High-mannose N-linked Glycans Research Articles

In vivo tracking of unlabelled mesenchymal stromal cells by mannose-weighted chemical exchange saturation transfer MRI.

The tracking of the in vivo biodistribution of transplanted human mesenchymal stromal cells (hMSCs) relies on reporter genes or on the addition of exogenous imaging agents. However, reporter genes and exogenous labels may require bespoke manufacturing and regulatory processes if used in cell therapies, and the labels may alter the cells' properties and are diluted on cellular division. Here we show that high-mannose N-linked glycans, which are abundantly expressed on the surface of hMSCs, can serve as a biomarker for the label-free tracking of transplanted hMSCs by mannose-weighted chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI). For live mice with luciferase-transfected hMSCs transplanted into their brains, post-mortem fluorescence staining with a mannose-specific lectin showed that increases in the CEST MRI signal, which correlated well with the bioluminescence intensity of viable hMSCs for 14 days, corresponded to the presence of mannose. In vitro, osteogenically differentiated hMSCs led to lower CEST MRI signal intensities owing to the concomitantly reduced expression of mannose. The label-free imaging of hMSCs may facilitate the development and testing of cell therapies.

Toward Engineering the Mannose 6-Phosphate Elaboration Pathway in Plants for Enzyme Replacement Therapy of Lysosomal Storage Disorders.

Mucopolysaccharidosis (MPS) I is a severe lysosomal storage disease caused by α-L-iduronidase (IDUA) deficiency, which results in accumulation of non-degraded glycosaminoglycans in lysosomes. Costly enzyme replacement therapy (ERT) is the conventional treatment for MPS I. Toward producing a more cost-effective and safe alternative to the commercial mammalian cell-based production systems, we have produced recombinant human IDUA in seeds of an Arabidopsis mutant to generate the enzyme in a biologically active and non-immunogenic form containing predominantly high mannose N-linked glycans. Recombinant enzyme in ERT is generally thought to require a mannose 6-phosphate (M6P) targeting signal for endocytosis into patient cells and for intracellular delivery to the lysosome. Toward effecting in planta phosphorylation, the human M6P elaboration machinery was successfully co-expressed along with the recombinant human IDUA using a single multi-gene construct. Uptake studies using purified putative M6P-IDUA generated in planta on cultured MPS I primary fibroblasts indicated that the endocytosed recombinant lysosomal enzyme led to substantial reduction of glycosaminoglycans. However, the efficiency of the putative M6P-IDUA in reducing glycosaminoglycan storage was comparable with the efficiency of the purified plant mannose-terminated IDUA, suggesting a poor in planta M6P-elaboration by the expressed machinery. Although the in planta M6P-tagging process efficiency would need to be improved, an exciting outcome of our work was that the plant-derived mannose-terminated IDUA yielded results comparable to those obtained with the commercial IDUA (Aldurazyme® (Sanofi, Paris, France)), and a significant amount of the plant-IDUA is trafficked by a M6P receptor-independent pathway. Thus, a plant-based platform for generating lysosomal hydrolases may represent an alternative and cost-effective strategy to the conventional ERT, without the requirement for additional processing to create the M6P motif.

Glycan Profiling Analysis of Equine Amniotic Progenitor Mesenchymal Cells and Their Derived Extracellular Microvesicles.

Equine amniotic mesenchymal cells (eAMCs) are involved in many mechanisms in tissue regenerative processes. Their secreted vesicles are important effectors in a wide array of biological processes, and contribute to in vivo healing of equine tendon lesions and endometrial inflammation. Glycoconjugates are involved in cellular recognition and in the efficient uptake of extracellular vesicles (EVs) by recipient cells. In this study, we evaluated the surface glycosylation pattern of eAMCs and their EVs from the eAMCs released in conditioned medium. We used a microarray procedure in which eAMCs and eAMC-EVs were spotted on microarray slides, and incubated with a panel of 14 biotinylated lectins and Cy3-conjugated streptavidin. Signal intensity was detected using a microarray scanner. Both eAMC and eAMC-EV microarrays interacted with all the lectins, indicating the presence of N- and O-linked glycans. With respect to eAMCs, eAMC-EVs, were found to be (1) enriched in Galβ1,3GalNAc terminating O-glycans, α2,3-linked sialoglycans, and high-mannose N-glycans (Con A); (2) diminished in N-acetyllactosamine, GalNAc, Gal, GlcNAc, and fucose terminating glycans; and (3) unchanged in α2,6 linked sialoglycans content. These results suggest that eAMC-EVs emerge from a specific eAMC microdomain, and that the high simultaneous presence of Galβ1,3GalNAc, α2,3 sialic acid, and high-mannose N-linked glycans may constitute markers of the eAMC-EVs. The role of these sugars in equine regenerative medicine requires further investigation.

