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. Author manuscript; available in PMC: 2016 Oct 1.
Published in final edited form as: Biomol NMR Assign. 2015 Feb 14;9(2):317–319. doi: 10.1007/s12104-015-9600-8

1H, 13C and 15N resonance assignment of the anti-HIV lectin from Oscillatoria agardhii

Marta G Carneiro 1, Leonardus M I Koharudin 2, Christian Griesinger 1, Angela M Gronenborn 2, Donghan Lee 1,*
PMCID: PMC4537409  NIHMSID: NIHMS664427  PMID: 25680849

Abstract

Lectins from different sources are known to interfere with HIV infection. The antiviral activity is mediated by binding to high mannose sugars present on the viral envelope, thereby inhibiting cell entry. The lectin from Oscillatoria agardhii (OAA) specifically recognizes a unique substructure of high mannose sugars and exhibits broad anti-HIV activity. Here we report the assignment of backbone and side-chain 1H, 13C and 15N resonances of free OAA.

Keywords: anti-HIV lectins, Oscillatoria agardhii agglutinin, NMR assignment

Biological context

In the last decade lectins from different sources have been found to prevent infection by HIV. In general, lectins block virus entry into the host cells by binding with high specificity and affinity to high-mannose carbohydrates (Man-9) present on the viral envelope. Interestingly, the binding epitopes on Man-9 vary for different lectins (Balzarini 2007; Huskens and Schols 2012), and application of such lectins as anti-HIV agents requires detailed characterization of their biophysical properties. Here we focus on the 14 kDa Oscillatoria agardhii agglutinin (OAA) (Sato et al. 2000; Sato et al. 2007). Compared to other carbohydrate binding agents with anti-HIV activity, OAA was found to specifically recognize a unique substructure of the Man-9 glycan found on the viral protein gp120 and to inhibit infection on a broad range of HIV-1 clinical isolates (Férir et al. 2014; Koharudin and Gronenborn 2011).

Methods and experiments

OAA was expressed and purified as described by (Koharudin et al. 2011). The NMR samples consisted of 2mM 15N or 15N, 13C labeled OAA in 20 mM sodium acetate, 20 mM sodium chloride, 3 mM sodium azide, 90/10 % H2O/D2O (pH 5) or 100 % D2O. Sequence specific assignments of the backbone resonances were obtained using 2D [15N,1H]-HSQC and 3D HNCA (Bax et al. 1990; Grzesiek and Bax 1992) measured on a Bruker 800 MHz AVANCE III spectrometer. Ambiguities were resolved using sequential NOEs from a 3D 15N-resolved [1H,1H] NOESY (Marion et al. 1989a; Marion et al. 1989b; Zuiderweg and Fesik 1989), measured on a Bruker 800 MHz AVANCE III spectrometer, using a mixing time of 60 ms. Complete 1H and 13C assignments of aliphatic side-chains were achieved using 2D [1H,13C]-HSQC, 3D hCCH-TOCSY (Bax et al. 1990), measured on a Bruker 600 MHz AVANCE III spectrometer and 3D 13Caliphatic-resolved [1H,1H] NOESY (Muhandiram et al. 1993), measured on a Bruker 800 MHz AVANCE III spectrometer with a mixing time of 80 ms. Nearly complete assignments of aromatic side-chains were accomplished using 2D [1H,13C]-HSQC and 3D 13Caromatic-resolved [1H,1H] NOESY (Muhandiram et al. 1993) measured on an Oxford 700 MHz AVANCE III spectrometer with a mixing time of 80 ms. Amide side-chains were assigned using the 2D [15N,1H]-HSQC , 2D [1H,13C]-HSQC, 3D 15N-resolved [1H,1H] NOESY and 3D 13Caliphatic-resolved [1H,1H] NOESY. The spectrometers operating at 600 and at 700 MHz were equipped with 5 mm TXI room-temperature probes and the spectrometer operating at 800 MHz was equipped with a 5 mm TCI cryogenic probe. Backbone carbonyl resonance assignments were obtained using 3D HNCO (Grzesiek and Bax 1992; Kay et al. 1994; Schleucher et al. 1993) measured on a Bruker 900 MHz AVANCE spectrometer equipped with a 5 mm TCI cryogenic probe. All experiments were performed at 298 K and all spectra were processed using NMRPipe (Delaglio et al. 1995) and analyzed with CARA (Keller 2004).

Assignments and data deposition

Complete backbone assignment was achieved (Fig. 1). Of note are the unusual proton chemical shifts of the G26 and G93 amide resonances caused by ring current effects from the aromatic side chains of W90 and W23, respectively (Koharudin et al. 2011). All 1H, 13C and 15N side-chain assignments were obtained, except for the aromatic side-chain and backbone carbonyl resonances of Y71, and the carbonyl backbone resonance of W23. Based on the chemical shifts, 10 β-strands were predicted using the program TALOS-N (Shen and Bax 2013) and the positions in the sequence are consistent with the published crystal structure (Koharudin et al. 2011), as shown in Fig. 2. It should be noted that the OAA homolog lectin family have an unique fold (Koharudin et al. 2012) compared to other lectins and thus, only intimate correlations among OAA homolog lectin family in the chemical shifts of 13Cα and 13Cβ and their secondary structural elements are expected.

Figure 1.

