Journal of Molecular Biology
New Carbohydrate Specificity and HIV-1 Fusion Blocking Activity of the Cyanobacterial Protein MVL: NMR, ITC and Sedimentation Equilibrium Studies
Introduction
The human immunodeficiency virus (HIV) enters a cell by way of an orchestrated series of recognition events between invading virus and target host cell.1 These include binding of the surface Envelope (Env) glycoprotein gp120 to the target cell receptor CD4,2 followed by subsequent interactions between gp120 and a chemokine receptor, CXCR4 or CCR5, depending on viral tropism.3 The conformational changes brought about by this multi-protein assembly facilitates gp41-mediated membrane fusion.
Active areas of current research in pharmaceutical companies and universities include engineering Env-derived or receptor-derived peptides or proteins that can block Env-mediated fusion as well as identifying completely novel proteins that inhibit diverse strains of HIV. Depending on their size and stability, such inhibitors have the potential to be used as antivirals in vivo, or as microbicides ex vivo. Examples of engineered proteins and peptides that inhibit viral entry include those that target the pre-fusogenic form of gp41 and those that target receptor-binding sites on gp120. Examples of nanomolar gp41 inhibitors include the peptides Fuzeon™,4 C345 and N36[Mut],6 and the engineered proteins NCCG-gp41,7 N35CCG-N138 and 5-helix.9 Other than antibodies, examples of natural proteins that potently inhibit HIV-1 Env-mediated fusion include cyanovirin-N (CVN),10 scytovirin,11 and actinohivin.12 Interestingly, all of these proteins were isolated from prokaryotic organisms, with CVN and scytovirin coming from the cyanobacteria Nostoc ellipsosporum and Scytonema varium, respectively, and actinohivin from an actinomycete strain. CVN, in particular, has been studied extensively at the structural and biochemical levels, and shown to recognize a stacked conformation of α-(1→2)-linked mannobiose13., 14. or mannotriose structures.15., 16. Consequently, CVN binds with high affinity GlcNAc2Man8 D1D3 and GlcNAc2Man9,13., 17., 18. the two mammalian high-mannose oligosaccharides that present these structures in an accessible manner; and it is through these interactions that CVN potently blocks HIV-1 Env-mediated fusion.13., 18. While the mechanisms by which scytovirin and actinohivin inhibit HIV have not been elucidated fully, early studies suggest that these bacterially derived proteins may exert their antiviral activity through high-affinity, carbohydrate-mediated interactions with gp120.
Little is known about the prevalence or function of cyanobacterial carbohydrate-binding proteins. Recently, we have been engaged in both structural and functional studies of these proteins for a variety of fundamental reasons: they represent novel proteins in terms of amino acid sequence homology and three-dimensional structures, they can recognize precise carbohydrate structures and conformations with unusually high (nanomolar) affinity, and at least some of these proteins potently block HIV-1 Env-mediated fusion. Perhaps most importantly, however, complexes formed between these proteins and their carbohydrate ligands represent novel templates for protein–carbohydrate recognition, and increase our understanding of the interactions required to achieve high-affinity protein–carbohydrate binding. Here, we report on the HIV-1 fusion blocking activity of a novel carbohydrate-binding protein, MVL, which shares no detectable sequence homology with any known protein families, and demonstrate carbohydrate specificity and stoichiometry of binding through NMR chemical shift mapping experiments, and isothermal titration calorimetry (ITC) and sedimentation equilibrium experiments.
