Differences in the mannose oligomer specificities of the closely related lectins from Galanthus nivalis and Zea mays strongly determine their eventual anti-HIV activity

<p>Abstract</p> <p>Background</p> <p>In a recent report, the carbohydrate-binding specificities of the plant lectins <it>Galanthus nivalis </it>(GNA) and the closely related lectin from <it>Zea mays </it>(GNA_maize) were determined by glycan array analysis and indicated that GNA_maizerecognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity.</p> <p>Results</p> <p>GNA_maizeappeared to be ~20- to 100-fold less inhibitory than GNA against HIV infection, syncytia formation between persistently HIV-1-infected HuT-78 cells and uninfected CD4⁺T-lymphocyte SupT1 cells, HIV-1 capture by DC-SIGN and subsequent transmission of DC-SIGN-captured virions to uninfected CD4⁺T-lymphocyte cells. In contrast to GNA, which preferentially selects for virus strains with deleted high-mannose-type glycans on gp120, prolonged exposure of HIV-1 to dose-escalating concentrations of GNA_maizeselected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR) analysis revealed that GNA and GNA_maizeinteract with HIV III_Bgp120 with affinity constants (K_D) of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNA_maizeselectively binds complex-type GlcNAcβ1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNA_maizeto gp120, whereas GNA_maizecannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcβ1,2man specific) or SNA (NeuAcα2,6X specific).</p> <p>Conclusion</p> <p>The markedly reduced anti-HIV activity of GNA_maizecompared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNA_maizethat have high affinity for mannose oligomers. These findings underscore the need for mannose oligomer recognition of therapeutics to be endowed with anti-HIV activity and that mannose, but not complex-type glycan binding of chemotherapeutics to gp120, may result in a pronounced neutralizing activity against the virus.</p

Interference with glycosaminoglycan-chemokine interactions with a probe to alter leukocyte recruitment and inflammation in vivo

In vivo leukocyte recruitment is not fully understood and may result from interactions of chemokines with glycosaminoglycans/GAGs. We previously showed that chlorite-oxidized oxyamylose/COAM binds the neutrophil chemokine GCP-2/CXCL6. Here, mouse chemokine binding by COAM was studied systematically and binding affinities of chemokines to COAM versus GAGs were compared. COAM and heparan sulphate bound the mouse CXC chemokines KC/CXCL1, MIP-2/CXCL2, IP-10/CXCL10 and I-TAC/CXCL11 and the CC chemokine RANTES/CCL5 with affinities in the nanomolar range, whereas no binding interactions were observed for mouse MCP-1/CCL2, MIP-1α/CCL3 and MIP-1β/CCL4. The affinities of COAM-interacting chemokines were similar to or higher than those observed for heparan sulphate. Although COAM did not display chemotactic activity by itself, its co-administration with mouse GCP-2/CXCL6 and MIP-2/CXCL2 or its binding of endogenous chemokines resulted in fast and cooperative peritoneal neutrophil recruitment and in extravasation into the cremaster muscle in vivo. These local GAG mimetic features by COAM within tissues superseded systemic effects and were sufficient and applicable to reduce LPS-induced liver-specific neutrophil recruitment and activation. COAM mimics glycosaminoglycans and is a nontoxic probe for the study of leukocyte recruitment and inflammation in vivo

Role of the HIV-1 gp120 envelope glycans in the interaction and antiviral activity of carbohydrate-binding agents

