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. Author manuscript; available in PMC: 2022 Oct 15.
Published in final edited form as: ACS Chem Biol. 2021 Aug 5;16(10):2026–2035. doi: 10.1021/acschembio.1c00474

Synthesis of 3-O-Sulfated Heparan Sulfate Oligosaccharides Using 3-O-Sulfotransferase Isoform 4

Jine Li 1, Guowei Su 2, Yongmei Xu 3, Katelyn Arnold 4, Vijayakanth Pagadala 5, Chunyu Wang 6, Jian Liu 7
PMCID: PMC8526403  NIHMSID: NIHMS1740025  PMID: 34351732

Abstract

Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.

Graphical Abstract

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Heparan sulfate (HS), a member of the glycosaminoglycan family, is present ubiquitously on the cell surface and in the cellular matrix. HS influences multiple biological, physiological, and pathogenic processes.1 Heparin is a highly sulfated form of HS and has been widely used as an anticoagulant drug. HS is composed of repeating disaccharide units of N-acetylglucosamine (GlcNAc) α(1 → 4) glucuronic acid β(1→ or N-sulfoglucosamine (GlcNS) α(1 → 4)iduronic acid (IdoA) α(1 →. Each saccharide residue could be modified by a different sulfation. The common sulfation modifications are 2-O-sulfation of the IdoA residue (or to a lesser extent on the GlcA residue) and N-sulfation and 6-O-sulfation of glucosamine residues. The sulfation patterns and the conformations of IdoA and GlcA residues play critical roles in dictating the biological functions of HS.2

Glucosamine 3-O-sulfation is a rare modification in HS isolated from biological sources, but it is closely related to the biological functions. For example, 3-O-sulfation is key for the anticoagulant activity of heparin. 3-O-Sulfated HS serves as a cell entry receptor for herpes simplex virus to establish infection.3,4 3-O-Sulfated HS has also been shown to contribute to cancer pathogenesis, neural development, and regulation.5–8 The distinct biological functions observed for 3-O-sulfated HS are due to the unique saccharide sequences around the 3-O-sulfation site.9

The biosynthesis of 3-O-sulfated HS is accomplished by a family of 3-O-sulfotransferases (3-OSTs), which includes seven isoforms in the human genome.9,10 These isoforms yield different 3-O-sulfation saccharide domains. 3-O-Sulfotransferase isoform 1 (3-OST-1) is widely expressed in endothelial cells of many organs, and it primarily generates HS with the antithrombin-binding domain of -GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-, which is necessary for anticoagulant activity. The 3-O-sulfotransferase isoform 3 (3-OST-3), including 3-OST-3A and -3B, is expressed in many organs to produce HS with the -IdoA2S-GlcNS3S6S- domain. 3-OST-3A and 3-OST-3B have identical amino acid sequences in the sulfotransferase catalytic domain, but their sequences differ in the N-terminal transmembrane domains.11 The functions of 3-OST-3 modified HS are reported to be related to virus infections, antioncogenesis, and tumor-promoting effects.4,12,13 Studies with HS modified by 3-OST-3 have been shown to prevent herpes simplex virus type-1 (HSV-1) entry by its ability to bind to the envelope glycoprotein D (gD) with high affinity.14 Recently, an octasaccharide modified by 3-OST-3 containing the IdoA2S-GlcNS3S6S-IdoA2S- motif has been shown to bind to antithrombin and display anticoagulant activity.15 The 3-O-sulfotransferase isoform 5 (3-OST-5) is expressed in placenta, brain, and spinal cord.16 3-OST-5 can generate anticoagulant HS with the -GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S- domain as well as the gD-binding HS with the -IdoA2S-GlcNS3S6S- domain. The 3-O-sulfotransferase isoform 6 (3-OST-6) is expressed in liver and kidney, and the 3-OST-6 modified HS can mediate the cellular entry of herpes simplex virus 1 (HSV-1).17

HS modified by 3-OST-2 or 3-OST-4 plays an important function in the central nervous system.18 In a zebrafish model, the HS modified by 3-OST-2 and 3-OST-4 facilitates neurotropic HSV-1 entry and spreads via binding to HSV-1 envelope glycoprotein D (gD).19,20 Both 3-OST-2 and 3-OST-4 have increased expression in the brains with Alzheimer’s disease (AD).21 In addition, 3-OST-2 and 3-OST-4 are involved in synaptogenesis in neuronal development7 through 3-O-sulfated HSs.7,8 The current method to characterize the structure of HS from biological sources is merely to conduct disaccharide compositional analysis.22 However, the disaccharide analysis method is unsuited to determine the level of 3-O-sulfated HS modified by 3-OST-2 and 3-OST-4 isolated from brain tissues because 3-O-sulfated saccharide domains are resistant to the heparin lyases degradation.23 Consequently, little is known about the saccharide structure of the neural HS modified by 3-OST-2 and 3-OST-4. An alternative approach to investigate the structure of HS modified by 3-OST-2 and 3-OST-4 is to prepare 3-O-sulfated oligosaccharides using corresponding enzymes. The resulting 3-O-sulfated oligosaccharides, which may resemble the functional domain of the neural HS, are used as molecular probes to investigate the functions of these HS in different biochemical and animal models.

