Actinohivin Research Articles

Engineering Lactobacillus rhamnosus GG and GR-1 to express HIV-inhibiting griffithsin

Probiotic bacteria are being explored for the in situ delivery of various therapeutic agents. In this study, we aimed to express two HIV-inhibiting lectins, actinohivin (AH) and griffithsin (GRFT), in the probiotic strains Lactobacillus rhamnosus GG and L. rhamnosus GR-1 for gastrointestinal and vaginal mucosal delivery, respectively. Constructs were generated for the intracellular and extracellular production of AH and GRFT under the control of the promoter of their Major Secreted Protein Msp1. Also, intracellular expression of GRFT was investigated under the control of the nisA promoter from the inducible nisin-controlled expression (NICE) system. For the extracellular localization, the signal leader peptide of Msp1/p75 from L. rhamnosus GG was translationally fused with the genes encoding AH and GRFT. Construction of recombinant strains expressing the AH monomer and dimer was unsuccessful, probably due to the intracellular toxicity of AH for the lactobacilli. On the other hand, recombinant strains for intra- and extracellular production of GRFT by L. rhamnosus GG and GR-1 were successfully constructed. The highest expression levels of recombinant GRFT were observed for the constructs under the control of the inducible nisA promoter and we demonstrated anti-HIV activity against an M-tropic and a T-tropic HIV-1 strain. We can conclude that recombinant Lactobacillus expressing anti-HIV lectins could contribute to the development of enhanced probiotic strains that are able to inhibit HIV transmission and subsequent replication, although further research and development are required.

The characteristic structure of anti-HIV actinohivin in complex with three HMTG D1 chains of HIV-gp120.

The anti-HIV lectin actinohivin (AH) specifically interacts with HMTG (high-mannose-type glycan), which is attached to the glycoprotein gp120 of HIV-1 in a process in which the three branched mannotriose chains (D1, D2, and D3) of HMTG exhibit different binding affinities, it being estimated that that of D1 is the strongest, that of D3 is weaker, and that of D2 is undetectable. These properties have been ascribed to the stereochemical differences in linkages between the second and the third mannose residues of the three chains. In order to clarify the interaction geometry between AH and the major target D1, an X-ray determination of the crystal structure of AH in complex with D1-which is α(1,2)mannotriose composed of three mannose (Man) residues linked together only by α(1,2) bonding-has been performed. In each of the three D1-binding pockets of AH, two Man residues of D1 are accommodated at zones 1 and 2 in the pocket, in the same way as those found in the α(1,2)mannobiose-bound AH crystals. However, an OMIT map shows poor densities at both ends of the two residues. This suggests the existence of positional disorder of D1 in the pocket: the two zones are each occupied by two Man residues in two different modes, with mode A involving the Man1 and Man2 residues and mode B the Man2 and Man3 residues. In each mode, D1 is stabilized by adopting a double-bracket-shaped conformation through CH⋅⋅⋅O interactions. In mode B, however, the Man1 residue, which is the most sensitive residue to AH binding, protrudes wholly into the solvent region without contacts with AH. In mode A, in contrast, the Man3 residue interacts with the essential hydrophobic amino acid residues (Tyr and Leu conserved between the three pockets) of AH. Therefore, mode A is likely to be the one that occurs when whole HMTG is bound. In this mode, the two hydroxy groups (O3 and O4) of the Man2 residue are anchored in zone 2 by four hydrogen bonds with Asp, Asn, and Tyr residues of AH. In addition, it has been found that an isolated water molecule buried in the hydrophobic long loop bridges between Asp of AH and the hydroxy group of Man2 through hydrogen bonds. The most interesting feature is found in the interaction of the Man1 and Man3 residues with AH. All eight hydroxy groups of the two residues are completely exposed in the solvent region, whereas their hydrophobic parts make contacts with a Leu residue and two Tyr residues so that the shape of D1 and the surface of AH fit well over a wide area. These structural characteristics are potentially useful for development of AH to produce more effective antiretroviral drugs to suppress the infectious expansion of HIV/AIDS and to help expedite an end to the HIV/AIDS pandemic in the near future.

Peculiarity in crystal packing of anti-HIV lectin actinohivin in complex with α(1–2)mannobiose

Previously, the anti-HIV lectin actinohivin (AH) was cocrystallized with the target α(1-2)mannobiose (MB) in the apparent space group P213. However, three MB-bound AH rotamers generated by ±120° rotations around the molecular pseudo-threefold rotation axis are packed randomly in the unit cell according to P212121 symmetry [Hoque et al. (2012). Acta Cryst. D68, 1671-1679]. It was found that the AH used for crystallization contains short peptides attached to the N-terminus [Suzuki et al. (2012). Acta Cryst. F68, 1060-1063], which cause packing disorder. In the present study, the fully mature homogeneous AH has been cocrystallized with MB into two new crystal forms at different pH. X-ray analyses of the two forms reveal that they have peculiar character in that the space groups are the same, P22121, and the unit-cell parameters are almost the same with the exception of the length of the a axis, which is doubled in one form. The use of homogeneous AH resulted in the absence of disorder in both crystals and an improvement in the resolution, thereby establishing the basis for AH binding to the target MB. In addition, the two crystal structures clarify the interaction modes between AH molecules, which is important knowledge for understanding the multiple binding effect generated when two AH molecules are linked together with a short peptide [Takahashi et al. (2011). J. Antibiot. 64, 551-557].