Figure 1

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2D 1H-15N HSQC spectrum of uniformly 15N-labeled OAA in 90% H2O/10% D2O recorded at 800 MHz and 298 K. Backbone resonance assignments are indicated by the one-letter amino acid code and the sequence number. The amide resonances of G26 and G93 exhibit unusual proton chemical shifts and are displayed in the inset. All side chain resonances are labeled in red and amino groups of N and Q are connected with a dotted line. Arginine side chain resonances are folded in the 15N dimension and contoured in grey.

Figure 2.

Figure 2

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Secondary structure elements predicted by TALOS-N (Shen and Bax 2013). The locations of predicated β-strands are indicated by arrows.

The chemical shifts have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) database under the accession number 25306.

Acknowledgements

This work was supported by the Max Planck Society, the EU (ERC grant agreement number 233227 to CG), and the National Institute of Health grant GM080642 (to AMG).

Footnotes

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. Balzarini J. Targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy. Nat Rev Microbiol. 2007;5:583–597. doi: 10.1038/nrmicro1707. doi:10.1038/nrmicro1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bax A, Clore GM, Gronenborn AM. 1H-1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J Magn Reson. 1990;88:425–431. doi:10.1016/0022-2364(90)90202-K. [Google Scholar]
  3. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995;6:277–293. doi: 10.1007/BF00197809. doi:10.1007/BF00197809. [DOI] [PubMed] [Google Scholar]
  4. Férir G, Huskens D, Noppen S, Koharudin LMI, Gronenborn AM, Schols D. Broad anti-HIV activity of the Oscillatoria agardhii agglutinin homologue lectin family. J Antimicrob Chemother. 2014 doi: 10.1093/jac/dku220. doi:10.1093/jac/dku220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grzesiek S, Bax A. Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein. J Magn Reson. 1992;96:432–440. doi:10.1016/0022-2364(92)90099-S. [Google Scholar]
  6. Huskens D, Schols D. Algal Lectins as Potential HIV Microbicide Candidates. 2012 doi: 10.3390/md10071476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mar Drugs 10:1476–1497. doi: 10.3390/md10071476. doi:10.3390/md10071476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kay LE, Xu GY, Yamazaki T. Enhanced-sensitivity triple-resonance spectroscopy with minimal H2O saturation. J Magn Reson A. 1994;109:129–133. doi:10.1006/jmra.1994.1145. [Google Scholar]
  9. Keller RLJ. The computer aided resonance assignment tutorial. CANTINA Verlag. 2004 [Google Scholar]
  10. Koharudin LMI, Furrey W, Gronenborn AM. Novel fold and carbohydrate specificity of the potent anti-HIV cyanobacterial lectin from Oscillatoria agardhii. J Biol Chem. 2011;286:1588–1597. doi: 10.1074/jbc.M110.173278. doi:10.1074/jbc.M110.173278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Koharudin LMI, Gronenborn AM. Structural basis of the anti-HIV activity of the cyanobacterial Oscillatoria agardhii agglutinin. Structure. 2011;19:1170–1181. doi: 10.1016/j.str.2011.05.010. doi:10.1016/j.str.2011.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Koharudin LMI, Kollipara S, Aiken C, Gronenborn AM. Structural insights into the anti-HIV activity of the Oscillatoria agardhii agglutinin homolog lectin family. J Biol Chem. 2012;287:33796–33811. doi: 10.1074/jbc.M112.388579. doi:10.1074/jbc.M112.388579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Marion D, Driscoll PC, Kay LE, Wingfield PT, Bax A, Gronenborn AM, Clore GM. Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1β. Biochemistry. 1989a;28:6150–6156. doi: 10.1021/bi00441a004. doi:10.1021/bi00441a004. [DOI] [PubMed] [Google Scholar]
  14. Marion D, Kay LE, Sparks SW, Torchia DA, Bax A. Three-dimensional heteronuclear NMR of 15N labeled proteins. J Am Chem Soc. 1989b;111:1515–1517. doi:10.1021/ja00186a066. [Google Scholar]
  15. Muhandiram DR, Farrow NA, Xu GY, Smallcombe SH, Kay LE. A gradient 13C NOESY-HSQC experiment for recording NOESY spectra of 13C-labeled proteins dissolved in H2O. J Magn Reson B. 1993;102:317–321. doi:10.1006/jmrb.1993.1102. [Google Scholar]
  16. Sato Y, Murakami M, Miyazawa K, Hori K. Purification and characterization of a novel lectin from a freshwater cyanobacterium, Oscillatoria agardhii. Comp Biochem Physiol B: Biochem Mol Biol. 2000;125:169–177. doi: 10.1016/s0305-0491(99)00164-9. doi:10.1016/S0305-0491(99)00164-9. [DOI] [PubMed] [Google Scholar]
  17. Sato Y, Okuyama S, Hori K. Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii. J Biol Chem. 2007;282:11021–11029. doi: 10.1074/jbc.M701252200. doi:10.1074/jbc.M701252200. [DOI] [PubMed] [Google Scholar]
  18. Schleucher J, Sattler M, Griesinger C. Coherence selection by gradients without signal attenuation: Application to the three-dimensional HNCO experiment. Angw Chem, Int Ed Engl. 1993;32:1489–1491. doi:10.1002/anie.199314891. [Google Scholar]
  19. Shen Y, Bax A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J Biomol NMR. 2013;56:227–241. doi: 10.1007/s10858-013-9741-y. doi:10.1007/s10858-013-9741-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Zuiderweg ERP, Fesik SW. Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a. Biochemistry. 1989;28:2387–2391. doi: 10.1021/bi00432a008. doi:10.1021/bi00432a008. [DOI] [PubMed] [Google Scholar]

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