In this work, 3-OST-4 was successfully expressed in insect cells using the baculovirus expression approach. Using purified 3-OST-4, we synthesized ten 3-O-sulfated oligosaccharides with the -GlcA2S-GlcNS3S ± 6S unit (Figure 1). One of these oligosaccharides, HSoligo 7 (HS oligosaccharide 7), displayed lower anticoagulant activity. HSoligo 7 showed increased binding to tau protein, a key protein in the pathology of Alzheimer’s disease, and high mobility group box 1 (HMGB1), a nuclear protein that has pro-inflammatory activity. Access to the 3-OST-4 enzyme has expanded the capability of the chemoenzymatic synthesis of 3-O-sulfated oligosaccharides. Our findings offer key reagents in the form of structurally unique HS oligosaccharides to probe the biological functions of HS, including the HS roles in regulating neural processes.

Figure 1.

Figure 1.

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Chemical structures of oligosaccharides prepared in this study. All these oligosaccharides contain 3-O-sulfated glucosamine residue. The 3-O-sulfation site is shown in red color for emphasis.

RESULTS AND DISCUSSION

Expression and Purification of 3-O-Sulfotransferase Isoform 4 from SF9 Insect Cells.

A library of 3-O-sulfated hexasaccharides was recently synthesized via a chemical approach, but it does not cover the -GlcA2S-GlcNS3S ± 6S-disaccharide domain.24 In a separate report, only three hexasaccharides containing the disaccharide domain of -GlcA2S-GlcNS3S ± 6S- were chemical synthesized.25 To overcome the limitation for the synthesis of 3-O-sulfated oligosaccharides, the recombinant 3-OST-4 was successfully expressed in SF9 insect cells using the baculovirus approach. The protein was purified to homogeneity using a heparin-Sepharose column followed by a Ni-Sepharose column (Supplementary Figure S1A). Additional biochemical characterizations were carried out to compare the activity under different pHs, divalent ions, and temperatures (Supplementary Figure S1B–S1D).

We next determined the reactivities of structurally defined HS dodecasaccharides (12-mers) and a structurally heterogeneous HS polysaccharide toward 3-OST-4 modification (Table 1). Compared to a HS polysaccharide substrate, our data suggest that the enzyme preferably sulfates the 12-mer that contains the -GlcNS-IdoA2S- disaccharide repeating unit (12-mer NS2S). Lower reactivity was detected for 12-merNS (with the -GlcNS-GlcA- disaccharide repeating domain). 12merNAc, 12merNS6S, and 12-mer NS2S6S were not substrates for 3-OST-4. Our data suggests that the enzyme does not react with oligosaccharides that carry 6-O-sulfo groups or are nonsulfated. This conclusion guided our subsequent efforts to synthesize 3-O-sulfated oligosaccharides using the 3-OST-4 enzyme. A total of 10 different oligosaccharides were synthesized on the scale of 9–227 mg (Figure 1 and Supplementary Table S1). The ten 3-O-sulfated oligosaccharides differed in the number of 3-O-sulfated glucosamine residues and positions of GlcA2S and IdoA2S residues. Different synthetic routes were employed to synthesize these 3-O-sulfated oligosaccharides as described below.

Table 1.

Reactivity of Different Dodecasaccharides (12-mers) and HS to 3-OST-4 Modification

substrates abbreviated saccharide sequence 35S-labeled 12-mer or HS (cpm)
control no substrate 140
12-mer NAc GlcNAc-(GlcA-GlcNAc)5-GlcA-pNP 222
12-mer NS GlcNS-(GlcA-GlcNS)5-GlcA-pNP 2,670
12-mer NS2S GlcNS-GlcA-(GlcNS-IdoA2S)4-GlcNSGlcA-pNP 22,664
12-mer NS6S GlcNS6S-(GlcA-GlcNS6S)5-GlcA-pNP 765
12-mer NS2S6S GlcNS6S-GlcA-(GlcNS6S-IdoA2S)4-GlcNS6S-GlcA-pNP 368
HS polysaccharide a mixture of polysaccharides with different sulfation patterns and size of the sugar chain; no defined saccharide sequence 45,050
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3-OST-4 Sulfates HS Oligosaccharides with -IdoA2S-GlcNS-IdoA2S-but Does Not Sulfate the -GlcA-GlcNS-IdoA2S- Domain.

Our attempt to synthesize HSoligo 1 from 8-mer substrate C was successful as depicted in Figures 2A and 2C. After the modification by 3-OST-4, about 71% of 8-mer substrate C was converted to HSoligo 1 (Figure 2C). The structure of HSoligo 1 was confirmed by electrospray ionization mass spectrometry (ESI-MS) (Supplementary Table S1) as well as NMR (Supplementary Figures S3–S6). The site of the 3-O-sulfation site was determined to be at the GlcNS residue that is flanked by two IdoA2S residues to form the -IdoA2S-GlcNS3S-IdoA2S- trisaccharide sequence. Indeed, the chemical shift of the C-3 proton of the GlcNS residue (residue d) was assigned to be 4.40 ppm (Supplementary Figure S3). The chemical shift of the C-3 proton of residues b, f, and h was assigned to be 3.67, 3.69, and 3.60 ppm, respectively (Supplementary Figure S3). The downfield shift in the proton signal from residue d compared to the C-3 proton from three other GlcNS residues is indicative of this GlcNS residue with a 3-O-sulfo group (Supplementary Figure S3). However, 3-OST-4 failed to sulfate 6-mer substrate A and 8-mer substrate B. There is a difference in the saccharide sequences between 8-mer substrate C and 6-mer substrate A and 8-mer substrate B (Figure 2A). Neither the 6-mer nor the 8-mer contains the trisaccharide domain of -IdoA2S-GlcNS-IdoA2S-. Our data suggest that the 3-OST-4 enzyme recognizes the trisaccharide domain of IdoA2S-GlcNS-IdoA2S to carry out the 3-O-sulfation reaction.