Structural insights into the specific anti-HIV property of actinohivin: structure of its complex with the α(1-2)mannobiose moiety of gp120.

Actinohivin (AH) is an actinomycete lectin with a potent specific anti-HIV activity. In order to clarify the structural evidence for its specific binding to the α(1-2)mannobiose (MB) moiety of the D1 chains of high-mannose-type glycans (HMTGs) attached to HIV-1 gp120, the crystal structure of AH in complex with MB has been determined. The AH molecule is composed of three identical structural modules, each of which has a pocket in which an MB molecule is bound adopting a bracket-shaped conformation. This conformation is stabilized through two weak C-H...O hydrogen bonds facilitated by the α(1-2) linkage. The binding features in the three pockets are quite similar to each other, in accordance with the molecular pseudo-threefold symmetry generated from the three tandem repeats in the amino-acid sequence. The shape of the pocket can accept two neighbouring hydroxyl groups of the O(3) and O(4) atoms of the equatorial configuration of the second mannose residue. To recognize these atoms through hydrogen bonds, an Asp residue is located at the bottom of each pocket. Tyr and Leu residues seem to block the movement of the MB molecules. Furthermore, the O(1) atom of the axial configuration of the second mannose residue protrudes from each pocket into an open space surrounded by the conserved hydrophobic residues, suggesting an additional binding site for the third mannose residue of the branched D1 chain of HMTGs. These structural features provide strong evidence indicating that AH is only highly specific for MB and would facilitate the highly specific affinity of AH for any glycoprotein carrying many HMTGs, such as HIV-1 gp120.

Crystallographic study of the interaction of the anti-HIV lectin actinohivin with the α(1-2)mannobiose moiety of gp120 HMTG.

Actinohivin (AH) is a new potent anti-HIV lectin of microbial origin. In order to modify it to produce a more efficient drug, its three-dimensional structure has previously been determined with and without the target α(1-2)mannobiose moiety of the high-mannose-type glycan (HMTG) attached to HIV-1 gp120. However, ambiguity remained in the structures owing to packing disorder that was possibly associated with peptide fragments attached at the N-terminus. To resolve these problems, the duration of cultivation of the AH-producing strain was examined and it was found that in a sample obtained from a 20 d culture the heterogeneous fragments were completely removed to produce mature AH with high homogeneity. In addition, the purification procedures were simplified in order to increase the yield of AH and the addition of solvents was also examined in order to increase the solubility of AH. AH thus obtained was successfully crystallized with high reproducibility in a different form to the previously obtained crystals. The crystal diffracted well to beyond 1.90 Å resolution and the crystallographic data suggested that it contained no packing disorder.

Broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells.

BackgroundDendritic cells (DC), present in the skin, are the first target cells of dengue virus (DENV). Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is present on DC and recognizes N-glycosylation sites on the E-glycoprotein of DENV. Thus, the DC-SIGN/E-glycoprotein interaction can be considered as an important target for inhibitors of viral replication. We evaluated various carbohydrate-binding agents (CBAs) against all four described serotypes of DENV replication in Raji/DC-SIGN+ cells and in monocyte-derived DC (MDDC).Methodology/Principal FindingsA dose-dependent anti-DENV activity of the CBAs Hippeastrum hybrid (HHA), Galanthus nivalis (GNA) and Urtica dioica (UDA), but not actinohivin (AH) was observed against all four DENV serotypes as analyzed by flow cytometry making use of anti-DENV antibodies. Remarkably, the potency of the CBAs against DENV in MDDC cultures was significantly higher (up to 100-fold) than in Raji/DC-SIGN+ cells. Pradimicin-S (PRM-S), a small-size non-peptidic CBA, exerted antiviral activity in MDDC but not in Raji/DC-SIGN+ cells. The CBAs act at an early step of DENV infection as they bind to the viral envelope of DENV and subsequently prevent virus attachment. Only weak antiviral activity of the CBAs was detected when administered after the virus attachment step. The CBAs were also able to completely prevent the cellular activation and differentiation process of MDDC induced upon DENV infection.Conclusions/SignificanceThe CBAs exerted broad spectrum antiviral activity against the four DENV serotypes, laboratory-adapted viruses and low passage clinical isolates, evaluated in Raji/DC-SIGN+ cells and in primary MDDC.