Section snippets
Background
Microcystis viridis NIES-102 is a freshwater bloom-forming cyanobacterium that was observed to have transient hemagglutinating activity when grown in the laboratory under anaerobic conditions.19 This activity was traced to a 113 amino acid residue, 13 kDa protein, termed MVL. The amino acid sequence of MVL was determined by enzymatic digestion and sequencing, allowing for cloning of the mvl gene from template total genomic M. viridis DNA. The amino acid sequence of MVL was found to comprise two
Discussion
Protein–carbohydrate interactions mediate a host of interactions that facilitate recognition and attachment among macromolecules, cells and foreign pathogens.26., 27. For this reason, molecules that block deleterious carbohydrate-mediated interactions, such as those governing viral entry or bacterial pathogenesis, have the potential to be useful therapeutics and/or mechanistic probes. In practice, however, one fundamental obstacle to realizing this concept is the intrinsically low affinities
Cells
B-SC-1 cells (American Type Culture Collection) grown in DMEM 10% (Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum) 2 mM l-glutamine and 50 mg ml−1 of gentamycin (all from Gibco BRL, Bethesda, MD) were used for all assays.
Reagents
Recombinant vaccinia viruses used in this study were obtained from the AIDS Research and Reference program, Division of AIDS, NIAID, National Institutes of Health, and include the following recombinants (donor in parentheses): vCB-32 encoding
Acknowledgements
We thank C. Broder for the CCR5-encoding recombinant vaccinia virus, I. Hamachi for Manα(1-2)Manα-containing trisaccharides, M. Clore & D. Williams for helpful discussions, and the Intramural AIDS Targeted Antiviral Program of the Office of the Director, NIH (C.A.B.) for financial support.
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Cyanobacterial lectins: potential emerging therapeutics
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2022, International Journal of Biological MacromoleculesCitation Excerpt :Also, the recent studies categorized algal and cyanobacterial lectins into three main classes, high mannose (HM) specific lectins, N-glycan specific lectins, and those display specificity towards both depending on carbohydrate-binding specificity [4]–[6]. The lectins showed specificity towards the high mannose (HM) class included lectins purified from blue-green algae, Microcystis aeruginosa PCC7806 (Microvirin or MVN) [7], Microcystis Viridis NIES-102 (MVL) [8], Nostoc ellipsosporum (Cyanovirin-N or CV-N) [9], Scytonema varium (Scytovirin or SVN) [10] and Oscillatoria agardhii NIES-204 (OAA) [11] and those purified from marine algae, Kappaphycus alvarezii (KAA) [5,12], Boodlea coacta (BCA) [12] Griffithsia sp. (Griffithsin or GRFT) [13] and Eucheuma Serra (ESA) [6]. The marine algal lectins and cyanobacterial lectins have been attracting researchers' attention owing to their unique carbohydrate specificity which gained them their antiviral activity.
Antibacterial and antiviral metabolites from cyanobacteria: Their application and their impact on human health
2021, Current Research in BiotechnologyCyanobacterial lectins characteristics and their role as antiviral agents
2017, International Journal of Biological MacromoleculesCitation Excerpt :However, in contrast to CVN, no cross linking or aggregation occurs upon interaction of Cyt-CVNH with Man-9 [93]. M. viridis lectin (MVL) exhibits specificity towards chitobiose core containing oligomannosides exemplified by Man3GalNAc2 and Man6GalNAc2, thus it recognizes 4–5 units of oligomannoside core [94–96]. Scytovirin (SVN) recognizes D3 arm of Man9GlcNAc2 and exhibits high affinity towards tetrasaccharide structure of Manα(1–2)Manα(1–6)Manα(1–6)Man, thus specified as Man4 [97].
Antiviral lectins: Selective inhibitors of viral entry
2017, Antiviral ResearchCitation Excerpt :This domain organization results in a cleft that contains the CRD, which accommodates pentasaccharides through direct interactions with 4 sugar monomers. MVL has been reported to have anti-HIV-1 and HCV activities at 30–37 nM and 14–34.3 nM, respectively (Williams et al., 2005; Kachko et al., 2013; Bewley et al., 2004). Pre-incubation of HCV with Man9GlcNAc2 prevented viral inhibition by MVL in a dose dependent manner, which is consistent with MVL inhibiting enveloped viruses through the interaction with glycans of glycoproteins (Kachko et al., 2013).