Abstract
OAA, the potent anti-HIV protein from Oscillatoria agardhii NIES-204 belongs to a new lectin family, shows strict binding specificity for high-mannose N-glycans, and has an extremely high association constant in the picomolar range for recombinant gp120, an envelope protein of HIV. In this study we have cloned the gene encoding OAA from the genomic DNA of the cyanobacterium, and efficiently expressed the recombinant lectin (rOAA) in Escherichia coli. The rOAA expressed as a His-tagged fusion protein was recovered in a soluble form and purified in high yield (48 mg/1 l-culture) by metal chelate chromatography. The fusion protein was cleaved with factor Xa, and the resulting rOAA was isolated in a final yield of 14.8 mg/1 l-culture by reversed-phase HPLC. Both the N-terminal sequence and the molecular mass of rOAA were found to be identical with those of OAA. The rOAA was fully functional with the same properties as OAA, as evidenced by hemagglutination activity, hapten-inhibition test, and binding specificity for high-mannose-type N-glycans. This rOAA should be applicable as a specific probe for high-mannose N-glycans and should contribute to elucidation of the molecular basis of its strict carbohydrate-binding specificity and potent anti-HIV activity.
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References
Sharon N, Lis H (eds) (2003) Lectins, 2nd edn. Kluwer Academic Publishers, Netherlands
Boyd MR, Gustafson KR, Mcmahon JB, Shoemarker RH, O’Keefe BR, Mori T, Gulakowski RJ, Wu L, Rivera MI, Laurencot CM, Currens MJ, Cardellina JHII, Buckhett RW Jr, Nara PL, Pannell LK, Sowder RCII, Henderson LE (1997) Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob Agents Chemother 41:1521–1530
Yamaguchi M, Ogawa T, Muramoto K, Kamio Y, Jimbo M, Kamiya H (1999) Isolation and characterization of a mannan-binding lectin from the freshwater cyanobacterium (Blue-green algae) Microcystis viridis. Biochem Biophys Res Commun 265:703–708
Hori K, Matsubara K, Miyazawa K (2000) The primary structure of a low molecular weight peptidic agglutinin from the marine red alga, Hypnea japonica. Biochim Biophys Acta 1474:226–236
Calvete JJ, Costa FHF, Saker-Sampaio S, Murciano MPM, Nagano CS, Cavada BS, Grangeiro TB, Ramos MB, Bloch C Jr, Freitas BT, Sampaio AH (2000) The amino acid sequence of the agglutinin isolated from the red alga Bryothamnion triquetrum defines a novel lectin structure. Cell Mol Life Sci 57:343–350
Bokesch HR, O’Keefe BR, McKee TC, Pannell LK, Patterson GM, Gardella RS, Sowder RCII, Turpin J, Watson K, Buckheit RW Jr, Boyd MR (2000) A potent novel anti-HIV protein from the cultured cyanobacterium Scytonema varium. Biochemistry 42:2578–2584
Wang S, Zhong F-D, Zhang Y-J, Wu Z-J, Lin Q-Y, Xie LH (2004) Molecular characterization of a new lectin from the marine alga Ulva pertusa. Acta Biochim Biophys Sin 36:111–117
Mori T, O’Keefe BR, Sowder RCII, Bringans S, Gardella R, Berg S, Cochram P, Turpin JA, Buckheit RW Jr, McMahon JB, Boyd MR (2005) Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp. J Biol Chem 280:9345–9353
Nagano CS, Debray H, Nascimento KS, Pinto VPT, Cavada BS, Sampaio SS, Farias WRL, Sampaio AH, Calvete JJ (2005) HCA and HML isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis define a novel lectin family. Protein Sci 14:2167–2176
Hori K, Sato Y, Ito K, Fujiwara Y, Iwamoto Y, Makino H, Kawakubo A (2007) Strict specificity for high-mannose type N-glycans and primary structure of a red alga Eucheuma serra lectin. Glycobiology 17:479–491