Figure 2.

Figure 2.

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Synthesis of HSoligo 1, 2, and 3 using the 3-OST-4 enzyme. Panel A shows the synthetic scheme for HSoligo 1. The synthesis was completed by incubating 8-mer substrate C with the 3-OST-4 enzyme. The trisaccharide domain of -IdoA2S-GlcNS-IdoA2S- in 8-mer substrate C is highlighted in blue. The site of 3-O-sulfation in HSoligo 1 is highlighted in green. Panel B shows the synthetic schemes for HSoligo 2 and 3. The sites of 3-O-sulfation in HSoligo 2 and 3 are highlighted in green. Panel C shows the HPLC profiles of 8-mer substrate C with or without 3-OST-4 modification. Panel D shows HPLC profiles of 6-mer substrate D with or without 3-OST-4 modification. The oligosaccharides are presented in shorthand symbols. The actual chemical structures for each shorthand symbols are shown under “Keys for the symbols”. Structurally defined oligosaccharides, including 6-mer substrate A, 8-mer substrate B, 8-mer substrate C, 6-mer substrate D, and 6-mer substrate E, were synthesized by the chemoenzymatic method, and they are commercially available from Glycan Therapeutics (www.glycantherapeutics.com).

Synthesis of HS Oligosaccharides with the -GlcA2S-GlcNS3S- Disaccharide Domain.

Next, the synthesis of oligosaccharides containing both 2-O-sulfated glucuronic acid (GlcA2S) and GlcNS3S residues was achieved. Previously, it was reported that 3-O-sulfotransferase isoform 2 (3-OST-2) generates the disaccharide sequence of -GlcA2S-GlcNS3S-using a HS polysaccharide substrate.26 With high quality recombinant 3-OST-4 in hand, we explored the synthesis of the -GlcA2S-GlcNS3S6S domain from 6-mer substrate D and 6-mer substrate E, GlcA2S-containing hexasaccharides. The 6-mer substrate D has a single GlcA2S residue, and 6-mer substrate E has two GlcA2S residues (Figure 2B). Both 6-mer substrates D and E were highly reactive to the 3-OST-4 modification to yield HSoligo 2 and HSoligo 3, respectively. For example, 6-mer substrate D was nearly quantitatively converted to HSoligo 2 after 3-OST-4 modification as determined by anion exchange HPLC analysis (Figure 2D). The full structural characterization of HSoligo 2 and 3 was completed by ESI-MS and NMR analysis (Supplementary Figures S7–S16). HSoligo 2 and HSoligo 3 were further modified by 6-O-sulfotransferase to install the 6-O-sulfation, yielding HSoligo 4 and HSoligo 5, respectively (Figure 3A). The structures of HSoligo 4 and HSoligo 5 were also confirmed by ESI-MS and NMR (Supplementary Figures S17–26). It should be noted that both GlcA2S and GlcNS3S ± 6S residues are rare in HS.27 Therefore, the disaccharide domain of -GlcA2S-GlcNS3S ± 6S- that combines both rare individual saccharide residues is very low in abundance. This domain has been only reported in the HS from basement glomerular membrane two decades ago.28 Lack of effective methods for identifying such a specific -GlcA2S-GlcNS-domain from biological samples may also contribute to the perception for the rarity of this 3-O-sulfated domain.

Figure 3.

Figure 3.

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Schemes for the synthesis of 3-O-sulfated HS oligosaccharides with diversified saccharide sequences. Panel A shows the synthetic schemes for HSoligo 4 and 5. The synthesis of HSoligo4 and 5 was completed in the one step reaction from HSoligo 2 and 3, respectively. Panel B shows the synthetic scheme for the synthesis of HSoligo 6 and HSoligo 7. Panel C shows the synthetic scheme for the synthesis of HSoligo 8 and HSoligo 9. Panel D shows the synthetic scheme for the synthesis of HSoligo 10.

We synthesized HSoligo 6 and HSoligo 7, octasaccharides that contain a domain sequence of -GlcA2S-GlcNS3S ± 6S-GlcA2S-GlcNS3S ± 6S- (Figure 3B). The synthesis was initiated from HSoligo 3, followed by elongation to 8-mer and 2-O-sulfation. To this point, 3-OST-4 successfully installed the second 3-O-sulfation to the designated saccharide residue to yield HSoligo 6. The 6-O-sulfation of HSoligo 6 yielded HSoligo 7. ESI-MS analysis confirmed that HSoligo 6 is an octasaccharide carrying two 3-O-sulfo groups and the NMR analysis proved 3-O-sulfation sites (Supplementary Figures 27 and 28). Confirmation of the four 6-O-sulfo groups on HSoligo 7 was performed by ESI-MS and NMR (Supplementary Figures S32 and S33). Additional structural analysis data for HSoligo 6 and 7 are shown in Supplementary Figures S29–S31 and S34–S36. To this end, we conclude that our method can prepare a saccharide consisting of more than one -GlcA2S-GlcNS3S ± 6S- disaccharide repeat.

Diversification of the Synthesis of 3-O-Sulfated Oligosaccharides.

