8), heparitinase II (no EC number), heparinase (EC 4.2.2.7) were obtained from Seikagagu; heparitinase IV was from Dr. Yoshida, Seikagagu Corp., Tokyo, Japan. Heparitinase I recognizes the following sequences: GlcNAc/NS±6S(3S?)-down-arrow GlcUA-GlcNAc/NS±6S. The arrow indicates the cleavage site. Heparitinase II has broad sequence recognition, GlcNAc/NS±6S(3S?)-down-arrow GlcUA/IdceA±2S-GlcNAc/NS±6S. Heparinase and heparitinase IV recognizes the sequences: GlcNAc/NS±3S±6S-down-arrow IdceA2S-GlcNAc/NS±6S. The reaction products and references can be found in the Seikagagu's catalog.

The digestion of 3-O-sulfate-tagged HSact and HSinact precursors was carried out in 100 µl of 40 mM ammonium acetate (pH 7.0) containing 1 mM CaCl2 with 2 milliunits of enzyme or 2 milliunits of each heparitinase I, heparitinase II, and heparinase. The digestion was incubated at 37 °C overnight unless otherwise indicated.

PAMN High Performance Liquid Chromatography-- Separation and characterization of 3-O-sulfate-tagged tetra- and hexasaccharides were carried out by HPLC using an amine-bound silica PA01 column (0.46 × 25 cm) (PAMN-HPLC) (YMC). 100-µl aliquots containing 2 nmol of each six known heparan sulfate disaccharide standards (Seikagagu) were included with all analytic runs. The PAMN-HPLC was eluted with H2O for 5 min followed by 0-100% KH2PO4 linear gradient for 100 min at a flow rate of 1 ml/min. The elution of cold disaccharide standards was detected by on-line UV monitoring, and radiolabeled disaccharides were monitored by on-line liquid scintillation spectrometry (Packard Instrument Co.). Each fraction represents a 0.5-ml elution volume.

Sequential Enzymatic Degradation of Delta 4,5-Tetrasaccharides-- Delta 4,5Glycuronidase (no EC number) and Delta 4,5glycuronate 2-O-sulfatase (no EC number) were from Dr. Yoshida, Seikagagu Corp., Tokyo, Japan. alpha -N-Acetylglucosamine 6-O-sulfate sulfatase (6-O-sulfatase) (no EC number) and alpha -N-acetylglucosaminidase (no EC number) were purified from bovine kidney in our laboratory. No other enzymatic activity was detected in these enzyme preparations. GlcUA-[3H]anManR3S and GlcUA-[3H]anManR3S6S standards were prepared as described previously (31). GlcUA-anManR335S and GlcUA-anManR335S6S standards were prepared from 3-O-35S-sulfate-tagged cold F9 HS. In brief, after hydrazinolysis, 3-O-35S-sulfate-tagged F9 HS was treated with high pH nitrous acid (pH 4.0) and low pH nitrous acid (pH 1.5). The resultant disaccharides were reduced with NaBH4 and desalted by Bio-Gel P2 chromatography. 3-O-35S-Sulfate-tagged disaccharides were collected by ion pairing reverse phase HPLC as previously reported (37). The identity of GlcUA-anManR335S and GlcUA-anManR335S6S was further confirmed by co-chromatography on ion pairing reverse phase HPLC with GlcUA-[3H]anManR3S and GlcUA-[3H]anManR3S6S standards. GlcUA-anManR335S and GlcUA-anManR335S6S are the only disaccharides generated by 3-O-ST-1 and PAP35S tagging of cold F9 HS.

3-O-35S-Sulfate-tagged tetrasaccharides were digested with 10 milliunits of Delta 4,5glycuronate 2-O-sulfatase or Delta 4,5glycuronidase in a total volume of 100 µl of 50 mM imidazole HCl buffer (pH 6.5) at 37 °C overnight. 5% (5 µl) of the reaction mixture was combined with six HS disaccharide UV standards and GlcUA-[3H]anManR3S6S standard (2000 cpm) in a total volume of 100 µl and analyzed by polyamine HPLC. Completion of the digestion was confirmed by polyamine HPLC. 40 µl of 0.5 M sodium acetate (pH 5.5), 55 µl of H2O, and 10 µl 6-O-sulfatase (8 µg) were added to 95 µl of reaction mixture. The 6-O-sulfatase digestion was completed in 2 days at 37 °C as monitored by polyamine HPLC as described above. 100 µl of 6-O-sulfatase-treated materials was diluted with 300 µl of H2O. 2 µl of 16 M acetic acid and then 5 µl of alpha -N-acetylglucosaminidase (3.5 µg) were added (pH 4.2). alpha -N-Acetylglucosaminidase digestion was completed overnight at 37 °C as monitored by polyamine HPLC.

    RESULTS

The HSact Precursors Contain Six Potential AT-binding Sites Whose Structure Is Completed by in Vitro 3-O-Sulfation-- We have previously reported that F9 cells make 1% HSact and 29% HSact precursors, and the metabolically labeled GlcUA-anManR335S (6%) and GlcUA-anManR335S635S (12%) accounted for about 18% of O-sulfated disaccharides in 1% F9 HSact (36). The high percentage of 3-O-sulfated disaccharides in the biosynthesized HSact chain suggests that there are multiple potential 3-O-sulfation sites in the F9 HSact precursor chain. To estimate this parameter, [6-3H]GlcN metabolically labeled HSact precursor was isolated by exhaustive conversion to HSact by exposure in vitro to pure 3-O-ST-1 as well as PAPS and subsequent captured by AT affinity chromatography. The 3-O-sulfate-tagged HSact precursors were subjected to a mixture of heparitinase I, heparitinase II, and heparinase treatment. According to a previous publication (38), the combined heparitinase I, heparitinase II, and heparinase digestion should depolymerize all the 3-O-sulfate containing sites into tetrasaccharides and the remaining sugar residues into disaccharides. The depolymerized materials were sized by Bio-Gel P2 chromatography (Fig. 1). 15% of 3H counts are resistant to digestion and remained as tetrasaccharides. In contrast, 0.5% of 3H counts are resistant to digestion and remained as tetrasaccharides in [6-3H]GlcN metabolically labeled F9 HS control (data not shown).


