ECB-ART-48957
J Biol Chem
2021 Oct 01;2974:101207. doi: 10.1016/j.jbc.2021.101207.
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Structural and kinetic analyses of holothurian sulfated glycans suggest potential treatment for SARS-CoV-2 infection.
Dwivedi R
,
Samanta P
,
Sharma P
,
Zhang F
,
Mishra SK
,
Kucheryavy P
,
Kim SB
,
Aderibigbe AO
,
Linhardt RJ
,
Tandon R
,
Doerksen RJ
,
Pomin VH
.
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Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10-60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-β-GalNAcX-(1→4)-β-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO3-. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC50 values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (KD) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 103 nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC50 values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2.
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Figure 1. Purification, electrophoretic mobility, and 1D1H NMR spectra of the polyanionic glycosidic fractions obtained from Pentacta pygmaea.A, crude polysaccharides (20 mg) obtained from the body wall of the sea cucumber P. pygmaea through nonspecific proteolytic (papain) digestion were fractionated in a DEAE cellulose column (2.5 × 20 cm) equilibrated with 100 mM sodium acetate buffer (pH 6.0). Multiple fractions (1 ml each) were obtained using a linear gradient of NaCl, from 0 to 3 M (---). Fractions were individually analyzed by Metachromasy using 1,9-dimethylmethylene blue (Abs 525 nm, ○) for the presence of sulfated glycan, by Dubois reaction (Abs 490 nm, •) for hexose, by Ehrlich assay (Abs 420 nm, Δ) for sialic acid, and by Carbazole reaction (Abs 550 nm, ▲) for uronic acid. The fractions corresponding to the respective peaks, labeled as nonsulfated glycan and the P. pygmaea-derived fucosylated chondroitin sulfate (PpFucCS), were pooled, dialyzed, and lyophilized for further characterization. B, molecular weight distribution of the purified PpFucCS was analyzed by polyacrylamide gel electrophoresis along with a series of molecular markers: low-molecular-weight heparin (LMWH) (∼8 kDa), unfractionated heparin (UFH) (∼15 kDa), chondroitin sulfate-A (CS-A) (∼40 kDa), chondroitin sulfate-C (∼60 kDa), and the sea cucumber Isostichopus badionotus-derived fucosylated chondroitin sulfate (IbFucCS) and sulfated fucan (IbSF). Samples (10 μg/each) were loaded on a 12% polyacrylamide gel and stained by 0.1% (w/v) toluidine blue (in 1% acetic acid) after electrophoretic migration. C, one-dimensional (1D) 1H NMR spectrum (δH expansion 6.0–0.0 ppm) of the purified PpFucCS were recorded in D2O, at 50 °C, on a 600 MHz Bruker NMR instrument. The NMR signals corresponding to characteristic peaks were properly labeled, as seen for the four composing α-fucose (Fuc) units (2,4S, 2,4S, 4S, and 0S) and the three composing β-N-acetylgalactosamine (GalNAc, N) units (4S, 6S, and 0S), all based on the diagnostic 1H signals present in the anomeric region, glucuronic acid (U) and GalNAc and Fuc methyl protons. S denotes sulfation in which the preceding number indicates the site or lack of occurrence. |
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Figure 2. 2D1H-1H and1H-13C NMR spectra of the composing units of PpFucCS.A, the overlayed COSY (green), TOCSY (magenta), and 150 ms mixing time NOESY (gray) spectral strip from the anomeric region (δH/δH expansions 5.8–5.0/6.0–3.25 ppm) shows the presence of four spin systems (vertical dashed lines) corresponding to four α-fucose (Fuc) units labeled as A–D, respectively, Fuc2,4S, Fuc2,4S, Fuc 4S, and Fuc. U denotes signals corresponding to glucuronic acid. B, δH/δH expansions 4.8 to 4.5/6.0 to 3.25 ppm in TOCSY spectrum covering the anomeric region of N-acetylgalactosamine (GalNAc) units, showing the presence of three spin systems (vertical dashed lines) corresponding to the non-, 6-, and 4-sulfated GalNAc units labeled, respectively, as N0S, N6S, and N4S. Pairs of numbers between parentheses indicate position of the 1H nuclei assigned in the cross-peaks, marked by circles. C and D, 1H-13C HSQC spectrum (δH/δC expansions 6.0–3.25/112.0–45.0 ppm, and δH/δC expansions 2.5–1.0/30.0–10.0 ppm), showing all 1H-13C cross-peaks of PpFucCS. All 2D NMR spectra were acquired at 50 °C on a 600 MHz Bruker NMR instrument. Labels on 1H-13C cross-peaks of (C) and (D) follow the same patterns of (A) and (B). |