We further expanded the use of 3-OST-4 to synthesize oligosaccharides with complex sulfation patterns. In one form of diversification, we synthesized HSoligo 8. This oligosaccharide contains a trisaccharide domain of -GlcA2S-GlcNS3S-IdoA2S-, where that the IdoA2S residue is located at the reducing end of the trisaccharide. The synthesis was initiated from an octasaccharide, and the conversion to HSoligo 8 was completed in two enzymatic steps (Figure 3C). Further modification by 6-O-sulfotransferase yields HSoligo 9. In another form of diversification, we synthesized HSoligo 10. HSoligo 10 has a tetrasaccharide sequence of -IdoA2S-GlcNS-GlcA2S-GlcNS3S-, where the IdoA2S is located at the nonreducing end. The process was initiated from HSoligo 3, and the entire synthesis was completed in six chemoenzymatic steps (Figure 3D). Taken together, we demonstrate diverse synthesis of 3-O-sulfated oligosaccharides through arrangement of the order of the enzymatic modification steps. Data for the structural characterization of HSoligo 8, 9, and 10 are shown in Supplementary Figures S37–S51.

Contribution to the Anticoagulant Activity of the Saccharide Residues around the 3-O-Sulfation Site.

We chose five oligosaccharides to investigate the roles of GlcA2S in contributing to the anticoagulant activity as measured by the ability to inhibit the activity of factor Xa (FXa) or factor IIa (FIIa). The FXa inhibition potency was compared to fondaparinux, an approved anticoagulant drug.29 We chose four oligosaccharides carrying 6-O-sulfation, including HSoligo 4, 5, 7, and 9, to evaluate the anticoagulant activity, provided that 6-O-sulfation is known to be required for the anticoagulant activity.15,30 HSoligo 8, an octasaccharide lacking 6-O-sulfation, is a negative control. We found that HSoligo 9 displayed an IC50 (half maximal inhibition concentration) value of 22.5 ng/mL, which is comparable to fondaparinux (14.7 ng/mL), a positive control in our study (Table 2). Compared to the structure of the nonreducing end saccharide of the GlcNS3S6S residue in HSoligo 9 and fondaparinux, a GlcA2S residue is in HSoligo 9, and a GlcA residue is in fondaparinux. Our data suggest that a GlcA2S residue on the nonreducing end of the GlcNS3S6S residue does not significantly affect the anti-Xa activity.

Table 2.

IC50 Values for the Anti-FXa and Anti-FIIa Activities of the Synthesized Compounds

compounds abbreviated sequences anti-FXa (ng/mL)a anti-IIa (ng/mL)b
HSoligo 4 GlcNS6S-GlcA-GlcNS6S-GlcA2S-GlcNS3S6S-GlcA-pNP 311.4 no inhibition
HSoligo 5 GlcNS6S-GlcA-GlcNS6S-GlcA2S-GlcNS3S6S-GlcA2S-pNP 154.4 no inhibition
HSoligo 7 GlcNS6S-GlcA-GlcNS6S-GlcA2S-GlcNS3S6S-GlcA2S-GlcNS3S6S-GlcA2S-pNP 79.5 no inhibition
HSoligo 8 GlcNS-GlcA-GlcNS-GlcA2S-GlcNS3S-IdoA2S-GlcNS-GlcA-pNP no inhibition no inhibition
HSoligo 9 GlcNS6S-GlcA-GlcNS6S-GlcA2S-GlcNS3S6S-IdoA2S-GlcNS-GlcA-pNP 22.5 no inhibition
fondaparinux GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-OMe 14.7 no inhibition
unfractionated no defined saccharide sequences not determined 23.0
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a

Representative inhibition curves for FXa activity for fondaparinux and HSoligos 5, 7, and 8 are shown in Supplementary Figure S52A.

b

Representative inhibition curves for FIIa activity for heparin and HSoligos 5 and 7 are shown in Supplementary Figure S52B.

Next, we compared the anti-FXa activity when a GlcA2S or a GlcA residue is located at the reducing end of the GlcNS3S6S residue. The anti-FXa IC50 value for HSoligo 7 was increased by 3.5-fold compared to HSoligo 9. In HSoligo 9, an IdoA2S residue is present on the reducing end of the GlcNS3S6S residue. Despite HSoligo 7 having two GlcNS3S6S residues, a GlcA2S residue is at the reducing ends of both GlcNS3S6S residues. We also discovered that HSoligo 4 and 5 both are hexasaccharides, displayed a 6.9-fold and 13.8-fold increase in the IC50 value compared to HSoligo 9. In HSoligo 4, a GlcA residue is at the reducing end of the GlcNS3S6S residue; whereas in HSoligo 5, a GlcA2S residue is at the reducing end of the GlcNS3S6S residue. The data suggest that the presence of IdoA2S at the reducing end plays a role in contributing to the anti-FXa activity, and the presence of IdoA2S is more important in hexasaccharides than octasaccharides to display the anti-FXa activity. HSoligo 8 displayed no inhibition, which is expected as it does not carry 6-O-sulfo groups.15,30 A lower anti-FXa activity for HSoligo 4, 5, and 7 is consistent with previous reports on the role of IdoA2S contributing to anticoagulant activity.27,31 An IdoA2S residue can display both chair 4C1 conformation and skew boat 2SO conformation, but GlcA2S can only display chair 1C4 conformation in solution.32 The lack of conformational flexibility from the 2SO conformation may reduce anticoagulant potency.

We measured the anti-FIIa activity of the oligosaccharides (Table 2 and Supplementary Figure S52B). However, we did not observe any inhibition effect on the activity of FIIa, suggesting that HSoligo 9, like fondaparinux, is a FXa specific inhibitor. HSoligo 7 also lacks anti-FIIa activity due to it being short compared to the size of the sugar chain. Heparin was used as a positive control for the anti-FIIa assay.

HSoligo 7 Binds to HMGB1 and Tau.