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Fig. 1.   Bio-Gel P2 fractionation of heparitinase I-, heparitinase II-, and heparinase-digested 3-O-sulfate-tagged HSact precursors. [6-3H]GlcN metabolic HS were treated with 3-O-ST-1 and PAPS. 3-O-Sulfate-tagged [3H]HS was AT affinity fractionated into HSact and HSinact. 3-O-Sulfate-tagged [3H]HSact precursors were digested with 2 milliunits each of heparitinase I, heparitinase II, and heparinase overnight. The products were analyzed by Bio-Gel P2 chromatography ("Experimental Procedures"). The fractions indicated by solid bars were pooled as tetrasaccharides and disaccharides.

The di- and tetrasaccharides indicated by solid bar in Fig. 1 were collected and subjected to polyamine HPLC chromatography (Fig. 2). Each disaccharide peak on HPLC was assigned by comparing to standards ("Experimental Procedures"). The two tetrasaccharide peaks that are resistant to the combined treatment of heparitinase I, heparitinase II, and heparinase digestion are likely to represent the 3-O-sulfate-containing tetrasaccharides. We will subsequently show that the first tetrasaccharide peak has the structure Delta UA-GlcNAc6S-GlcUA-GlcNS3S and the second peak has the structure Delta UA-GlcNAc6S-GlcUA-GlcNS3S6S.


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Fig. 2.   Polyamine HPLC profile of di- and tetrasaccharides. [3H]Di- and [3H]tetrasaccharides from 3-O-sulfatetagged HSact precursors collected after a Bio-Gel P2 column (Fig. 1) were further resolved by polyamine HPLC. The elution times of the disaccharide peaks were compared with those of authentic standards and numerically labeled accordingly. The tetrasaccharide fractions indicated by solid bars were pooled as tetrasaccharide I (1) and tetrasaccharide II (2). The numbers correspond with numbers and names in Table I, which summarizes the relative abundance of each of the di- and tetrasaccharides.

The di- and tetrasaccharides from Fig. 2 were further analyzed to estimate the number of 3-O-sulfation sites per HSact precursor chain. We first assessed the molecular weight for F9 HS by SDS-polyacrylamide gel electrophoresis as described previously (39), where Mr = 2.112 × protein M-21027. HS exhibits an average molecular weight (Mr) of 42,000 (data not shown). Based on this and the average disaccharide molecular mass of 500 Da, there are about 84 disaccharides per chain, i.e. 82 disaccharides in the repetitive -(UA-GlcNAc/S)n- region and one tetrasaccharide GlcUA-Gal-Gal-Xyl- in the linkage region. Assuming the average HS consists of 82 disaccharides in the repetitive -(UA-GlcNAc/S)n- region, the number of di- and tetrasaccharides per 3-O-sulfated HSact precursor chain are summarized in Table I. There are 4 × Delta UA-GlcNAc6S-GlcUA-GlcNS3S and 2 × Delta UA-GlcNAc6S-GlcUA-GlcNS3S6S per chain, therefore a total of six 3-O-sulfate sites per HSact precursor chain.

                              
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Table I
Di- and tetrasaccharide compositions of 3-O-sulfate-tagged HSact precursors
In vitro 3-O-sulfated [3H]HSact precursors were depolymerized with a mixture of heparitinase I, heparitinase II, and heparinase. The resulting unsaturated di- and tetrasaccharides were originally separated on a Bio-Gel P2 column (Fig. 1) and were then further resolved by polyamine HPLC (Fig. 2). The number of each di- and tetrasaccharide per chain was determined in two steps. Initially, we determined the radioactivity per di- or tetrasaccharide. To this end, the sum of radioactivity in all di- and tetrasaccharide peaks was divided by 82 to determine the average radioactivity per disaccharide and 41 to determine the average radioactivity per tetrasaccharide. Subsequently, the total radioactivity in each of various di- or tetrasaccharide peak was divided by the average radioactivity per di- or tetrasaccharide to determine the number of a particular di- or tetrasaccharide in a specific peak. The numbers represent the average of two separate experiments. Total N-sulfates, 2-O-sulfate, 6-O-sulfates, and 3-O-sulfates per chain were added according to the number of these sulfates in each di- and tetrasaccharide species. It should be noted that these data are consistent with previously published data from our laboratory, which indicates that 3-O-sulfated disaccharides comprise 18% of all metabolic labeled O-sulfated disaccharides in F9 HSact (GlcUA-anManR335S, 6%, and GlcUA-anManR335S635S, 12%) (36). In addition, direct comparison of the number of 3-O-sulfate incorporations per HSact precursor chain (6 pmol 335S/pmol F9 HS) are also consistent with the values provided.