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Figure 3. Structural representation of sulfated glycans.A, PpFucCS is composed of a chondroitin sulfate backbone of alternating N-acetylgalactosamine (GalNAc) and glucuronic acid (GlcA) in repeating disaccharide units of [→3)-β-GalNAc-(1→4)-β-GlcA-(1→], where the GalNAc units are mostly 4-sulfated (80%), and, to a much lesser extent, 4,6-disulfated (10%) or nonsulfated (10%). The GlcA units are substituted at the C3 position by three types of α-fucose (Fuc) branches: 40% Fuc2,4S-(1→ (A unit in NMR), 30% Fuc2,4S-(1→4)-Fuc-(1→ (B-D units in NMR), and 30% Fuc4S-(1→ (C unit in NMR), where S = SO3−. B, unfractionated heparin (UFH) is mostly composed of alternating α-glucosamine (GlcN) and α-iduronic acid (IdoA) in repeating disaccharide units of [→4)-GlcN-(1→4)-IdoA-(1→]. Sulfation occurs mostly at the C6 and N-positions of GlcN and C2 position of IdoA. Although more rare, other substitutions like sulfation at the C3 position and acetylation at the N-position of the GlcN unit as well as nonsulfation in both monosaccharides can also occur. C, the sulfated α-fucan isolated from Isostichopus badionotus (IbSF) is composed of the following tetrasaccharide unit [→3)-Fuc2,4S-(1→3)-Fuc2S-(1→3)-Fuc2S-(1→3)-Fuc-(1→]. |
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Figure 4. Anti-SARS-CoV-2 activity of different sulfated glycans. Plots showing (A) quantitation of GFP-transduced cells in the presence of different mammalian GAGs: unfractionated heparin, UFH (black); chondroitin sulfates CS-A, CS-B (dermatan sulfate), and CS-C (purple); and marine sulfated glycans: algal BoSG and sea urchin LvSF (gray), and holothurian PpFucCS (blue), IbFucCS (red), and IbSF (green) at an examined concentration of 50 mg/l. The average for the vehicle (control) is indicated with a dotted line. The numbers between parentheses on top of each bar indicate the average value. B, curves show percentage of SARS-CoV-2 inhibition of the three holothurian sulfated glycans and unfractionated heparin in a concentration-dependent manner. Numbers in parentheses indicate the increasing times observed in the IC50 values of the curves compared with the curve of the lowest IC50 value. C, plots showing percentage cell viability of HEK293T cells when treated with holothurian sulfated glycans at the highest concentration of 50 mg/l. ns stands for nonsignificant. |
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Figure 5. SPR bar plots. The bar plots (based on triplicate experiments for standard deviation) indicate normalized S-protein (A and C–F) or N501Y mutant (B) binding to surface heparin inhibited with different sulfated glycans and concentrations. Control bars are in gray, UFH in black, PpFucCS in blue, IbFucCS in red, and IbSF in green. The numbers on top of each bar (except for control bars used for normalization) indicate the average value obtained in the experiments. |
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Figure 6. Molecular modeling of the oligosaccharide building blocks from the holothurian sulfated glycans in their unbound and bound states with SARS-CoV-2 S-protein RBD.A and B, PpFucCS1, (C and D) PpFucCS2, (E and F) PpFucCS3, (G and H) IbSF. A, C, E, and G, 2D plots of the distribution of glycosidic torsion angles of the marine glycans in their unbound state, from 1 μs MD simulations. B, D, F, and H, 2D plots of the distribution of glycosidic torsion angles of the marine glycans in their bound state with S-protein RBD, from 200 ns MD simulations. 3D conformations of the composing oligosaccharide structures of the holothurian sulfated glycans are shown in sticks. Bound glycan glycosidic torsional plots are shown in blue (WT) and orange (N501Y). |
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Figure 7. Predicted binding poses of sulfated glycans bound to wildtype (left panel) and N501Y mutant (right panel) of SARS-CoV2 S-protein RBD. Docked glycan in WT (pink) and in N501 mutant (green) and selected neighboring interacting residues (gray) are shown in sticks for (A) PpFucCS1, (B) PpFucCS2, (C) PpFucCS3, (D) IbSF, and (E) heparin. Dashed lines indicate polar interactions between the RBD and glycan. F, average docking scores from five independent docking runs as obtained from AutoDock Vina. Error bars represent ±standard deviation. |
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Figure 8. Anticoagulant concentration-dependent response of holothurian sulfated glycans.A, aPTT, (B) AT-mediated factor IIa inhibition, (C) AT-mediated factor Xa inhibition, and (D) HCII-mediated factor IIa inhibition. The sulfated glycans tested (in a concentration range up to 100 μg/ml) were unfractionated heparin (UFH; in black), chondroitin sulfate B-type (CS-B, also known as DS; in purple), and the three holothurian sulfated glycans, PpFucCS (blue), IbFucCS (red), and IbSF (green). Values in parentheses in (A) indicate the percentage of IU/mg calculated from the aPTT curves related to the standard for heparin of 180 IU/mg. Numbers in parentheses in B–D indicate the increasing times observed in the IC50 values of the curves compared with the curve of the lowest IC50 value. The concentrations of coagulation (co)factors used in the experiments were 10 nM of AT or HCII and 2 nM of factors IIa or Xa. |
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