Molecular Cloning of Actinohivin, a Novel Anti-HIV Protein from an Actinomycete, and Its Expression in Escherichia coli

Inokoshi, Junji; Chiba, Harumi; Asanuma, Satoshi; Takahashi, Atsushi; Ōmura, Satoshi; Tanaka, Haruo

doi:10.1006/bbrc.2001.4496

Cited by 21 publications

(16 citation statements)

References 8 publications

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“…17 The proteins were highly purified by metal-chelating Sepharose 4B and HPLC (as shown in Figure 2) as single bands on SDS-polyacrylamide gel electrophoresis. The syncytium formation assay (Table 2) shows that the substitutions of Ala for Asn17, Tyr18, Asn19, Asn21 and Asn31 have minimal affect on activity, compared with that of His-AH, suggesting that these residues are not involved in HMTG binding.…”

Section: Resultsmentioning

confidence: 99%

“…PCR was carried out using a 0.2 mM deoxynucleoside triphosphate mixture, Takara LA Taq buffer (Takara Bio, Tokyo, Japan), 0.25 mM MgCl 2 , 5% DMSO, 50 pmol of each primer and 0.5 U Takara LA Taq DNA polymerase (Takara Bio), and 10 ng of pBluescript II SK+: 2A3K (pBSK+: 2A3K) 17 or PCR products as a template. The reaction was run for 30 cycles, incorporating denaturing at 94 1C for 30 s, annealing at 60 1C for 30 s and polymerization at 72 1C for 1 min for each cycle.…”

Section: Materials and Methods Pcrmentioning

confidence: 99%

“…Finally, mutant AH was purified by HPLC using an ODS column (Senshu Pak Pegasil ODS, 6Â250 mm, Senshu Scientific, Tokyo, Japan) under the same conditions, as previously described. 17 …”

Section: Expression and Purification Of Mutant Ahsmentioning

confidence: 99%

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Actinohivin: specific amino acid residues essential for anti-HIV activity

et al. 2010

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Actinohivin (AH) is a microbial lectin containing 114 amino acids, which inhibits human immunodeficiency virus (HIV) infection. This effect is brought about by its specific binding to Man-a(1-2)-Man unit(s) of high-mannose type glycan (HMTG) bound to HIV gp120. The recently determined crystal structure of AH suggests that three repeated segments (the residue numbers 1-38, 39-76 and 77-114 for segments 1, 2 and 3, respectively) form three sugar-binding pockets to accommodate Man-a(1-2)-Man units. The strong specific binding of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the 'cluster effect' of lectin. It remains to be seen which residues of the sugar-binding pockets are essential for acceptance of Man-a(1-2)-Man. To identify the amino acid residues critical for anti-HIV effect, we performed mutational analysis. Mutant AHs were subjected to enzyme-linked immunosorbent assay testing for gp120-binding activity and to syncytium formation assay. As a result, it was revealed that Asp15, Tyr23, Leu25, Asn28 and Tyr32 in segment 1, Tyr61 in segment 2 and Tyr99 in segment 3 are essential for anti-HIV activity. The conserved residues, Asp53, Leu63, Asn66 and Tyr70, in segment 2 and, Asp91, Leu101, Asn104 and Tyr108, in segment 3 are also necessary. Furthermore, aromatic residues at positions 23 and 32 are required for creation of potency. These data will be useful for predicting the detailed mechanism of AH-Man-a(1-2)-Man/HMTG/gp120 interaction by computational analysis and for possible development of more potent microbicides for prevention of HIV transmission.

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Section: Resultsmentioning

confidence: 99%

Section: Materials and Methods Pcrmentioning

confidence: 99%

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Actinohivin: specific amino acid residues essential for anti-HIV activity

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“…Actinohivin (AH) is an anti-HIV protein that has been isolated from a prokaryotic microorganism (the actinomycete Longispora albida K97-0003 T ) (14,27). AH consists of 114 amino acids (12,524.3 Da) and exhibits a unique highly conserved internal sequence triplication (60% identity and ϳ70% homology on each segment).…”

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confidence: 99%

Actinohivin, a Broadly Neutralizing Prokaryotic Lectin, Inhibits HIV-1 Infection by Specifically Targeting High-Mannose-Type Glycans on the gp120 Envelope

Hoorelbeke

Huskens

Férir

et al. 2010

Antimicrob Agents Chemother

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The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydratebinding sites. AH strongly interacts with gp120 derived from different X4 and R5 human immunodeficiency virus (HIV) strains, simian immunodeficiency virus (SIV) gp130, and HIV type 1 (HIV-1) gp41 with affinity constants (K D ) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (␣1,2-mannose) 3 oligosaccharide but not by ␣1,3/␣1,6-mannose-or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of ␣1,2-mannose-specific monoclonal antibody 2G12, and AH covers a broader epitope on gp120 than 2G12. Prolonged exposure of HIV-1-infected CEM T-cell cultures with escalating AH concentrations selects for mutant virus strains containing N-glycosylation site deletions (predominantly affecting high-mannose-type glycans) in gp120. In contrast to 2G12, AH has a high genetic barrier, since several concomitant N-glycosylation site deletions in gp120 are required to afford significant phenotypic drug resistance. AH is endowed with broadly neutralizing activity against laboratory-adapted HIV strains and a variety of X4 and/or R5 HIV-1 clinical clade isolates and blocks viral entry within a narrow concentration window of variation (ϳ5-fold). In contrast, the neutralizing activity of 2G12 varied up to 1,000-fold, depending on the virus strain. Since AH efficiently prevents syncytium formation in cocultures of persistently HIV-1-infected HuT-78 cells and uninfected CD4؉ T lymphocytes, inhibits dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated capture of HIV-1 and subsequent virus transmission to CD4 ؉ T lymphocytes, does not upregulate cellular activation markers, lacks mitogenic activity, and does not induce cytokines/chemokines in peripheral blood mononuclear cell cultures, it should be considered a potential candidate drug for microbicidal use.

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“…T (6)(7)(8). AH exhibits potent anti-HIV activity against various strains of T-tropic and M-tropic HIV-1 and HIV-2 (IC 50 ϭ 2-110 nM) and inhibits viral entry to cells by binding to the high-Man type glycans (HMTGs) of gp120 (9).…”

mentioning

confidence: 99%

Mechanism by which the lectin actinohivin blocks HIV infection of target cells

Tanaka

Chiba²,

Inokoshi³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Man␣(1-2)Man, Man␣(1-2)Man␣(1-2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (K d ‫؍‬ 3.4 ؋ 10 ؊8 M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high-and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the ''cluster effect'' of lectin. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.action mechanism ͉ anti-HIV ͉ gp120 ͉ high-mannose type glycan ͉ cyanovirin-N

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Molecular Cloning of Actinohivin, a Novel Anti-HIV Protein from an Actinomycete, and Its Expression in Escherichia coli

Cited by 21 publications

References 8 publications

Actinohivin: specific amino acid residues essential for anti-HIV activity

Actinohivin: specific amino acid residues essential for anti-HIV activity

Actinohivin, a Broadly Neutralizing Prokaryotic Lectin, Inhibits HIV-1 Infection by Specifically Targeting High-Mannose-Type Glycans on the gp120 Envelope

Mechanism by which the lectin actinohivin blocks HIV infection of target cells

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