Tsai CC, Emau P, Jiang Y, Agy MB, Shattock RJ, Schmidt A, Morton WR, Gustafson KR, Boyd MR (2004) Cyanovirin-N inhibits AIDS virus infections in vaginal transmission models. AIDS Res Hum Retroviruses 20:11–18
Bewley CA, Gustafson KR, Boyd MR, Covell DG, Bax A, Clore GM, Groneborn AM (1998) Solution structure of cyanovirin-N, a potent HIV-inactivating protein. Nature Struct Biol 5:571–578
Yang F, Bewley CA, Louis JM, Gustafson KR, Boyd MR, Gronenborn AM, Clore GM, Wlodawer A (1999) Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping. J Mol Biol 288:403–412
McFeeters RL, Xiong C, O’Keefe BR, Bokesch HR, McMahon JB, Ratner DM, Castelli R, Seeberger PH, Byrd RA (2007) The novel fold of scytovirin reveals a new twist for antiviral entry inhibitors. J Mol Biol 369:451–461
Bewley CA, Cai M, Ray S, Ghirlando R, Yamaguchi M, Muramoto K (2004) New carbohydrate specificity and HIV-fusion blocking activity of the cyanobacterial protein MVL: NMR, ITC and sedimentation equilibrium studies. J Mol Biol 339:901–914
Williams DC Jr, Lee JY, Cai M, Bewley CA, Clore GM (2005) Crystal structures of the HIV-1 inhibitory cyanobacterial protein MVL free and bound to Man3GlcNAc2. J Biol Chem 280:29269–29276
Ziółkowska NE, O’Keefe BR, Mori T, Zhu C, Giomarelli B, Vojdani F, Palmer KE, McMahon JB, Wlodawer A (2006) Domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding. Structure 14:1127–1135
Botos IA, Wlodawer A (2005) Proteins that bind high-mannose sugars of the HIV envelope. Progr Biophys Mol Biol 88:233–282
Balzarini J (2006) Inhibition of HIV entry by carbohydrate-binding proteins. Antiviral Res 71:237–247
Ziółkowska NE, Wlodawer A (2006) Structural studies of algal lectins with anti-HIV activity. Acta Biochim Pol 53:617–626
Sato Y, Murakami M, Miyazawa K, Hori K (2000) Purification and characterization of a novel lectin from a freshwater cyanobacterium, Oscillatoria agardhii. Comp Biochem Physiol 125B:169–177
Sato Y, Okuyama S, Hori K (2007) Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii. J Biol Chem 282:11021–11029
Romeo JM, Esmon B, Zusman DR (1986) Nucleotide sequence of the myxobacterial hemagglutinin gene contains four homologous domains. Proc Natl Acad Sci USA 83:6332–6336
Ochman H, Medhora MM, Garza D, Hartl DL (1990) In PCR protocols. In: Innis MA, Gelfand DH, Sninsky DH, White TJ (eds) Amplification of flanking sequences by inverse PCR. Academic Press, San Diego, California
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948
Shagger H, Jagou G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Hori K, Miyazawa K, Ito K (1986) Preliminary characterization of agglutinins from seven marine algal species. Bot Mar 31:323–331
Shine J, Dalgarno L (1974) The 3’-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346
Stevens RC (2000) Design of high-throughput methods of protein production for structural biology. Struct Fold Des 8:R177–R185
Acknowledgments
We thank Dr Makoto Hirayama for his critical reading of the manuscript. We are grateful to Dr Haruyuki Iefuji for analyzing the N-terminal amino acid sequence of rOAA. This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science.
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Sato, T., Hori, K. Cloning, expression, and characterization of a novel anti-HIV lectin from the cultured cyanobacterium, Oscillatoria agardhii . Fish Sci 75, 743–753 (2009). https://doi.org/10.1007/s12562-009-0074-4
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DOI: https://doi.org/10.1007/s12562-009-0074-4