We tested the binding of the oligosaccharides to high mobility group box 1 (HMGB-1), a ubiquitous nuclear protein that acts as a proinflammatory protein when it is released to extracellular space.33 HMGB1 is reportedly implicated as a pathogenic mediator in neuroinflammation, traumatic brain injury, cognitive impairments, and epileptogenesis.34,35 Previously, we have shown that neutralizing the proinflammatory activity of HMGB1 by an HS 18-mer prevents acute liver damage caused by acetaminophen overdose.36 We chose to evaluate two oligosaccharides, HSoligo 4 and 7. These two oligosaccharides carry the highest number of sulfo groups per disaccharide unit and contain GlcA2S residues without IdoA2S residues. To this end, we prepared biotinylated HSoligo 4 and 7 by attaching a biotin tag to the reducing end of the oligosaccharides (Supplementary Figures S53 and S54). The oligosaccharides were mixed with mouse liver lysate that contains HMGB1. The complex of oligosaccharide and HMGB1 was captured from the solution by an avidin-affinity column and analyzed by Western (Figure 4A). Compared to the negative control oligosaccharide (NS6S 8-mer), HSoligo 4 showed a 6-fold increase, and HSoligo 7 showed a 15-fold increase in binding to HMGB-1. The extent of the binding of HSoligo 7 to HMGB1 is comparable to that of 18-mer AXa, a positive control to demonstrate the binding between HS and HMGB1 (Figure 4A).36 We also tested if HSoligo 7 displays hepatoprotection in acetaminophen overdosed mice, but we did not find a decrease in the plasma level of alanine aminotransferase (ALT) (Supplementary Figure S55). We attribute the lack of the hepatoprotection effect to its mild anti-FXa activity from HSoligo 7 as it was previously reported that anticoagulant HS 18-mer AXa or heparin does not display the hepatoprotection effect in the model.36

Figure 4.

Figure 4.

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Bindings of HSoligo 4 and 7 to HMGB1 and tau. Panel A shows the binding of oligosaccharides to HMGB1. Four biotinylated oligosaccharides, including HSoligo 4 (lane 1), 8-mer NS6S (lane 2), HSoligo 7 (lane 3), and 18-mer AXa (lane 4), were used in the experiment. Each biotinylated oligosaccharide was incubated with mouse liver lysate that contains HMGB1. The mixture was then subjected to affinity purification using an avidin-column. The top portion shows the image of Western analysis after affinity purification. HMGB1 is migrated at around 30 KDa. The bottom portion shows the relative intensity of the HMGB1 signal from each oligosaccharide that is normalized to 8-mer NS6S (lane 2). 18-mer AXa was used as a positive control. Panel B shows the binding of oligosaccharides to tau. Each oligosaccharide was incubated with Cy5-labeled tau. The mixture was then subjected to affinity purification using an avidin-column. The top portion shows the fluorescence image of SDS-gel analysis after affinity purification. The bottom shows the relative intensity of fluorescently labeled tau protein, which was migrated at 60 kDa. The intensity was normalized to HSoligo 4 (lane 1). The synthesis of 8-mer NS6S, with the abbreviated sequence of GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-Biotin, was reported previously.43 The synthesis of 18-mer AXa, with the abbreviated sequence of GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-Biotin, was published elsewhere.36 The data is represented as the mean ± SD (n ≥ 3).

Next, we examined the binding of the oligosaccharides to tau protein (Figure 4B), which forms an intraneuronal neurofibrillary tangle, a pathological hallmark of Alzheimer’s disease. Previously, we have demonstrated that 3-O-sulfated HS enhances the binding to tau and its cellular uptake,37 raising the possibility of using 3-O-sulfated oligosaccharides as a decoy receptor to prevent the internalization of tau. To this end, biotinylated HSoligo 4 and 7 along with NS6S 8-mer and 18-mer AXa were mixed with fluorescently labeled tau protein. The complex of oligosaccharide and tau protein was captured from the solution by an avidin-agarose column and analyzed by SDS gel. We observed that the binding of HSoligo 7 to tau is 4-fold higher than that for HSoligo 4, suggesting that the size of oligosaccharide plays a role in binding to tau. Compared with 8-mer NS6S, an octasaccharide that does not contain neither GlcA2S residue nor 3-O-sulfation, the binding of HSoligo 7 to tau is 2-fold higher, suggesting that the sulfation pattern also plays a role. The level of the binding between tau and HSoligo 7 is comparable to 18-mer AXa, suggesting that the unique sulfation pattern in HSoligo 7 compensates the size requirement of the sugar chain to display high binding to tau protein.

CONCLUSION

In this study, recombinant 3-OST-4 was successfully expressed in insect cells, and the enzyme was applied to the synthesis of defined HS oligosaccharides for the first time. The 3-OST-4 differs from other isoforms in its tissue expression and the substrate preference in terms of the saccharide sequence surrounding the 3-O-sulfation site. The 3-OST-4 enzyme is predominantly expressed in the human brain.38 Understanding the structures of HS modified by 3-OST-4, in principle, sheds lights on the unique structural features of HS from human brain. The enzyme installs the 3-O-sulfo group in a disaccharide domain of -GlcA2S-GlcNS- to form the -GlcA2S-GlcNS3S- sequence. The 3-O-sulfated disaccharide sequence is rarely found in HS isolated from natural sources, including human brain. Whether this rare 3-O-sulfated saccharide domain is present in human brain under normal or pathological positions remains to be investigated. It is premature to conclude that the 3-O-sulfated HS generated by the 3-OST-4 enzyme has unique functions at the present time. Our success in synthesizing the 3-O-sulfated oligosaccharides opens the possibility for further evaluation. HSoligo 7 has low anticoagulant activity but displays increased binding to both tau and HMGB-1, both proteins known be involved in the pathogenic processes of Alzheimer’s disease.39,40 The availability of the structurally defined 3-O-sulfated oligosaccharides synthesized by the 3-OST-4 enzyme adds a new set of chemical tools to investigate the function of HS in biological systems.