Since its identification in the early eighties, HIV became a global health problem of unprecedented dimensions. Although at least 25 anti-HIV-1 compounds against 5 different targets (the virus-encoded reverse transcriptase, protease, envelope gp41 and integrase, and the cellular co-receptor CCR5) are currently available for treatment, toxic side-effects and drug resistance development still compromise a long-term efficient treatment, and viral eradication is a big challenge. Therefore, it is important to design and define novel antiviral compounds with a different mechanism of action by preferentially acting on new antiviral targets. HIV entry is a multi-stage process that would be susceptible to therapeutic intervention. It is carried out by the envelope glycoproteins of HIV (gp120 and gp41) which mediate attachment and viral entry into susceptible target cells. The HIV-1 gp120 envelope glycoprotein is highly glycosylated. Approximately half of its molecular weight is contributed by N-linked carbohydrate structures, which act as a shield masking conserved and potentially neutralizable epitopes. Several studies have shown that carbohydrate-binding agents (CBAs) act as efficient inhibitors of HIV entry. The aim of our research was to get a more detailed insight in the antiviral mode of action of a selection of interesting CBAs.The importance of the nature of the target carbohydrates led us to investigate the prokaryotic actinohivin (AH) and the plant lectin GNAmaize. We performed in Chapter 3 a detailed investigation of AH regarding its anti-HIV activity spectrum, potential side-effects, kinetic interaction with the HIV-1 envelope protein gp120 and its resistance spectrum. AH has a pronounced and consistent neutralizing activity against a wide variety of X4 and R5 HIV-1 strains in the nanomolar range, likely due to mannose oligomer recognition of AH on gp120 and does not markedly induce cytokines/chemokines in peripheral blood mononuclear cell cultures. Surface plasmon resonance (SPR) analysis revealed that the interaction of AH with gp120 is selectively reversed by (alfa1,2-mannose)3 oligosaccharides. Further in Chapter 3, we also studied the plant lectin Galanthus nivalis (GNA) and the closely related lectin from Zea mays (GNAmaize) and could demonstrate that the markedly reduced anti-HIV activity (up to ~100-fold) of GNAmaize compared to GNA is explained by the shift in glycan recognition from high-mannose (for GNA) to complex-type (for GNAmaize) glycans, and underscores the importance of efficient mannose-oligomer recognition of therapeutics as a prerequisite to exert significant anti-HIV activity.The red algae-derived griffithsin (GRFT) is the most potent HIV entry inhibitor reported so far among the CBAs and has been shown to bind to high-mannose type glycans on gp120. We investigated in Chapter 4 the mechanisms of HIV inhibition by GRFT. In order to understand the role of the three individual carbohydrate-binding sites (CBS) present in each monomer of the GRFT dimer, mutationswere made at each CBS (collaboration with P. LiWang, Merced, USA). We found a discrepancy between HIV-1 gp120 binding ability and HIV inhibitory potency for the mutant GRFT variants compared to wild-type GRFT. This suggests that the antiviral mechanism of GRFT action is not simply related to gp120 binding but a functional cross-linking ability of GRFT which seemed to be compromised in the mutant GRFT variants proved of crucial importance for eventual potent antiviral action. It has also earlier been reported that CBAs are able to prevent DC-SIGN-mediated capture of HIV-1 and transmission of the DC-SIGN-captured virions to CD4+ T lymphocyte cells. DC-SIGN is a receptor expressed on dendritic cells (DCs), which are residing in peripheral tissues and are among the first cells to encounter HIV-1 during sexual transmission. In Chapter 4, we demonstrated that GRFT is inhibitory against HIV-1 gp120 binding to DC-SIGN, efficiently prevents DC-SIGN-mediated transfer of HIV-1 to CD4+ T-lymphocytes and is able to competitively expel gp120 from the gp120-DC-SIGN complex (a property that was not observed for HHA, another mannose-specific potent anti-HIV-1 CBA). We could also demonstrate that functionally intact CBSs of GRFT are important for the optimal inhibition of DC-SIGN-mediated virus capture and transmission of HIV.CBAs that consist of proteins or peptides might have a variety of disadvantages to be applied for systemic use, including difficulties to purify bulk amounts, susceptibility to proteases and potential generation of immune responses. Therefore, we also focused our attention on pradimicin antibiotics that represent a unique class of nonpeptidic CBAs which efficiently inhibit HIV infection. In Chapter 5 we investigated the antiviral properties of pradimicin S (PRM-S) and next, we investigated the role of several cations in the interaction of PRM-A with gp120. PRM-S could prevent infection of CD4+ T-lymphocytes by a variety of HIV-1 clades, and DC-SIGN-directed HIV-1 capture and DC-SIGN-dependent transmission of HIV-1 to uninfected CD4+ T cells. It showed affinity for HIV-1 gp120 presumably and predominantly through a1,2-mannose oligomer interaction and such interaction was Ca2+-dependent. We also found that, whereas Ca2+ and to a lesser extent Ba2+, Sr2+ and Cd2+ can trigger PRM-A multimerization and coordinates binding of these [PRM-A-cation] complexes to the glycans on gp120, the cations Zn2+, Mg2+ and Mn2+ can only form [PRM-A-cation] complexes but cannot further coordinate binding of such complexes with the gp120 glycans. All the above mentioned binding studies as well as most gp120 binding studies performed by other investigators were performed with recombinant monomeric HIV-1 envelope protein gp120. However, in the native form, Env is a trimer comprised of three gp120 and three gp41 subunits. Therefore, comparative SPR-based binding kinetics of monomeric gp120 and trimeric gp140 was investigated for a broad variety of envelope-binding agents in Chapter 6. An obligatory trimeric gp140 was constructed with a D7324 tag (in collaboration with R.W. Sanders, Amsterdam, the Netherlands) and captured to a sensor chip pre-immobilized with D7324 antibody. Virtual similar SPR-derived binding kinetics for CBAs was observed, irrespective of the oligomeric state of gp120. These findings indicate that monomeric gp120 is a good surrogate molecule for estimating native HIV-1 Env trimer binding affinities of Env-binding CBAs. Therefore, these studies confirm the relevance of the SPR data obtained in our investigations.status: publishe