Genetically engineered cell factories produce glycoengineered vaccines that target antigen-presenting cells and reduce antigen-specific T-cell reactivity

Genetically engineered cell factories produce glycoengineered vaccines that target antigen-presenting cells and reduce antigen-specific T-cell reactivity

GalNAc-Specific Soybean Lectin Inhibits HIV Infection of Macrophages through Induction of Antiviral Factors.

Although it has been shown that some mannose-binding lectins (MBLs) exhibit significant activity against HIV infection, little is known about whether N-acetylgalactosamine (GalNAc)-binding lectins have the ability to inhibit HIV infection. Here, we demonstrate that a soybean-derived lectin (SBL) with GalNAc-binding affinity could potently suppress HIV infection of macrophages in a dose-dependent fashion. Unlike the MBLs, which block HIV only through binding to the glycosylated envelope proteins (gp120 and gp41) of the virus, SBL inhibited HIV at multiple steps of the virus infection/replication cycle. SBL could activate the beta interferon (IFN-β)-STAT signaling pathway, resulting in the upregulation of a number of antiviral interferon-stimulated genes (ISGs) in macrophages. In addition, SBL treatment of macrophages induced the production of C-C chemokines, which bind to HIV entry coreceptor CCR5. Deglycosylation of cell surface galactosyl moieties or presaturation of GalNAc-binding capacity could compromise SBL-mediated induction of the antiviral factors. Furthermore, SBL exerted its anti-HIV activity in the low nanomolar range with no mitogenic effect on CD4+ T cells, a major advantage in the development of SBL as a potential anti-HIV agent compared with MBLs. These data indicate a necessity to further investigate SBL as an alternative and cost-effective anti-HIV natural product.IMPORTANCE Mannose-binding lectins (MBLs) can block the attachment of HIV to target cells and have been suggested as anti-HIV microbicides. However, the mitogenic effect of MBLs on CD4+ T cells limits this potential in clinical settings. Lectins with galactose (Gal)- or N-acetylgalactosamine (GalNAc)-binding specificity are another important category of carbohydrate-binding proteins (CBP). Compared to high-mannose N-linked glycans, GalNAc-type glycans present much less in HIV gp120 or gp41 glycosylation. Here, we demonstrate that GalNAc-specific soybean lectin (SBL) triggers antiviral signaling via recognition of the cell surface galactosyl group of macrophages, which results in the suppression of HIV at multiple steps. More importantly, SBL has no mitogenic effect on the activation of CD4+ T cells, a major advantage in the development of Gal/GalNAc-specific lectins as naturopathic anti-HIV agents.

Purification, characterization and biological significance of mannose binding lectin from Dioscorea bulbifera bulbils

Dioscorea bulbifera or air potato has been used as a folk remedy to treat cancer. A mannose binding lectin from bulbils of D. bulbifera was purified in a single step by affinity chromatography on mucin coupled Sepharose 4B column, determined by its fine sugar specificity by glycan array analysis and studied for its clinical potential in cancer and HIV research.SDS-PAGE showed that lectin is a monomer of Mr 24kDa. DBL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, fetuin, asialofetuin and transferrin but not by any monosaccharides. Glycan array analysis of DBL revealed its affinity toward high mannose N-linked glycans with enhanced affinity for terminal mannose including N-linked glycans of HIV envelope glycoprotein gp120 and has strong anti-reverse transcriptase activity. DBL showed strong binding to non-metastatic human colon epithelial cancer HT 29, metastatic SW 620 and hepatocellular HepG2 cell lines. DBL showed dose and time dependent growth inhibitory effects on all the three cell lines HT 29, SW 620 and HepG2 with IC50 of 110μg, 9.8μg, 40μg respectively at 72h. Inhibitory effect of DBL was effectively blocked in presence of competing glycans like mucin. DBL has promising clinical potential both in cancer and HIV research.

