The role of <Emphasis Type="Italic">N</Emphasis>-glycans of HIV-1 gp41 in virus infectivity and susceptibility to the suppressive effects of carbohydrate-binding agents

Background

Carbohydrate-binding agents (CBAs) are potent antiretroviral compounds that target the N-glycans on the HIV-1 envelope glycoproteins. The development of phenotypic resistance to CBAs by the virus is accompanied by the deletion of multiple N-linked glycans of the surface envelope glycoprotein gp120. Recently, also an N-glycan on the transmembrane envelope glycoprotein gp41 was shown to be deleted during CBA resistance development.

Results

We generated HIV-1 mutants lacking gp41 N-glycans and determined the influence of these glycan deletions on the viral phenotype (infectivity, CD4 binding, envelope glycoprotein incorporation in the viral particle and on the transfected cell, virus capture by DC-SIGN⁺ cells and transmission of DC-SIGN-captured virions to CD4⁺ T-lymphocytes) and on the phenotypic susceptibility of HIV-1 to a selection of CBAs. It was shown that some gp41 N-glycans are crucial for the infectivity of the virus. In particular, lack of an intact N616 glycosylation site was shown to result in the loss of viral infectivity of several (i.e. the X4-tropic III_B and NL4.3 strains, and the X4/R5-tropic HE strain), but not all (i.e. the R5-tropic ADA strain) studied HIV-1 strains. In accordance, we found that the gp120 levels in the envelope of N616Q mutant gp41 strains NL4.3, III_B and HE were severely decreased. In contrast, N616Q gp41 mutant HIV-1_ADA contained gp120 levels similar to the gp120 levels in WT HIV-1_ADA virus. Concomitantly deleting multiple gp41 N-glycans was often highly detrimental for viral infectivity. Using surface plasmon resonance technology we showed that CBAs have a pronounced affinity for both gp120 and gp41. However, the antiviral activity of CBAs is not dependent on the concomitant presence of all gp41 glycans. Single gp41 glycan deletions had no marked effects on CBA susceptibility, whereas some combinations of two to three gp41 glycan-deletions had a minor effect on CBA activity.

Conclusions

We revealed the importance of some gp41 N-linked glycans, in particular the N616 glycan which was shown to be absolutely indispensable for the infectivity potential of several virus strains. In addition, we demonstrated that the deletion of up to three gp41 N-linked glycans only slightly affected CBA susceptibility.

     

N-glycosylation and biological activity of recombinant human alpha1-antitrypsin expressed in a novel human neuronal cell line

Human alpha‐1‐antitrypsin (A1AT) is a protease inhibitor that is involved in the protection of lungs from neutrophil elastase enzyme that drastically modifies tissue functioning. The glycoprotein consists of 394 amino acids and is N‐glycosylated at Asn‐46, Asn‐83, and Asn‐247. A1AT deficiency is currently treated with A1AT that is purified from human serum. In view of therapeutic applications, rA1AT was produced using a novel human neuronal cell line (AGE1.HN®) and we investigated the N‐glycosylation pattern as well as the in vitro anti‐inflammatory activity of the recombinant glycoprotein. rA1AT (300 mg/L) was biologically active as analyzed using elastase assay. The N‐glycan pool, released by PNGase F digestion, was characterized using 2D‐HPLC, MALDI‐TOF mass spectrometry, and by exoglycosidase digestions. A total of 28 N‐glycan structures were identified, ranging from diantennary to tetraantennary complex‐type N‐glycans. Most of the N‐glycans were found to be (α1–6) core‐fucosylated and part of them contain the Lewis X epitope. The two major compounds are a monosialylated diantennary difucosylated glycan and a disialylated diantennary core‐fucosylated glycan, representing 25% and 18% of the total N‐glycan pool, respectively. Analysis of the site‐specificity revealed that Asn‐247 was mainly occupied by diantennary N‐glycans whereas Asn‐46 was occupied by di‐, and triantennary N‐glycans. Asn‐83 was exclusively occupied by sialylated tri‐ and tetraantennary N‐glycans. Next, we evaluated the anti‐inflammatory activity of rA1AT using A1AT purified from human serum as a reference. rA1AT was found to inhibit the production of TNF‐α in neutrophils and monocytes as commercial A1AT does. Biotechnol. Bioeng. 2011;108:2118–2128. © 2011 Wiley Periodicals, Inc.     