Role of the carbohydrate-binding sites of Griffithsin in the prevention of DC-SIGN-mediated capture and transmission of HIV-1

Background The glycan-targeting C-type DC-SIGN lectin receptor is implicated in the transmission of the human immunodeficiency virus (HIV) by binding the virus and transferring the captured HIV-1 to CD4+ T lymphocytes. Carbohydrate binding agents (CBAs) have been reported to block HIV-1 infection. We have now investigated the potent mannose-specific anti-HIV CBA griffithsin (GRFT) on its ability to inhibit the capture of HIV-1 to DC-SIGN, its DC-SIGN-directed transmission to CD4+ T-lymphocytes and the role of the three carbohydrate-binding sites (CBS) of GRFT in these processes. Findings GRFT inhibited HIV-1(IIIB) infection of CEM and HIV-1(NL4.3) infection of C8166 CD4+ T-lymphocytes at an EC50 of 0.059 and 0.444 nM, respectively. The single mutant CBS variants of GRFT (in which a key Asp in one of the CBS was mutated to Ala) were about ~20 to 60-fold less potent to prevent HIV-1 infection and ~20 to 90-fold less potent to inhibit syncytia formation in co-cultures of persistently HIV-1 infected HuT-78 and uninfected C8166 CD4+ T-lymphocytes. GRFT prevents DC-SIGN-mediated virus capture and HIV-1 transmission to CD4+ T-lymphocytes at an EC50 of 1.5 nM and 0.012 nM, respectively. Surface plasmon resonance (SPR) studies revealed that wild-type GRFT efficiently blocked the binding between DC-SIGN and immobilized gp120, whereas the point mutant CBS variants of GRFT were ~10- to 15-fold less efficient. SPR-analysis also demonstrated that wild-type GRFT and its single mutant CBS variants have the capacity to expel bound gp120 from the gp120-DC-SIGN complex in a dose dependent manner, a property that was not observed for HHA, another mannose-specific potent anti-HIV-1 CBA. Conclusion GRFT is inhibitory against HIV gp120 binding to DC-SIGN, efficiently prevents DC-SIGN-mediated transfer of HIV-1 to CD4+ T-lymphocytes and is able to expel gp120 from the gp120-DC-SIGN complex. Functionally intact CBS of GRFT are important for the optimal action of GRFT.status: publishe

Role of the Carbohydrate-Binding Sites of Griffithsin in the Prevention of DC-SIGN-Mediated Capture and Transmission of HIV-1