Sequential fragment ion filtering and endoglycosidase-assisted identification of intact glycopeptides.

Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans. Thirdly, a fraction of the Endo H-treated glycopeptides was further subjected to PNGase F treatment in 18O water to remove the remaining complex glycans. The intact glycopeptides and deglycosylated peptides were analyzed by nano-RPLC-MS/MS, and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools. Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptide, thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification. We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line. We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells. The intensity of different glycoforms at a specific glycosite was obtained to reach the occupancy ratios of site-specific glycoforms. These results indicate that our method can be used for characterizing site-specific protein glycosylation in complex samples. Graphical abstract Through integrating the information of intact glycopeptide, fragment ions filters and endoglycosidase digestion, the reliability of the identification could be significantly improved. We quantified the site-specific glycoforms occupancy ratios through the MS response signaling of each glycopeptide at the same time.

DC-SIGN as an attachment factor mediates Japanese encephalitis virus infection of human dendritic cells via interaction with a single high-mannose residue of viral E glycoprotein

The skin-resident dendritic cells (DCs) are thought to be the first defender to encounter incoming viruses and likely play a role in Japanese encephalitis virus (JEV) early infection. In the current study, following the demonstration of JEV productive infection in DCs, we revealed that the interaction between JEV envelope glycoprotein (E glycoprotein) and DC-SIGN was important for such infection as evidenced by antibody neutralization and siRNA knockdown experiments. Moreover, the high-mannose N-linked glycan at N154 of E glycoprotein was shown to be crucial for JEV binding to DC-SIGN and subsequent internalization, while mutation of DC-SIGN internalization motif did not affect JEV uptake and internalization. These data together suggest that DC-SIGN functions as an attachment factor rather than an entry receptor for JEV. Our findings highlight the potential significance of DC-SIGN in JEV early infection, providing a basis for further understanding how JEV exploits DC-SIGN to gain access to dendritic cells.

Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4.

Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) is a 120 kDa acute-phase glycoprotein produced primarily in the liver, secreted into the blood, and identified in serum. ITIH4 is involved in liver development and stabilization of the extracellular matrix (ECM), and its expression is altered in liver disease. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4. First, we confirmed that the four ITIH4 N-X-S/T sequons (N81, N207, N517, and N577) were glycosylated by treating ITIH4 tryptic/GluC glycopeptides with PNGaseF in the presence of 18O water. Next, we performed glycosidase-assisted LC–MS/MS analysis of ITIH4 trypsin-GluC glycopeptides enriched via hydrophilic interaction liquid chromatography to characterize ITIH4 N-glycoforms. While microheterogeneity of N-glycoforms differed between ITIH4 protein expressed in HEK293 cells and protein isolated from serum, occupancy of N-glycosylation sites did not differ. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4. We also identified isoform-specific ITIH4 O-glycoforms and documented that utilization of O-glycosylation sites on ITIH4 differed between the cell line and serum.

F-box Only Protein 2 (Fbxo2) Regulates Amyloid Precursor Protein Levels and Processing