The sum of N-sulfates, 2-O-sulfate, 6-O-sulfates, and 3-O-sulfates for each sugar residues is provided in Table I and equals 100% of the total sulfate groups per 3-O-sulfate-tagged chain, which corresponds to a total of 71 sulfate groups. Of these, 37 sulfate groups or 52% are O-sulfates. Furthermore, since 3-O-sulfates comprise 14% of all metabolically labeled O-sulfates (36). We can calculate that each endogenous HSact chain contains 5.2 3-O-sulfation sites (37 × 14%).

By treating cold F9 HS with PAP35S and 3-O-ST-1, we found that 6 pmol of [35S]sulfates from PAP35S are transferred to 3-O-sulfate positions in 1 pmol of F9 HSact precursors (data not shown). Thus, from the evidence presented above, we concluded that there are six 3-O-sulfate acceptor sites per HSact precursor chain, which can be captured as 3-O-sulfated tetrasaccharides as indicated above.

We then estimated the number of 3-O-sulfate acceptor sites per HSinact precursor chain. We previously reported that metabolically labeled GlcUA-anManR335S (5%) and GlcUA-anManR335S635S (5%) accounted for 10% of O-sulfated disaccharides in retinoic acid and dibutyryl cAMP plus theophilline F9 HSinact. However, endogenous retinoic acid and dibutyryl cAMP plus theophilline F9 HSinact are composed of 3-O-sulfated HSinact as well as HSact and HSinact precursors that have never been 3-O-sulfated (36). To obtain a more accurate estimate, cold F9 HS was exhaustively converted with PAP35S and 3-O-ST-1. After AT affinity fractionation to remove all 3-O-sulfate-tagged HSact precursors, we found that 6 pmol of [35S]sulfates from PAP35S are transferred to 3-O-sulfate positions in 1 pmol of F9 HSinact precursors (data not shown). Therefore, we conclude that there are six 3-O-sulfate acceptor sites in HSinact precursors as well.

3-O-Sulfate-tagged Tetrasaccharide Structures-- To characterize the two tetrasaccharide structures in Fig. 2B, we repeated the experiment to obtain the tetrasaccharides using different F9 HS preparations. To this end, either cold F9 HS was tagged with radiolabeled 3-O-sulfates or 35SO4 metabolically labeled HS was tagged with cold 3-O-sulfates in vitro by PAPS/PAP35S and purified 3-O-ST-1. Affinity purified 3-O-sulfate-tagged HSact precursors were subjected to heparitinase I, heparitinase II, and heparinase digestion. The radiolabeled tetrasaccharides were collected from Bio-Gel P2 chromatography. The identical two tetrasaccharide peaks with the same 2 to 1 ratio of tetrasaccharide I to tetrasaccharide II were found after polyamine HPLC chromatography among the three 3-O-sulfate-tagged tetrasaccharide sources (data not shown). This result suggests that we can characterize the tetrasaccharides from any 3-O-sulfate-tagged F9 HSact precursors.

The 3-O-35S-tagged tetrasaccharide I and tetrasaccharide II from cold F9 HSact were separated by polyamine HPLC chromatography and collected for the tetrasaccharide sequencing analysis. A fraction of each tetrasaccharide collected was re-run on polyamine HPLC (Fig. 3, A and B). Tetrasaccharide I and tetrasaccharide II were first treated with low pH nitrous acid, which cleaves the GlcNS±6S-GlcUA/IdceA±2S linkage but not the GlcNAc±6S-GlcUA/IdceA±2S linkage in the tetrasaccharides. However, after low pH nitrous acid treatment and NaBH4 reduction, both tetrasaccharide peaks remained as tetrasaccharides as judged by the Bio-Gel P2 profile (data not shown), but the elution time on polyamine HPLC was altered (Fig. 3, C and D). The alteration in this parameter corresponds to the lost of one N-sulfate and ring contraction from the reducing end GlcN residue of both tetrasaccharides.


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Fig. 3.   Polyamine HPLC analysis of sequential treated 3-O-35S-tagged tetrasaccharides. 3-O-35S-Tagged HSact precursor tetrasaccharides collected from polyamine HPLC were sequentially treated with low pH nitrous acid, Delta 4,5glycuronidase, 6-O-sulfatase, and N-acetylglucosaminidase and were resolved by polyamine HPLC chromatography (see "Experimental Procedures"). A, tetrasaccharide I; B, tetrasaccharide II; C and D, after low pH nitrous acid and NaBH4 treatment; E and F, followed by Delta 4,5glycuronidase; G and H, 6-O-sulfatase; I and J, N-acetylglucosaminidase treatments. The arrow indicates the elution position of the internal GlcUA-[3H]anManR3S6S standard. GlcA indicates GlcUA.

The tetrasaccharides collected were then treated with Delta 4,5glycuronate 2-O-sulfatase. The retention time of tetrasaccharide I and tetrasaccharide II remained the same, whereas the 2-O-sulfate in the internal Delta 4,5UA2S-GlcNS UV standard was completely removed as judged by the shift of UV absorbance on polyamine HPLC. This result indicates that there are no 2-O-sulfates on Delta 4,5UA in either tetrasaccharide (data not shown). Indeed, Delta 4,5glycuronidase removed a sugar residue from the non-reducing end in both tetrasaccharides. The polyamine HPLC profiles of Delta 4,5glycuronidase-treated samples are shown in Fig. 3, E and F.