<div><p>Background</p><p>The glycan-targeting C-type DC-SIGN lectin receptor is implicated in the transmission of the human immunodeficiency virus (HIV) by binding the virus and transferring the captured HIV-1 to CD4⁺ T lymphocytes. Carbohydrate binding agents (CBAs) have been reported to block HIV-1 infection. We have now investigated the potent mannose-specific anti-HIV CBA griffithsin (GRFT) on its ability to inhibit the capture of HIV-1 to DC-SIGN, its DC-SIGN-directed transmission to CD4⁺ T-lymphocytes and the role of the three carbohydrate-binding sites (CBS) of GRFT in these processes.</p><p>Findings</p><p>GRFT inhibited HIV-1(III_B) infection of CEM and HIV-1(NL4.3) infection of C8166 CD4⁺ T-lymphocytes at an EC₅₀ of 0.059 and 0.444 nM, respectively. The single mutant CBS variants of GRFT (in which a key Asp in one of the CBS was mutated to Ala) were about ∼20 to 60-fold less potent to prevent HIV-1 infection and ∼20 to 90-fold less potent to inhibit syncytia formation in co-cultures of persistently HIV-1 infected HuT-78 and uninfected C8166 CD4⁺ T-lymphocytes. GRFT prevents DC-SIGN-mediated virus capture and HIV-1 transmission to CD4⁺ T-lymphocytes at an EC₅₀ of 1.5 nM and 0.012 nM, respectively. Surface plasmon resonance (SPR) studies revealed that wild-type GRFT efficiently blocked the binding between DC-SIGN and immobilized gp120, whereas the point mutant CBS variants of GRFT were ∼10- to 15-fold less efficient. SPR-analysis also demonstrated that wild-type GRFT and its single mutant CBS variants have the capacity to expel bound gp120 from the gp120-DC-SIGN complex in a dose dependent manner, a property that was not observed for HHA, another mannose-specific potent anti-HIV-1 CBA.</p><p>Conclusion</p><p>GRFT is inhibitory against HIV gp120 binding to DC-SIGN, efficiently prevents DC-SIGN-mediated transfer of HIV-1 to CD4⁺ T-lymphocytes and is able to expel gp120 from the gp120-DC-SIGN complex. Functionally intact CBS of GRFT are important for the optimal action of GRFT.</p></div

Identification of a gene coding for a deglycosylating enzyme in Hypocrea jecorina

An enzyme with mannosyl glycoprotein endo-N-acetyl-beta-D-glucosaminidase (ENGase)-type activity was partially purified from the extracellular medium of the mould Hypocrea jecorina (Trichoderma reesei). Internal peptides were generated and used to identify the gene in the T. reesei genome. The active enzyme is processed both at the N- and at the C-terminus. High-mannose-type glycoproteins are good substrates, whereas complex-type glycans are not hydrolysed. The enzyme represents the first fungal member of glycoside hydrolase family 18 with ENGase-type activity. Bacterial ENGases and the fungal chitinases belonging to the same family show very low homology with Endo T. Database searches identify several highly homologous genes in fungi and the activity is also found within other Trichoderma species. This ENGase activity, not coregulated with cellulase production, could be responsible for the extensive N-deglycosylation observed for several T. reesei cellulases

Anti-HIV-1 activity of GRFT and its mutant variants in different cell systems.

a<p>EC₅₀ required to inhibit virus-induced cytopathicity in CEM (HIV-1 III_B) cell cultures by 50%.</p>b<p>EC₅₀ required to inhibit virus (HIV-1 NL4.3)-induced cytopathicity in C8166 cell cultures by 50%.</p>c<p>50%-Effective concentration or compound concentration required to inhibit syncytia formation between persistently HIV-1(III_B)-infected HuT-78/HIV-1 cells and uninfected CD4⁺ T-lymphocyte Sup T1 cells by 50%.</p>d<p>50%-Effective concentration or compound concentration required to inhibit syncytia formation between persistently HIV-1(III_B)-infected HuT-78/HIV-1 cells and uninfected CD4⁺ T-lymphocyte C8166 cells by 50%.</p><p>The data from which the EC₅₀'s were derived are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s005" target="_blank">S5</a> and are the mean of at least 2 to 3 independent experiments.</p

Inhibitory activity of GRFT and its mutant variants against DC-SIGN-mediated virus transmission of HIV-1-captured virions to CD4⁺ T-lymphocyte cells.

<p>The HIV-1-captured Raji/DC-SIGN cultures used for virus transmission in the presence or absence of the wild-type and mutant GRFT variants contained following amounts of captured HIV-1: 5.50±2.09 ng p24/10⁶ cells (for the WT GRFT experiments), 4.30±0.15 ng p24/10⁶ cells (for the mutant D30A GRFT experiments), 3.66±0.59 ng p24/10⁶ cells (for the mutant D70A GRFT experiments), 4.04±0.72 ng p24/10⁶ cells (for the mutant D112A GRFT experiments), and 3.78±0.77 ng p24/10⁶ cells (for the mutant Triple A GRFT experiments). Data represent the mean of three independent experiments.</p