The high mannose-type glycan binding lectin actinohivin: dimerization greatly improves anti-HIV activity

The actinomycete-derived lectin actinohivin (AH) inhibits entry of HIV-1 to susceptible cells at low nM concentrations. The cooperative binding of three segments of AH to three high mannose-type glycans (HMTGs) of HIV-1 gp120 generates specific and strong anti-HIV activity. Dimerization of AH effectively improves anti-HIV activity by increasing the number of HMTG-binding pockets. AH dimers were prepared using an Escherichia coli expression system and their anti-syncytium formation and anti-HIV activities were evaluated. Each dimer was constructed by a head-to-tail fusion of two AH molecules, with or without a spacer. As a result, His-TEV-AH/RTB(132-143)/AH, which has the residues 132-143 of ricin toxin B-chain (RTB) as a spacer, had 20-fold higher anti-syncytium formation activity and also exhibited 2-30-fold higher anti-HIV activity than AH against various clinically isolated HIV-1 strains, including drug-resistant ones. Mutation analysis implies that all six HMTG-binding pockets of the dimer participated in HMTG binding. Several AH dimers with different spacer sequences showed diverse activities, suggesting that the spacer sequence is an important factor to create higher anti-HIV activity. A dimer with improved anti-HIV activity would be a good candidate for investigation as a potential microbicide to prevent HIV transmission.

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

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-α(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-α(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-α(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-α(1-2)-Man/HMTG/gp120 interaction by computational analysis and for possible development of more potent microbicides for prevention of HIV transmission.

HIV-1 Neutralization Profile and Plant-Based Recombinant Expression of Actinohivin, an Env Glycan-Specific Lectin Devoid of T-Cell Mitogenic Activity

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1′s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide.

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

The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydrate-binding 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 (KD) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (alpha1,2-mannose)3 oligosaccharide but not by alpha1,3/alpha1,6-mannose- or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of alpha1,2-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 (approximately 5-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.

Essential regions for antiviral activities of actinohivin, a sugar-binding anti-human immunodeficiency virus protein from an actinomycete

Actinohivin (AH) is a potent anti-human immunodeficiency virus (HIV) protein that consists of highly conserved three-tandem repeats (segments 1, 2, and 3). The molecular target of AH in its anti-HIV activity is high-mannose-type saccharide chains of HIV gp120. This article deals with sequence requirements for the anti-HIV activity of AH. The deleted or substituted DNAs encoding AH or His-AH were prepared using mutagenic oligonucleotide primers in PCR. The mutant constructs were expressed in Escherichia coli, and the activities of the recombinant protein products were examined by a syncytium-formation assay system that mimics anti-HIV activity. The single segment mutant His-AHs showed no anti-syncytium-formation activity, but the mutant His-AHs, which consists of 2 or 3 segments, retained reduced activities. His-AH(6–114) dramatically reduced the anti-syncytium-formation activity to that of His-AH(36–114) or His-AH(I5A). Furthermore, His-AH(Q33A), His-AH(Q71A), and His-AH(Q109A) in which glutamine residues were substituted into alanine showed reduced activities of 1/20, 1/10, and 1/30, respectively, in anti-syncytium formation compared with His-AH. These results indicate that three segments of AH are necessary for potent anti-syncytium-formation activity—that is, for potent anti-HIV activity and the cooperated involvement of each segment of AH increased the AH-gp120 interaction.

Actinohivin, a novel anti-human immunodeficiency virus protein from an actinomycete, inhibits viral entry to cells by binding high-mannose type sugar chains of gp120

We searched human immunodeficiency virus (HIV) entry inhibitors and found a novel anti-HIV protein, actinohivin (AH), in a culture filtrate of the newly discovered genus actinomycete Longispora albida gen. nov., sp. nov. This paper deals with the mechanism of action of the anti-HIV activity of AH. AH exhibited potent anti-HIV activities against various strains of HIV-1 and HIV-2. AH bound to the glycoprotein gp120 of various strains of HIV-1 and gp130 of simian immunodeficiency virus (SIV), but did not bind to non-glycosylated gp120 nor to cells having CD4 and coreceptors, suggesting that AH inhibits viral entry to cells by binding to the envelope glycoprotein. The investigation of the effects of various sugars on AH–gp120 binding by ELISA revealed that yeast mannan alone strongly inhibited the binding (IC 50=3.0 μg/ml). Experiments investigating the binding of AH to other glycoproteins revealed that AH binds to ribonuclease B and thyroglobulin that have a high-mannose type saccharide chain, but not to other glycoproteins having a N-glycoside type saccharide chain. The above results indicate that high-mannose type saccharide chains of gp120 are molecular targets of AH in its anti-HIV activity.