Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties
2022; Elsevier BV; Volume: 298; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2022.101856
ISSN1083-351X
AutoresSeon Beom Kim, Mary Zoepfl, Priyanka Samanta, Fuming Zhang, Ke Xia, Reena Thara, Robert J. Linhardt, Robert J. Doerksen, Michael A. McVoy, Vitor H. Pomin,
Tópico(s)Algal biology and biofuel production
ResumoSulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action. Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. 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Our results demonstrate that we can generate sulfated oligosaccharides from a marine alga with low residual of anticoagulant activity while retaining anti-SARS-CoV-2 activity. Crude polysaccharides were obtained from the body wall of the red alga B. occidentalis through nonspecific proteolytic digestion using papain followed by ethanol precipitation, as previously described (48Farias W.R.L. Valente A.-P. Pereira M.S. Mourão P.A.S. Structure and anticoagulant activity of sulfated galactans.J. Biol. Chem. 2000; 275: 29299-29307Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 54Zucker S. Buttle D.J. Nicklin M.J.H. Barrett A.J. The proteolytic activities of chymopapain, papain, and papaya proteinase III.Biochim. Biophys. Acta. 1985; 828: 196-204Crossref PubMed Scopus (96) Google Scholar, 55Tsou C.L. Cytochrome c modified by digestion with proteolytic enzymes. 1. Digestion.Biochem. J. 1951; 49: 362-367Crossref PubMed Scopus (68) Google Scholar). 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The MW distributions of the derivatives obtained were analyzed by PAGE (Fig. S2). Large-scale production of BoSG oligosaccharides was then made by 7 h of hydrolysis, since this time yielded a suitable MW range of medium-sized oligosaccharides to be filtered through the Bio-Gel P-10 column. Approximately, 30 mg of BoSG was employed for production of oligosaccharides. The SEC column of choice was based on previous work (57Pomin V.H. Pereira M.S. Valente A.-P. Tollefsen D.M. Pavão M.S.G. Mourão P.A.S. Selective cleavage and anticoagulant activity of a sulfated fucan: Stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity.Glycobiology. 2005; 15: 369-381Crossref PubMed Scopus (95) Google Scholar, 58Pomin V.H. Valente A.P. Pereira M.S. Mourão P.A.S. Mild acid hydrolysis of sulfated fucans: A selective 2-desulfation reaction and an alternative approach for preparing tailored sulfated oligosaccharides.Glycobiology. 2005; 15: 1376-1385Crossref PubMed Scopus (69) Google Scholar, 59Queiroz I.N.L. Vilela-Silva A.-C.E.S. Pomin V.H. Oligosaccharides from the 3-linked 2-sulfated alpha-L-fucan and alpha-L-galactan show similar conformations but different dynamics.Glycobiology. 2016; 26: 1257-1264PubMed Google Scholar, 60Pomin V.H. Park Y. Huang R. Heiss C. Sharp J.S. Azadi P. Prestegard J.H. Exploiting enzyme specificities in digestions of chondroitin sulfates A and C: Production of well-defined hexasaccharides.Glycobiology. 2012; 22: 826-838Crossref PubMed Scopus (30) Google Scholar, 61Queiroz I.N.L. Wang X. Glushka J.N. Santos G.R.C. Valente A.P. Prestegard J.H. Woods R.J. Mourão P.A.S. Pomin V.H. Impact of sulfation pattern on the conformation and dynamics of sulfated fucan oligosaccharides as revealed by NMR and MD.Glycobiology. 2015; 25: 535-547Crossref PubMed Scopus (16) Google Scholar). The Bio-Gel P-10 column has been reported to be effective for fractionation of medium- and/or small-sized sulfated oligosaccharides. Figure 1B shows the chromatographic profile of fractionation of BoSG derivatives monitored by metachromasy. Although no resolved peaks indicative of size-defined oligosaccharides were evident, four fractions (Fr1–Fr4) were randomly pooled. As analyzed by PAGE (Fig. 1C), oligosaccharide
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