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

Combined Deficiency of Coagulation Factors V and VIII: An Update

Combined deficiency of factor V (FV) and FVIII (F5F8D) is an autosomal recessive bleeding disorder characterized by simultaneous decreases of both coagulation factors. This review summarizes recent reports on the clinical presentations, treatments, and molecular mechanism of F5F8D. Genetic studies identified LMAN1 and MCFD2 as causative genes for this disorder, revealing a previously unknown intracellular transport pathway shared by the two important blood coagulation factors. LMAN1 and MCFD2 form a Ca2+-dependent cargo receptor complex that functions in the transport of FV/FVIII from the endoplasmic reticulum (ER) to the Golgi. Disrupting the LMAN1-MCFD2 receptor, complex formation is the primary molecular defect of missense mutations leading to F5F8D. The EF-hand domains of MCFD2 are necessary and sufficient for the interactions with both LMAN1 and FV/FVIII. Similarly, the carbohydrate recognition domain of LMAN1 contains distinct and separable binding sites for both MCFD2 and FV/FVIII. Therefore, FV and FVIII likely carry duel sorting signals that are separately recognized by LMAN1 and MCFD2 and necessary for the efficient ER-to-Golgi transport. FV and FVIII likely bind LMAN1 through the high-mannose N-linked glycans under the higher Ca2+ conditions in the ER and dissociate in the lower Ca2+ environment of the ER-Golgi intermediate compartment.

Modulation of CD147-induced matrix metalloproteinase activity: role of CD147 N-glycosylation

Degradation of the basement membrane by MMPs (matrix metalloproteinases) is one of the most critical steps in tumour progression. CD147 is a tumour-associated antigen that plays a key regulatory role for MMP activities. In the present study, mass spectrum analysis demonstrated that the purified native CD147 from human lung cancer tissue was N-glycosylated and contained a series of high-mannose and complex-type N-linked glycan structures. Moreover, native glycosylated CD147 existed exclusively as oligomers in solution and directly stimulated MMP production more efficiently than non-glycosylated prokaryotic CD147. The glycosylation site mutation results indicated that, among three N-glycan attachment sites, the N152Q mutants were retained in the endoplasmic reticulum and unfolded protein response signalling was activated. This improper intracellular accumulation impaired its MMP-inducing activity. Increased β1,6-branching of N-glycans as a result of overexpression of GnT-V (N-acetylglucosaminyltransferase V) plays an important role in tumour metastasis. In the present study, we identified CD147 as a target protein of GnT-V and found that overexpression of GnT-V resulted in an elevated level of CD147 at the plasma membrane and in cell-conditioned medium, thereby increasing the induction of MMPs. The present study reveals the important role of N-glycosylation of CD147 in its biological function and implied that targeting aberrant β1,6-branching of N-glycans on CD147 would be valuable for the development of novel therapeutic modalities against carcinoma.

2G12/PGT-binding yeast glycoprotein Gp38 elicits mannose-specific HIV-1 Env cross-reactive antibodies

Results Using the newly identified, glycan-specific PGT bNAbs to search for their binders from a triple mutant(TM) strain of Saccharomyces cerevisiae, we found that the PGT bNAbs not only bind to the previously identified 2G12reactive glycoproteins but also recognize several unknown proteins in TM yeast. One of them was identified as a short version of Gp38 with N-terminus truncation. Based on immunization of rabbits with various formulations and strategies, we found that a high titer of HIV-1 Env cross-reactive antibodies was induced when using a promiscuous T-cell epitope peptide conjugated Gp38 in a formulation with a Toll-like receptor 2 agonist and aluminum salts. The Gp38-elicited antibodies could bind to a broad range of monomeric gp120s from HIV and SIV. Moreover, the antibodies could also efficiently neutralize HIV-1 pseudoviruses when the viruses were produced in the presence of a mannosidase inhibitor kifunensine, which enriches high-mannose Man9GlcNAc2 N-linked glycans. Glycan microarray analysis showed that these antibodies bind to the synthetic Mana1,2-Mana1,2-Man containing oligosaccharides.

Energy-Dependent Electron Activated Dissociation of Metal-Adducted Permethylated Oligosaccharides

The effects of varying the electron energy and cationizing agents on electron activated dissociation (ExD) of metal-adducted oligosaccharides were explored, using permethylated maltoheptaose as the model system. Across the examined range of electron energy, the metal-adducted oligosaccharide exhibited several fragmentation processes, including electron capture dissociation (ECD) at low energies, hot-ECD at intermediate energies, and electronic excitation dissociation (EED) at high energies. The dissociation threshold depended on the metal charge carrier(s), whereas the types and sequence spans of product ions were influenced by the metal-oligosaccharide binding pattern. Theoretical modeling contributed insight into the metal-dependent behavior of carbohydrates during low-energy ECD. When ExD was applied to a permethylated high mannose N-linked glycan, EED provided more structural information than either collision-induced dissociation (CID) or low-energy ECD, thus demonstrating its potential for oligosaccharide linkage analysis.