Further GlcNAc 6-O-sulfatase treatment results in the loss of one sulfate as judged by polyamine HPLC (Fig. 3, G and H). After alpha -N-acetylglucosaminidase treatment, the resulting disaccharides coeluted with GlcUA-[3H]anManR3S and GlcUA-[3H]anManR3S6S standards, respectively (Fig. 3, I and J). From the above results, we concluded that the first tetrasaccharide peak has the structure Delta 4,5UA-GlcNAc6S-GlcUA-GlcNS3S (Fig. 3A) and the second peak has the structure Delta 4,5UA-GlcNAc6S-GlcUA-GlcNS3S6S (Fig. 3B).

We carried out a similar experiment to determine the 3-O-35S-tagged HSinact tetrasaccharide structures by using 3-O-35S-tagged F9 HSinact precursors. The combined treatment with heparitinase I, heparitinase II, and heparinase treatment generates both Delta 4,5di- (35%) and Delta 4,5tetrasaccharides (65%) on Bio-Gel P2 chromatography (data not shown). 40% of total 3-O-35S-sulfate-tagged F9 HSinact precursors remained as tetrasaccharides after low pH nitrous acid and NaBH4 treatment of the combined heparitinase I, heparitinase II, and heparinase-digested materials. The tetrasaccharides were resolved by polyamine HPLC. Three tetrasaccharide peaks were observed (Fig. 4).


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Fig. 4.   Polyamine HPLC profile of 3-O-35S-tagged HSinact precursor tetrasaccharides. 3-O-35S-Tagged HSinact precursors were digested with a mixture of heparitinase I, heparitinase II, and heparinase. Tetrasaccharides obtained after Bio-Gel P2 chromatography were treated with low pH nitrous acid and NaBH4 and subjected to Bio-Gel P2 chromatography again. Tetrasaccharides recovered after low pH nitrous acid and NaBH4 treatment were resolved by polyamine HPLC. Peak I, peak II, and peak III were collected for further structural analysis.

Peak II (56%) and peak III (29%) have the same retention time as the tetrasaccharides in Fig. 3, C and D. The identical polyamine HPLC profiles as in Fig. 3, E---J, were observed upon sequential Delta 4,5glycuronidase, 6-O-sulfatase, and alpha -N-acetylglucosaminidase treatments of the collected peak II and peak III (data not shown). Therefore, peak II has the structure Delta 4,5UA-GlcNAc6S-GlcUA-anManR3S and the peak III has the structure Delta 4,5UA-GlcNAc6S-GlcUA-anManR3S6S. This result indicates that 34% (85 × 40%) of the 3-O-sulfate acceptor sites in HSinact precursors are the same at the Delta 4,5tetrasaccharide level as in HSact precursors. The 3-O-sulfate-tagged HSact and HSinact precursor Delta 4,5tetrasaccharides do not bind to AT (data not shown). In view of the fact that GlcNAc6S-GlcUA-GlcNS3S6S-IdceA2S has 150-fold less affinity for AT (Kd = 4.6 × 10-6 M) compared with that of the pentasaccharide, GlcNAc6S-GlcUA-GlcNS3S6S-IdceA2S-GlcNS6S (Kd = 3.0 × 10-8 M) (40), 3-O-sulfate-tagged HSinact precursors with the critical 3-O and 6-O failed to bind to AT because they do not have proper sugar sequences toward the reducing end of the 3-O-sulfate-tagged GlcN residues.

The elution position of peak I on polyamine HPLC suggests that this tetrasaccharide may have only one sulfate group. After Delta 4,5glycuronidase treatment, the resulting products eluted at the same position as GlcNAc-GlcUA- anManR3S in Fig. 3G. After alpha -N-acetylglucosaminidase treatment, it coeluted with GlcUA-[3H]anManR3S standard. From the above evidence, we concluded that peak I has the structure of Delta 4,5UA-GlcNAc-GlcUA-anManR3S. The critical 6-O-sulfate group at residue 2 is missing in this tetrasaccharide. This observation explains the fact that a population of 3-O-sulfate-tagged HSinact precursors within the GlcNAc-GlcUA-GlcNS3S context failed to bind to AT. However, we cannot quantitate how many 6-O-sulfates are missing in the 3-O-sulfate-tagged HSinact precursors within the GlcNS-GlcUA-GlcNS335S context because heparitinase treatment does not preserve all the 3-O-sulfate acceptor sites as tetrasaccharides as compared with the 3-O-sulfate-tagged HSact precursors. Our inability to capture all of the 3-O-sulfate-tagged tetrasaccharides from the HSinact precursors is probably due to the heterogeneity in the structure of these precursors. This heterogeneity results from the fact that we have not, as yet, identified the ligands for 3-O-sulfated HSinact materials and hence cannot purify the 3-O-sulfated HSinact precursors.

Five of Six 3-O-Sulfate-tagged Oligosaccharides in HSact Precursors Bind to AT-- Both HSact and HSinact precursors contain about six 3-O-sulfation sites per chain. 3-O-Sulfate-tagged HSact precursors binds to AT, and 3-O-sulfate-tagged HSinact precursors do not. The next question we asked is how many of the 3-O-sulfate acceptor sites (potential AT-binding sites) in 3-O-sulfate-tagged HSact precursors actually bind to AT. We reasoned that heparitinase I digestion should leave AT-binding sites intact since all the 3-O-sulfate-tagged HSact precursors remained as 3-O-sulfate-tagged tetrasaccharide after heparitinase I, heparitinase II, and heparinase digestion (Fig. 1). If HS has the same AT-binding sequence as in heparin, i.e. GlcNAc6S-GlcUA-GlcNS3S±6S-IdceA2S-GlcNS6S, the reducing end IdceA2S-GlcNS6S cannot be cleaved by heparitinase I ("Experimental Procedures" (38)). To this end, 3-O-ST-1 and PAP35S-labeled, AT affinity purified HSact and HSinact were digested with 2 milliunits of heparitinase I overnight. A fraction of the digested materials from the 3-O-sulfate-tagged HSact and HSinact precursors were used in the AT affinity assay. We found that, for the digested HSact, the extent of AT binding was approximately equal to the undigested HSact control. To check the heparitinase I digestion reaction, the rest of the digested 3-O-sulfate-tagged HSact and HSinact materials were analyzed by Bio-Gel P-6 chromatography. The digestion patterns were reproducibly obtained, and sample profiles are provided (Fig. 5). For 3-O-sulfate-tagged HSact precursors, the data show that 85% remained larger than hexasaccharides, 14% were tetrasaccharides, and 0.5% were disaccharides (Fig. 5A). For 3-O-sulfate-tagged HSinact precursors, only 14% remained larger than hexasaccharides, 52% were tetrasaccharides, 33% were disaccharides (Fig. 5B).