MATERIALS AND EXPERIMENTAL PROCEDURES

Clone, Expression, and Purification of Recombinant 3-OST-4.

The 3-OST-4 coding gene (E163 to K456) was amplified using primer pairs 3OST-4-F (5′-CATCACGATTACGCGAATTCAGAGTCCAGCACCACCGACGAGG-3′) and 3OST-4-R (5′-GCCTCGAGACTGCAGGCTCTAGATCATTTATCACCCTCTTCCTGTTCC-3′) from the human cDNA template.41 The 0.9 kb PCR product was cloned into pFastBac-Mel-HT EcoRI/XbaI sites using HiFi DNA assembly master mix (NEB) to generate the recombinant plasmid pFastBa-Mel-HT-3OST4.42 The resultant plasmid was transformed into E. coli DH10Bac cells (Invitrogen) for Bacmid DNA preparation. The isolated Bacmid DNA was transfected to insect cells SF9 using Cellfectin Reagent (Invitrogen) following the instruction manual from Invitrogen. The cells were maintained in Sf900 III medium (Gibco) supplemented with 1% fetal bovine serum (Cytiva HyClone, Thermo Fisher Scientific). Infection of the recombinant virus for expressing 3-OST-4 was carried out when the insect cells’ density reached 2.0 × 106 cells/mL. After infection, the conditioned culture was incubated in the shaker incubator at 28 °C for 3 to 4 days. Next, the enzyme was harvested by centrifuge at 4,000 rpm for 10 min. One mM phenylmethanesulfonyl fluoride, 0.1% Triton X-100, and 2% glycerol were added to the supernatant. The pH was adjusted to 7.0, and the supernatant was centrifuged again to remove the precipitates. The supernatant was mixed with an equal volume of 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer containing 0.1% Triton X-100 and 2% glycerol, pH 7.0, and loaded to a heparin column (GE Healthcare). The target protein was eluted by a gradient of 0–1 M sodium chloride in 60 min at a flow rate of 6 mL/min in a buffer containing 20 mM MOPS, 2% glycerol, and 0.1% reduced Triton X-100, pH 7.0. Fractions containing the sulfotransferase activity were collected and dialyzed against a buffer containing 25 mM Tris, 500 mM NaCl, and 2% glycerol (pH 7.5). The dialyzed solution was further purified by a Ni-Sepharose column (GE Biosciences). The protein was eluted using a gradient of 0– 250 mM imidazole elution in a buffer containing 25 mM Tris, 500 mM NaCl, 0.1% reduced Triton X-100, and 2% glycerol, pH 7.5 for 50 min at a flow rate of 2 mL/min from the column. Glycerol was added to the collected fractions to the final concentration of 20%, and the solution was kept at −80 °C.

Determination of the Substrate Specificity of 3-OST-4 Using 12-mers.

Five HS dodecasaccharides, including 12merNAc, 12merNS, 12merNS2S, 12merNs6S, and 12merNS2S6S (from Glycan Therapeutics), were used. The 12-mer substrates (10 μg) were incubated with 5 μL of 3-OST-4 (0.8 mg mL−1) in the 100 μL reaction buffer containing 50 mM Tris-HCl (pH 7.5) and 1.2 × 105 cpm of [35S]PAPS (5 μM) at 37 °C for 1 h. DEAE column purification followed by 35S-radioactivity measurement was the same as described above.

Synthesis of Oligosaccharides.

To synthesize HSoligo 1, 20 mg of 8-mer substrate C (GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-GlcA-pNP from Glycan Therapeutics) was incubated with 0.15 mmol of PAPS and 2.5 mL of purified 3-OST-4 (0.8 mg mL−1) in a total volume of 200 mL of the reaction buffer containing 50 mM Tris (pH 7.2), 2 mM MnCl2, and 2 mM CaCl2. After incubation at 37 °C overnight, the product was purified by a 30 mL Giga Q column (from Tosohaas Bioscience) and eluted by 0 to 1 M NaCl in 20 mM NaOAc (pH 5.0) in 160 min at a flow rate at 1 mL/min. The product was subsequently dialyzed against water using a MWCO 1000 (molecular weight cutoff 1000 Da) membrane (Spectrum Laboratories, Inc. CA) to remove the salt.

HSoligos 2 and 3 were synthesized from NS 6-mer (GlcNS-GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP from Glycan Therapeutics). Briefly, in a total reaction volume of 1 L, NS 6-mer (1.6 g) was incubated with 2.4 mmol of PAPS, 25 mL of the 2-OST enzyme (2 mg mL−1), and 50 mM Tris-HCl buffer (pH 7.2) overnight at 37 °C. The 2-O-sulfated hexasaccharides, named as 6-mer substrate D (GlcNS-GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP) and 6-mer substrate E (GlcNS-GlcA-GlcNS-GlcA2S-GlcNS-GlcA2S-pNP), were purified by a 300 mL Q-Sepharose column. The product was eluted from the Q-Sepharose column with a linear gradient of 0 to 1 M NaCl in 20 mM NaOAc (pH 5.0) in 5 h at a flow rate of 6 mL/min. To synthesize HSoligo 2, 23 mg of 6-mer substrate D was incubated with 0.08 mmol of PAPS and 5 mL of purified 3-OST-4 (0.8 mg mL−1) in a total of 100 mL of the reaction buffer containing 50 mM Tris-HCl (pH 7.2) and 2 mM CaCl2. After overnight incubation at 37 °C, HSoligo 2 was purified by a 30 mL Q-Sepharose column.