High-Mannose Glycan-Dependent Epitopes Are Frequently Targeted in Broad Neutralizing Antibody Responses during Human Immunodeficiency Virus Type 1 Infection

Broad and potent neutralizing antibody (BNAb) responses are rare in people infected by human immunodeficiency virus type 1 (HIV-1). Clearly defining the nature of BNAb epitopes on HIV-1 envelope glycoproteins (Envs) targeted in vivo is critical for future directions of anti-HIV-1 vaccine development. Conventional techniques are successful in defining neutralizing epitopes in a small number of individual subjects but fail in studying large groups of subjects. Two independent methods were employed to investigate the nature of NAb epitopes targeted in 9 subjects, identified by the NIAID Center for HIV/AIDS Vaccine Immunology (CHAVI) 001 and 008 clinical teams, known to make a strong BNAb response. Neutralizing activity from 8/9 subjects was enhanced by enriching high-mannose N-linked glycan (HM-glycan) of HIV-1 glycoproteins on neutralization target viruses and was sensitive to specific glycan deletion mutations of HIV-1 glycoproteins, indicating that HM-glycan-dependent epitopes are targeted by BNAb responses in these subjects. This discovery adds to accumulating evidence supporting the hypothesis that glycans are important targets on HIV-1 glycoproteins for BNAb responses in vivo, providing an important lead for future directions in developing NAb-based anti-HIV-1 vaccines.

An endo-β-N-acetylglucosaminidase from Enterococcus faecalisV583 responsible for the hydrolysis of high-mannose and hybrid-type N-linked glycans

It has been demonstrated previously that Enterococcus faecalis produces secreted endoglycosidases that enable the bacteria to remove N-linked glycans from glycoproteins. One enzyme potentially responsible for this activity is EF0114, comprising a typical GH18 endoglycosidase domain and a GH20 domain. We have analyzed the other candidate, EF2863, and show that this predicted single domain GH18 protein is an endo-β-N-acetylglucosaminidase. EF2863 hydrolyzes the glycosidic bond between two N-acetylglucosamines (GlcNAc) in N-linked glycans of the high-mannose and hybrid type, releasing the glycan and leaving one GlcNAc attached to the protein. The activity of EF2863 is similar to that of the well known deglycosylating enzyme EndoH from Streptomyces plicatus. According to the CAZy nomenclature, the enzyme is designated EfEndo18A.

Removal of two high-mannose N-linked glycans on gp120 renders human immunodeficiency virus 1 largely resistant to the carbohydrate-binding agent griffithsin

High-mannose N-linked glycans recognized by carbohydrate-binding agents (CBAs) are potential targets for topical microbicides. To better understand the mechanisms by which CBAs inhibit human immunodeficiency virus (HIV)-1 infection at the molecular level, we systematically analysed the contribution of site-specific glycans to the anti-HIV activity of CBAs by site-directed mutagenesis. Our results demonstrate that a single deglycosylation at N295 or N448 in a range of primary and T-cell-line-adapted HIV-1 isolates resulted in marked resistance to griffithsin (GRFT) but maintained the sensitivity to cyanovirin (CV-N), Galanthus nivalis agglutinin (GNA) and a range of neutralizing antibodies. Unlike CV-N and GNA, the interaction between GRFT and gp120 appeared to be dependent on the specific trimeric 'sugar tower' including N295 and N448. This was further strengthened by the results of GRFT-Env binding experiments. Our study identifies GRFT-specific gp120 glycans and may provide information for the design of novel CBA antiviral strategies.