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Fig. 5.   Bio-Gel P6 fractionation of heparitinase I-digested 3-O-35S-tagged HSact and HSinact precursors. Cold F9 HS was treated with 3-O-ST-1 and PAP35S. The 3-O-35S-tagged HS were AT affinity fractionated into HSact and HSinact. 3-O-35S-Tagged HSact and HSinact precursors were digested with 2 milliunits of heparitinase I overnight. The products were analyzed by Bio-Gel P6 chromatography ("Experimental Procedures"). A, 3-O-35S-tagged HSact precursors; B, 3-O-35S-tagged HSinact precursors. n = the number of monosaccharide units in each peak.

Each fraction of oligosaccharides equal to or larger than hexasaccharides in 3-O-sulfate-tagged HSact and HSinact precursors were collected and lyophilized for the AT affinity assay. All the oligosaccharides from 3-O-sulfate-tagged HSact precursors bound to AT, but the oligosaccharides from 3-O-sulfate-tagged HSinact precursors did not. Since 85% of the 3-O-sulfate-tagged HSact oligosaccharides bind to AT and there are six 3-O-sulfation sites per chain, 6 × 85% = 5.1. It implies that at least five out of six 3-O-sulfate acceptor sites possess the correct positioning of all critical groups except for the 3-O-sulfate for AT binding in all HSact precursor chains. This experiment also suggests that HSact and HSinact precursors have distinct sulfation patterns around the potential 3-O-sulfate acceptor sites.

The majority of 3-O-sulfate-tagged HSinact precursors should have GlcUA at their reducing end, i.e. -GlcUA-GlcNS3S±6S-down-arrow GlcUA, since GlcUA residues at this position are apparently cleaved by heparitinase I in order to generate 85% of 3-O-sulfated di- (33%) and tetrasaccharides (52%). In contrast, GlcUA-GlcNS3S±6S-IdceA±2S should be the main 3-O-sulfate-tagged HSact sequence since it is uncleavable by heparitinase I.

It is interesting to note that 33% of 3-O-sulfate-tagged HSinact precursors can be degraded into disaccharides and 0.5% of 3-O-sulfate-tagged HSact precursors can be degraded into disaccharides. This result implies that 3-O-sulfation is not the only factor that determines whether 3-O-sulfated sequences are preserved as tetrasaccharides following the digestion with heparitinase.

Different Sulfation Distribution around 3-O-Sulfate Acceptor Sites in HSact and HSinact Precursors-- To compare further the sulfation patterns in HSact and HSinact precursors, [6-3H]GlcN metabolically labeled HS was treated with 3-O-ST-1 and PAP35S. The 3H and 35S double-labeled HS was AT affinity fractionated into 3-O-sulfate-tagged HSact and HSinact precursors. The low pH nitrous-treated and NaBH4 reduced products were analyzed by Bio-Gel P-6 chromatography (Fig. 6). These conditions depolymerize HS between GlcNS and GlcUA/IdceA residues, liberating oligosaccharides whose length reflects the spacing between N-sulfated GlcN units. The 3H radioactivity profiles are similar; therefore, the distribution of GlcNS residues are similar in HSact and HSinact precursors outside 3-O-sulfate acceptor sites. However, 3-O-sulfate acceptor sites in HSact and HSinact precursors are different because their 35S radioactivity profiles are distinct. The 3-O-sulfate acceptor sites in HSact precursors should be located in GlcUA/IdceA-GlcNAc6S-GlcUA-GlcNS335S±6S sequences since 88% of 3-O-sulfated materials remained as tetrasaccharides (Fig. 6A). In contrast, 3-O-sulfate acceptor sites in HSinact precursors should be located mainly in GlcUA/IdceA-GlcNS±6S-GlcUA-GlcNS335S±6S sequences since 60% of 3-O-sulfated materials was cleaved into disaccharides (Fig. 6B).


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Fig. 6.   Bio-Gel P6 fractionation of low pH nitrous-treated and NaBH4-reduced 3-O-35S-tagged [3H]HSact and [3H]HSinact precursors. [6-3H]GlcN metabolically labeled HS was treated with 3-O-ST-1 and PAP35S. The 3H- and 35S-doubled-labeled HS were AT affinity fractionated into HSact and HSinact. The low pH nitrous-treated and NaBH4-reduced 3-O-35S-tagged [3H]HSact and [3H]HSinact precursors were analyzed by Bio-Gel P6 chromatography (see "Experimental Procedures"). A, 3-O-35S-tagged [3H]HSact precursors; B, 3-O-35S-tagged [3H]HSinact precursors. , 3H cpm; open circle , 35S cpm. n = the number of monosaccharide units in each peak.