The synthesis of HSoligo 3 was started from 6-mer substrate E. The 6-mer substrate E (212 mg) was incubated with 0.3 mmol of PAPS and 20 mL of 3-OST-4 (0.8 mg mL−1) in a total volume of 500 mL of the reaction buffer containing 50 mM Tris-HCl (pH 7.2) and 2 mM CaCl2. The reaction was incubated at 37 °C overnight, and HSoligo 3 was purified as the product. The purification was carried out by a 150 mL Q-Sepharose column eluted with a linear gradient of NaCl from 0.5 to 1 M in 20 mM NaAcO (pH 5.0) in 4 h with a flow rate of 2 mL/min. HSoligos 2 and 3 were both subjected to dialysis against water using a MWCO 1000 membrane to remove the salt.

HSoligos 2 and 3 were further subjected to 6-O-sulfation to synthesize HSoligo 4 and 5, respectively. HSoligo 2 (15 mg) was incubated with 0.03 mmol of PAPS, 5 mL of the 6-OST enzyme (4 mg mL−1), and 50 mM Tris-HCl buffer (pH 7.2) in a total volume of 100 mL at 37 °C overnight. HSoligo 5 was obtained by incubation of 10 mg of compound 3, 0.09 mmol of PAPS, 3 mL of the 6-OST (4 mg mL−1) enzyme, and 50 mM Tris-HCl (pH 7.2) in a 100 mL volume overnight at 37 °C. Both HSoligos 4 and 5 were purified by a 30 mL Q-Sepharose column. The Q-Sepharose column was eluted with a linear gradient of NaCl from 0.4 to 0.8 M in 20 mM NaAcO (pH 5.0) in 240 min at a flow rate of 1 mL/min. Both HSoligo 4 and 5 were dialyzed twice using a MWCO 1000 membrane to remove the salt.

HSoligo 3 was elongated and sulfated to generate HSoligo 6 and 7. Briefly, HSoligo 3 (188 mg) was incubated with 0.2 mmol of UDP-GlcA in 500 mL of a buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM MnCL2, and PMHS2 (20 mg). The reaction was incubated at 37 °C overnight, and the product was purified by a Q-Sepharose column. Then, the product was further elongated with 0.15 mmol of UDP-GlcNTFA using PMHS2 in 500 mL under the same conditions to get the octasaccharide intermediate. The resultant octasaccharide intermediate was suspended in a 0.1 M LiOH solution at RT for 1 h to convert a GlcNTFA residue to a GlcNH2 residue. HPLC and ESI-MS were used to ensure the complete conversion of GlcNTFA to GlcNH2. Upon completion, the pH of the reaction was adjusted to 7.0 and then added into the reaction mixture with 25 mM Tris-HCl (pH 7.5), 0.12 mmol of PAPS, and 2 mL of purified N-sulfotransferase (3 mg mL−1) in a total of 500 mL. The N-sulfated product was purified by a Q-Sepharose column, diluted with 10 volumes of water. To the diluted eluant was added Tris to the final concentration 25 mM Tris-HCl, adjusted pH to 7.5, and then both 0.24 mmol of PAPS and 10 mL of 2-OST (2 mg mL−1) in the total reaction volume of 500 mL. The reaction was incubated at 37 °C overnight to generate the octasaccharide with three GlcA2S residues. The 27 mg resultant octasaccharide was added into a 500 mL reaction containing 50 mM Tris-HCl buffer (pH 7.2), 0.06 mmol of PAPS, 2 mM CaCl2, and 9 mL of 3-OST-4 (0.8 mg mL−1) to generate HSoligo 6. HSoligo 6 was purified after overnight incubation at 37 °C as the product and then subjected to 6-O-sulfation for HSoligo 7. The reaction containing 50 mM Tris-HCl (pH 7.5), 0.15 mmol of PAPS, 27 mg of HSoligo 6, and the 6-OST enzyme (6 mg) was incubated overnight at 37 °C. The formation of HSoligo 7 was detected by HPLC and purified by a Q-Sepharose column. HSoligo 7 was purified by a Q-Sepharose column (30 mL) and eluted using a linear gradient NaCl from 0.5 to 1 M in 200 min at a flow rate of 1 mL/min for 200 min.

HSoligo 8 was synthesized from 8-mer substrate B (GlcNS-GlcA-GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP from Glycan Therapeutics). The 8-mer substrate B (20 mg) was added into the 300 mL reaction buffer containing 0.03 mmol of PAPS, 50 mM Tris-HCl (pH 7.2), and 4 mL of the 2-OST enzyme (2 mg mL−1). The reaction was incubated overnight at 37 °C, and the product was purified by a Q-Sepharose column. About 10 mg of the product was mixed with 0.03 mmol of PAPS and 2 mL of 3-OST-4 (0.8 mg mL−1) in 100 mL of the buffer containing 50 mM Tris-HCl (pH 7.2) and 2 mM CaCl2 to produce HSoligo 8. The reaction was incubated at 37 °C overnight, and the formation of HSoligo 8 was detected by HPLC analysis. HSoligo 8 was purified by a Q-Sepharose column. Purified HSoligo 8 was further analyzed by MS and NMR after desalting. HSoligo 8 was then incubated with 0.03 mmol of PAPS, 2 mL of the 6-OST enzyme (4 mg mL−1), and 50 mM Tris-HCL buffer in a total volume of 100 mL overnight at 37 °C to generate HSoligo 9. HPLC was used to monitor the completion of the reaction. HSoligo 9 was purified using a 30 mL Q-Sepharose column, which was eluted with a gradient concentration of NaCl from 0.25 to 1 M in 20 mM NaOAc (pH 5.0) in 200 min at a flow rate of 1 mL/min. The 6-O-sulfation step was followed using the protocol as described under the synthesis of HSoligo 5.