Hemelipoglycoprotein from the ornate sheep tick, Dermacentor marginatus: structural and functional characterization

BackgroundTick carrier proteins are able to bind, transport, and store host-blood heme, and thus they function also as antioxidants. Nevertheless, the role of carrier proteins in ticks is not fully understood. Some of them are found also in tick males which do not feed on hosts to such an extent such as females (there are differences in male feeding in different tick species) and thus they are not dealing with such an excess of heme; some of the carrier proteins were found in salivary glands where the processing of blood and thus release of heme does not occur. Besides, the carrier proteins bind relatively low amounts of heme (in one case only two molecules of heme per protein) compared to their sizes (above 200 kDa).The main aim of this study is the biochemical characterization of a carrier protein from the ornate sheep tick Dermacentor marginatus, hemelipoglycoprotein, with emphasis on its size in native conditions, its glycosylation and identification of its modifying glycans, and examining its carbohydrate-binding specificity.ResultsHemelipoglycoprotein from D. marginatus plasma was purified in native state by immunoprecipitation and denatured using electroelution from SDS-PAGE separated plasma. The protein (290 kDa) contains two subunits with molecular weights 100 and 95 kDa. It is glycosylated by high-mannose and complex N-glycans HexNAc2Hex9, HexNAc2Hex10, HexNAc4Hex7, and HexNAc4Hex8. The purified protein is able to agglutinate red blood cells and has galactose- and mannose-binding specificity. The protein is recognized by antibodies directed against plasma proteins with hemagglutination activity and against fibrinogen-related lectin Dorin M from the tick Ornithodoros moubata.It forms high-molecular weight complexes with putative fibrinogen-related proteins and other unknown proteins under native conditions in tick plasma. Feeding does not increase its amounts in male plasma. The hemelipoglycoprotein was detected also in hemocytes, salivary glands, and gut. In salivary glands, the protein was present in both glycosylated and nonglycosylated forms.ConclusionA 290 kDa hemelipoglycoprotein from the tick Dermacentor marginatus, was characterized. The protein has two subunits with 95 and 100 kDa, and bears high-mannose and complex N-linked glycans. In hemolymph, it is present in complexes with putative fibrinogen-related proteins. This, together with its carbohydrate-binding activity, suggests its possible involvement in tick innate immunity. In fed female salivary glands, it was found also in a form corresponding to the deglycosylated protein.

Grifonin-1: A Small HIV-1 Entry Inhibitor Derived from the Algal Lectin, Griffithsin

BackgroundGriffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin's sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties.Methodology/ResultsThe new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC50 of 190.8±11.0 nM in in vitro TZM-bl assays and an EC50 of 546.6±66.1 nM in p24gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface.ConclusionIts substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1.

Yeast-Elicited Cross-Reactive Antibodies to HIV Env Glycans Efficiently Neutralize Virions Expressing Exclusively High-Mannose N-Linked Glycans

The HIV envelope (Env) protein uses a dense coat of glycans to mask conserved domains and evade host humoral immune responses. The broadly neutralizing antibody 2G12, which binds a specific cluster of high-mannose glycans on HIV Env, shows that the glycan shield can also serve as a target for neutralizing antibodies. We have described a triple mutant Saccharomyces cerevisiae strain that expresses high-mannose glycoproteins that bind to 2G12. When used to immunize rabbits, this yeast elicits antibodies that bind to gp120-associated glycans but fail to neutralize virus. Here we sought to determine the reason for these discordant results. Affinity purification of sera over columns conjugated with three 2G12-reactive yeast glycoproteins showed that these proteins could adsorb 80% of the antibodies that bind to gp120 glycans. Despite binding to monomeric gp120, these mannose-specific antibodies failed to bind cell surface-expressed trimeric Env. However, when Env was expressed in the presence of the mannosidase inhibitor kifunensine to force retention of high-mannose glycans at all sites, the purified antibodies gained the abilities to bind trimeric Env and to strongly and broadly neutralize viruses produced under these conditions. Combined, these data show that the triple mutant yeast strain elicits antibodies that bind to high-mannose glycans presented on the HIV envelope, but only when they are displayed in a manner not found on native Env trimers. This implies that the underlying structure of the protein scaffold used to present the high-mannose glycans may be critical to allow elicitation of antibodies that recognize trimeric Env and neutralize virus.