3-O-Sulfate-tagged HSact Hexasaccharide Structures-- The tetrasaccharide analysis, heparitinase I, and low pH nitrous acid treatment data imply that the dominant 3-O-sulfate acceptor sites in HSact precursor are GlcUA/IdceA-GlcNAc6S-GlcUA-GlcNS335S±6S-IdceA±2S, whereas the dominant 3-O-sulfate acceptor sites in HSinact precursor are GlcUA/IdceA-GlcNS/Ac±6S-GlcUA-GlcNS335S±6S-GlcUA. These results further clarify why 3-O-sulfate-tagged HSact precursors bind AT and 3-O-sulfate-tagged HSinact precursors do not. To obtain the minimum AT-binding sequence in 3-O-sulfate-tagged HSact precursors, the 3H and 35S double-labeled HSact and HSinact precursors were subjected to limited digestion with heparitinase I and heparitinase II. According to recently published work on the exolytic and processive mechanism of depolymerization of HS by heparitinase II (41), this digestion condition should preserve some of the AT-binding hexasaccharide structures. The digested products were analyzed by Bio-Gel P-6 chromatography (Fig. 7). The major hexasaccharide peak from 3-O-sulfate-tagged HSact precursors was collected and lyophilized. AT affinity purified, 3H and 35S double-labeled hexasaccharides were loaded on a polyamine HPLC column (Fig. 8). Two peaks (hexasaccharide I and hexasaccharide II) were found and collected for further analysis.


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Fig. 7.   Bio-Gel P6 fractionation of limit digested 3-O-35S-tagged [3H]HSact and [3H]HSinact precursors by heparitinase I and heparitinase II. 3-O-35S-Tagged [3H]HSact and [3H]HSinact precursors were digested with 1 milliunit each of heparitinase I and heparitinase II for 30 min. The products were analyzed by Bio-Gel P6 chromatography (see "Experimental Procedures"). The fractions indicated by the solid bar were pooled for further analysis. A, 3-O-35S-tagged [3H]HSact precursors; B, 3-O-35S-tagged [3H]HSinact precursors. , 3H cpm; open circle , 35S cpm. n = the number of monosaccharide units in each peak.


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Fig. 8.   Polyamine HPLC profile of AT-binding 3-O-sulfate-tagged hexasaccharides. Hexasaccharides produced by heparitinase I and heparitinase II limited depolymerization of 3-O-35S-tagged [3H]HSact precursors as in Fig. 7 were AT affinity purified and separated by polyamine HPLC. The hexasaccharide I and the hexasaccharide II resolved by polyamine HPLC were collected for further analysis.

Hexasaccharide I and hexasaccharide II were digested with heparitinase IV. All the hexasaccharides were degraded into di- and tetrasaccharides, which were separated and collected after Bio-Gel P2 chromatography (data not shown). The 3H-labeled disaccharides from hexasaccharide I and hexasaccharide II both co-eluted with Delta UA2S-GlcNS6S UV standard on polyamine HPLC (Fig. 9, A and B). The 3H and 35S double-labeled tetrasaccharide from hexasaccharide I has the structure Delta UA-[3H]GlcNAc6S-GlcUA-[3H]GlcNS335S. The tetrasaccharide from hexasaccharide II has the structure Delta UA-[3H]GlcNAc6S-GlcUA-[3H]GlcNS3S6S because they have the same retention time on polyamine HPLC as tetrasaccharide I in Fig. 3A and tetrasaccharide II in Fig. 3B. This experiment suggests that the AT-binding hexasaccharide I has the structure [6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS335S-UA2S-[6-3H]GlcNS6S and the hexasaccharide II has the structure [6-3H]GlcNAc6S -GlcUA-[6-3H]GlcNS335S6S-UA2S- [6-3H]GlcNS6S.


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Fig. 9.   Polyamine HPLC profiles of the disaccharides generated from the reducing ends of AT-binding 3-O-sulfate-tagged hexasaccharides. Hexasaccharide I and hexasaccharide II (Fig. 8) were digested with 2 milliunits of heparitinase IV overnight. The di- and tetrasaccharides were separated by Bio-Gel P2 chromatography. The disaccharides collected were resolved by polyamine HPLC. The arrow indicates the elution position of the internal Delta 4,5UA2S-GlcNS6S UV standard.

To determine the identity of the UA2S in both hexasaccharides, hexasaccharide I and hexasaccharide II were treated with low pH nitrous acid and NaBH4 reduced. The products were fractionated into disaccharide and tetrasaccharide by Bio-Gel P2 chromatography and collected for further analysis. The 3H-labeled disaccharides from both hexasaccharide I and hexasaccharide II coeluted with IdceA235S-anManR635S standard on ion pairing reverse phase HPLC (data not shown). Tetrasaccharide from hexasaccharide I eluted at the same position as Delta UA-GlcNAc6S-GlcUA-anManR3S on polyamine HPLC (Fig. 3C), and tetrasaccharide from hexasaccharide II eluted at the same position as Delta UA-GlcNAc6S-GlcUA-anManR3S6S on polyamine HPLC (Fig. 3D). Combining both hexasaccharide enzymatic and low pH nitrous acid analysis data, we concluded that hexasaccharide I has the structure Delta UA-GlcNAc6S-GlcUA-GlcNS3S-IdceA2S-GlcNS6S and hexasaccharide II has the structure Delta UA-GlcNAc6S-GlcUA-GlcNS3S6S-IdceA2S-GlcNS6S.