To produce HSoligo 10, about 40 mg of the N-sulfated octasaccharide elongated from compound 3 was incubated with 0.06 mmol of PAPS, 25 mM Tris-HCl (pH 7.5), 2 mL of C5-epimerase (about 0.5 mg mL−1), and 3 mL of 2-OST (2 mg mL−1) in a total volume of 500 mL. After overnight incubation at 37 °C, HSoligo 10 was purified by a 30 mL Q-Sepharose column, which was eluted with a gradient concentration of NaCl from 0.3 to 1 M in 20 mM NaOAc (pH= 5.0) in 200 min at a flow rate of 1 mL/min.

Determination of the Anti-FXa Activity.

The assays to determine the anti-FXa activity were based on a previous report. Briefly, human FXa (Enzyme Research Laboratories) was diluted to 50 U/mL with PBS. HS compounds (Fondaparinux, unfractionated heparin, HSoligo 4, 5, 7, 8, and 9) were dissolved in PBS at various concentrations (0–5000 ng/mL). Ten microliters of the sample was incubated with 60 μL of 35 μg/mL antithrombin (Cutter Biologics) for 2 min at 37 °C. Next, 100 μL of FXa (50 U/mL) was added and incubated for 4 min at 37 °C. Thirty microliters of diluted chromogenic substrate S-2765 (Diapharma) with 1 mg mL−1 was added, and the absorbance of the reaction mixture was measured at 405 nm continuously for 5 min. The maximum slope for each sample was converted to the percentage of FXa activity by dividing by the maximum slope for the control sample.

The Binding of HS Oligosaccharides to HMGB1 and Tau.

About 20 nmol of biotinylated HS oligosaccharides (NS6S 8-mer, 18-mer AXa, HSoligo 4 and 7) was mixed with 50 μL of fresh mouse liver lysate containing HMGB1 in 200 μL of a buffer containing 20 mM HEPES (pH 7.2), 100 mM NaCl, and 1% BSA and incubated overnight at 4 °C. Pierce High Capacity Streptavidin Agarose (Thermo Fisher) was used to isolate biotinylated HS oligosaccharide bound complexes. After washing 5 times with 20 mM HEPES and 250 mM NaCl at pH 7.2, samples were eluted with the LDS sample loading buffer (Life Technologies) and subjected for Western blot analysis as in a previous report.36 Anti-HMGB1 antibody (Abcam, rabbit monoclonal EPR3507) and goat anti-rabbit HRP (Abcam) were used as the primary and secondary antibodies for the Western blot.

To measure the binding of oligosaccharides to tau protein, about 10 nmol of biotinylated HS oligosaccharides (NS6S 8-mer, 18-mer AXa, biotinylated HSoligo 4 and 7) was mixed with 5 μg of Cy5-labeled tau protein in 100 μL of a buffer containing 20 mM HEPES (pH 7.2), 100 mM NaCl, and 1% BSA and incubated overnight at 4 °C. Pierce High-Capacity Streptavidin Agarose (Thermo Fisher) was used to isolated biotinylated HS oligosaccharide and tau protein complexes. After washing 3 times with 20 mM HEPES and 250 mM NaCl at pH 7.2 and 2 times with the PBS buffer, the samples were incubated with 50 μL of the PBS buffer with a mixture of heparin lyase II (14 μg) and heparin lyase III (20 μg) for 4 h at RT. The solution was harvested by centrifuge (12,000 rpm, 5 min) and resolved by NuPAGE 4–12% Bis-tris protein gels (Invitrogen). A Typhoon FLA 9500 fluorescent image analyzer scanner (GE Healthcare) was used to determine the labeled tau protein. Western blot and gel images were both quantified by ImageJ.

Supplementary Material

supplementary Inf

Funding

This work is supported in part by NIH grants (HL094463, HL144970, GM128484, GM134738, HL139187, GM123792, and AG069039) and Glycan Innovation grants from Eshelman Innovation Institute.

Footnotes

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.1c00474.

Experimental procedures, figures of biochemical characterization of 3-OST-4 and spectra, and table of summary of oligosaccharides synthesized and measured molecular weight (PDF)

Complete contact information is available at: https://pubs.acs.org/10.1021/acschembio.1c00474

The authors declare the following competing financial interest(s): J.Liu and Y.X. are founders of Glycan Therapeutics. V.P. and G.S. are employees of Glycan Therapeutics. J.Liu, Y.X., and V.P. have equity in Glycan Therapeutics. The J.Liu lab at UNC has received a gift from Glycan Therapeutics to support the research in glycoscience. J.Li, C.W., and K.A. declare no competing interests.

Contributor Information

Jine Li, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States.

Guowei Su, Glycan Therapeutics Corporation, Raleigh, North Carolina 27606, United States.

Yongmei Xu, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States.

Katelyn Arnold, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States.

Vijayakanth Pagadala, Glycan Therapeutics Corporation, Raleigh, North Carolina 27606, United States.

Chunyu Wang, Department of Biological Sciences, Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, United States;.

Jian Liu, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States;.

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