Based on the data presented in this paper, the differences between 3-O-sulfate acceptor sites in HSact and HSinact precursors are summarized in Fig. 10. It is important to note that the heterogeneity in the HSinact precursors results in a generation of a large number of potential 3-O-sulfate acceptor sites. Those structures for which we have evidence are provided in the figure. In this regard, the second sugar residue in HSinact precursors is either a GlcNS (60%) or GlcNAc (40%), and the fifth sugar residue in HSinact precursors is either a GlcUA (85%) or IdceA (15%). Furthermore, the 6-O-sulfate groups on residue 2 and 6 of the 3-O-sulfate acceptor site on the HSinact precursors can be present or absent. These alterations distinguish the potential 3-O-sulfate acceptor sites of the HSinact precursors from that of HSact precursors whose sequence is uniquely defined. The many possible combinations of alterations in the HSinact precursor acceptor sites reflect the underlying heterogeneity of the HSinact precursors. For this reason, we are unable to preserve the major 3-O-sulfate acceptor hexasaccharide sequence in HSinact precursors and cannot quantitatively provide the actual sequence of the 3-O-sulfate acceptor sites in HSinact precursors. However, it should be emphasized that the critical 6-O-sulfate is always present in HSact precursors, whereas it can be missing in HSinact precursors.


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Fig. 10.   3-O-Sulfate-tagged hexasaccharide structures in F9 HSact and HSinact precursors. The structures are based on the following data: 1) Six 3-O-sulfate acceptor sites per HSact and HSinact precursors; 2) Low pH nitrous acid, heparitinase I, and heparitinase II degradations of 3-O-sulfate-tagged HSact and HSinact precursors; 3) tetrasaccharide structures in 3-O-sulfate-tagged HSact and HSinact precursors; 4) hexasaccharide structures in 3-O-sulfate-tagged [3H]HSact precursors. The boxes in 3-O-sulfate-tagged HSinact precursors correspond to the sulfate residues existing in 3-O-sulfate-tagged HSact precursors. The 3-O-sulfate-tagged HSact and HSinact precursor sequences are different at all levels. IdA indicates IdceA; GlcA indicates GlcUA.


    DISCUSSION

This article presents an analysis of AT-binding HSact precursor sequences in F9 embryonal carcinoma cells. At least five out of six 3-O-sulfate acceptor oligosaccharides possess the correct positioning of all critical AT-binding groups except 3-O-sulfate in all HSact precursor chains. It is interesting to note that four AT-binding oligosaccharides in each LTA cell HSact chain are present as shown by an AT-protected heparin lyase assay in our laboratory.3 To possess multiple AT-binding sites in each HSact chain suggests that HSact biosynthesis is not a random process but a highly repetitive, highly organized operation. Based on the data presented in this paper, the schematic model for the structure of HSact precursors in F9 cells is advanced in Fig. 11. In this model, there are six potential AT-binding hexasaccharides that exhibited all groups necessary for AT binding except for the 3-O-sulfate moieties. Six potential AT-binding hexasaccharides are equal to 22% of the chain length of HSact precursors (18 of 82 disaccharides per chain) and contain 26 of 37 O-sulfates in 3-O-sulfate-tagged HSact precursors. Beyond the six potential AT-binding hexasaccharides, there are 5 × Delta UA-GlcNAc6S, 3 × Delta UA2S-GlcNS, 3 × Delta UA-GlcNS6S, 16 × Delta UA-GlcNS, and 37 × Delta UA-GlcNAc disaccharides to complete the structure of the HSact precursor chain (Table I). 5 × Delta UA-GlcNAc6S, 3 × Delta UA2S-GlcNS, and 3 × Delta UA-GlcNS6S are the 11 O-sulfated residues outside the AT-binding domain that need to be placed to complete the primary structure of HSact precursor (Fig. 11).


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Fig. 11.   Schematic model of 3-O-sulfate-tagged HSact precursor structures derived from the analysis HS from F9 cells. The model is based on the data presented in this paper, i.e. there are six 3-O-sulfation sites per chain and all 3-O-sulfate acceptor oligosaccharides bind to AT in 3-O-sulfate-tagged HSact precursors. It implies that HSact precursor formation involves correct N-sulfation, epimerization, 6-O-, and 2-O-sulfation at all six potential 3-O-acceptor sites per chain during the biosynthesis. IdA indicates IdceA; GlcA indicates GlcUA.

The reason we proposed that there are six instead of five potential AT-binding hexasaccharides in F9 HSact precursors is that we could place five out of six Delta UA2S-GlcNS6S residues in potential AT-binding domains in F9 HSact precursors. We suspect that heparitinase I may contain trace heparitinase II contaminants that generate 15% of the tetrasaccharides in 3-O-sulfate-tagged HSact precursors (Fig. 5). More likely, endo-D-glucuronidases may cleave mature HS chains limitedly in cultured F9 cells before HS chains were prepared for our structural studies (see "Experimental Procedures"). The general presence of endo-D-glucuronidases in variety of tissue and cells has been reported (41-48). Endo-D-glucuronidases recognize the sequences GlcUA/IdceA-GlcNAc/NS±6S-GlcUA-down-arrow GlcNAc/NS±3S±6S-IdceA2S-GlcNS±6S. The arrow indicates the cleavage site (49, 50). The cleavage eliminates both AT- and potential AT-binding sites (50). The endo-D-glucuronidases extensively cleave the newly synthesized heparin (Mr 60,000-100,000) to generate fragments that are stored in cytoplasmic granules of mast cells (Mr 5,000-25,000). This may explain why commercial heparin (Mr 5,000-25,000) contains limited numbers of AT-binding sites per chain (38). In contrast, endo-D-glucuronidases may cleave in a limited fashion the endogenous HS chains (48, 49). This limited cleavage may explain why we can place five out of six Delta UA2S-GlcNS6S residues in potential AT-binding domains in F9 HSact precursors.

It is apparent from our model that there is a template for HSact precursor formation that requires correct N-sulfation, epimerization, 2-O-, and 6-O-sulfation at all six sites along the chain during the biosynthesis. What remains to be explained is how this is accomplished. F9 HSact chain contains almost all the detectable 3-O-sulfated disaccharides (GlcUA-anManR3S and GlcUA-anManR3S6S) in HStotal. The 3-O-sulfated disaccharides accounted for about 18% of O-sulfated disaccharides in the HSact chain (36). Our current study showed that there are six 3-O-sulfate acceptor sites per HSact chain. Therefore, 3-O-sulfates are added in an all or none manner to the six 3-O-sulfate acceptor sites in HSact precursor in a concerted fashion to generate the AT-binding sites during biosynthesis. Based on this observation, our current model is that after chain polymerization, all other chain modification reactions, i.e. N-sulfation, epimerization, 2-O-, 6-O-sulfation, occur at all six regions in the same fashion as 3-O-sulfation of HSact precursors. To explain this concerted mechanism, we propose that N-STs recognize and sulfate a three-dimensional feature of the nascent polymerized HS chain (51) and therefore produce a definite spacing of N-sulfations. Epimerase may lay out different epimerization patterns due to different N-sulfation patterns, or 2-O-/6-O-sulfation occurring before epimerization. These patterns may be further amplified depending on whether the 6-O-sulfation or 2-O-sulfation occurs first (the initial action of 6-O-ST excludes subsequent function of 2-O-ST, whereas the initial action of the 2-O-ST allows subsequent function of 6-O-ST (52)). The structure of each subpopulation HS is determined by the sequential action of modification enzymes, i.e. pathway, in the Golgi apparatus. In other words, different pathways during HS modification generate different HS structures in the same cell.

This study shows that F9 cells produce different HS structures. The previous HSact-deficient mutants and 3-O-ST-1 studies in our laboratory suggested that the same cells make both HSact and HSinact precursors (35, 53), and the same HS proteoglycan core proteins carry both HSact and HSinact chains (54). A Chinese hamster ovary cell mutant defective in N-ST makes both fully sulfated and undersulfated HS chains (55). A human colon carcinoma cell makes both fully sulfated and undersulfated HS chains (56). All these observations suggest that different biosynthesis schemes that generate different HS structures occur in the same cells. In addition to the increasing numbers of specific HS domain structural studies in different tissues and cells (57-61), we suggest that different HS biosynthetic pathways exist, which generate HS with different structures and biologic functions. Evaluation of the different pathways will eventually delineate how the HS biosynthesis is regulated.

Currently we do not know where HSact and HSinact precursor biosynthesis pathways diverge. The constant presence of the critical 6-O-sulfate groups only in HSact precursors indicates that the pathway is either set up by or diverges before the critical 6-O-sulfation. It is possible that specific isoforms of 6-O-ST are involved in HSact precursor pathways, and the existing structures around the 3-O-sulfate acceptor site favor the action of a 6-O-ST isoform. Furthermore, the existence of auxiliary proteins that modulate the action of N-ST, epimerase, 2-O-ST, and 6-O-ST for HSact precursor formation have not been excluded.

In conclusion, the decision to synthesize HSact or HSinact depends on the presence of specific HS precursor intermediates, specific modification enzymes, and perhaps auxiliary factors in the Golgi biosynthesis machinery. Currently, we have obtained a series of Chinese hamster ovary mutants defective in HSact precursor formation. Complementation of these mutations will provide us with molecular details about the required elements for HSact precursor biosynthesis. This information should allow us to formulate a more definitive model of the HSact biosynthetic pathway. This model will be evaluated by reconstituting the sequential biosynthetic apparatus using different recombinant modification enzymes/proteins.

    ACKNOWLEDGEMENTS

We thank Peter Blaiklock for careful reading of this manuscript and members of the Rosenberg laboratory for their insightful comments.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants 5-P01-HL41484 and HL66385.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.

§ Recipient of National Research Service Award Postdoctoral Fellowship.

** To whom correspondence and reprint requests should be addressed: Massachusetts Institute of Technology, Bldg. 68-480, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-8804; Fax: 617-258-6553; E-mail: rdrrosen{at}MIT.EDU.

2 Liu, J., Shworak, N. W., Sinay, P., Schwartz, J. J., Zhang, L., Fritze, L. M. S., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, in press.

3 J. Liu and R. D. Rosenberg, unpublished results.

    ABBREVIATIONS

The abbreviations used are: HS, heparan sulfate; HSact, anticoagulantly active heparan sulfate; HSinact, anticoagulantly inactive heparan sulfate; CHAPS, 3-[(3-cholamidopropy)dimethylammonio]-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; GAG, glycosaminoglycan; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; AT, antithrombin; 3-O-ST-1, glucosaminyl 3-O-sulfotransferase-1; N-ST, N-deacetylase/N-sulfotransferase; 2-O-ST, iduronic/glucuronic acid 2-O-sulfotransferase; 6-O-ST, glucosaminyl 6-O-sulfotransferase; GlcUA, glucuronic acid; IdceA, iduronic acid; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
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Abstract
Introduction
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