Characterizing the release of bioactive N‐glycans from dairy products by a novel endo‐β‐N‐acetylglucosaminidase

Endo‐β‐N‐acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI‐1) is a novel enzyme that cleaves N‐N′‐diacetyl chitobiose moieties found in the N‐glycan core of high mannose, hybrid, and complex N‐glycans. These conjugated N‐glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45–8.45), temperature (27.5–77.5°C), reaction time (15–475 min), and enzyme/protein ratio (1:3,000–1:333) were evaluated on the release of N‐glycans from bovine colostrum whey by EndoBI‐1. A central composite design was used, including a two‐level factorial design (2⁴) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N‐glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan‐free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI‐1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1331–1339, 2015     

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

Background

In a recent report, the carbohydrate-binding specificities of the plant lectins Galanthus nivalis (GNA) and the closely related lectin from Zea mays (GNA_maize) were determined by glycan array analysis and indicated that GNA_maize recognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity.

Results

GNA_maize appeared 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_maize selected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR) analysis revealed that GNA and GNA_maize interact with HIV III_B gp120 with affinity constants (K_D) of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNA_maize selectively binds complex-type GlcNAcβ1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNA_maize to gp120, whereas GNA_maize cannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcβ1,2man specific) or SNA (NeuAcα2,6X specific).

Conclusion

The markedly reduced anti-HIV activity of GNA_maize compared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNA_maize that 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.     

Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn¹³⁶ in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N¹³⁹INN sequence, which ablates the overlapping Asn¹⁴¹-Asn¹⁴²-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu⁵⁹³, Trp⁵⁹⁶ and Lys⁶⁰¹. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.     

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid‐sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site‐specific characterization of the N‐ and O‐linked glycosylation of serum‐derived hSHBG. MS‐driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom‐up and top‐down manner to determine glycosylation sites, site‐specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum‐derived hSHBG is N‐glycosylated at Asn³⁵¹ and Asn³⁶⁷ with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi‐ and triantennary N‐Glycoforms with lactosamine‐type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N‐Glycoforms of Asn³⁶⁷ were slightly more branched and core fucosylated than Asn³⁵¹ N‐glycoforms due probably to a more surface‐exposed glycosylation site. The N‐terminal Thr⁷ was fully occupied by the two O‐linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N‐acetylneuraminic acid and GalNAc is N‐acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn³⁵¹ glycan occupancies and N‐glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.     

Dynamic responses of Bacteroides thetaiotaomicron during growth on glycan mixtures

Bacteroides thetaiotaomicron (Bt) is a human colonic symbiont that degrades many different complex carbohydrates (glycans), the identities and amounts of which are likely to change frequently and abruptly from meal‐to‐meal. To understand how this organism reacts to dynamic growth conditions, we challenged it with a series of different glycan mixtures and measured responses involved in glycan catabolism. Our results demonstrate that individual Bt cells can simultaneously respond to multiple glycans and that responses to new glycans are extremely rapid. The presence of alternative carbohydrates does not alter response kinetics, but reduces expression of some glycan utilization genes as well as the cell's sensitivity to glycans that are present in lower concentration. Growth in a mixture containing 12 different glycans revealed that Bt preferentially uses some before others. This metabolic hierarchy is not changed by prior exposure to lower priority glycans because re‐introducing high priority substrates late in culture re‐initiates repression of genes involved in degrading those with lower priority. At least some carbohydrate prioritization effects occur at the level of monosaccharide recognition. Our results provide insight into how a bacterial glycan generalist modifies its responses in dynamic glycan environments and provide essential knowledge to interpret related metabolic behaviour in vivo.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

A novel endo‐β‐N‐acetylglucosaminidase releases specific N‐glycans depending on different reaction conditions

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N‐glycans in protein functionality. Endo‐β‐N‐acetylglucosaminidase (EndoBI‐1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat‐stable enzyme that cleaves the N‐N′‐diacetyl chitobiose moiety found in the N‐glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI‐1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI‐1 is able to cleave a high diversity of N‐glycan structures. Nano‐LC‐Chip–Q‐TOF MS data also revealed that different reaction conditions resulted in different N‐glycan compositions released, thus modifying the relative abundance of N‐glycan types. In general, more sialylated N‐glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI‐1 is able to release a wide variety of N‐glycans, whose compositions can be selectively manipulated using different processing conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1323–1330, 2015     

Twoplex 12/13C6 aniline stable isotope and linkage‐specific sialic acid labeling 2D‐LC‐MS workflow for quantitative N‐glycomics

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease‐associated glycan alterations to the quantitative characterization of N‐glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI‐TOF‐MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run‐to‐run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC‐MS^E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N‐linked oligosaccharides using neutral ¹²C₆ and ¹³C₆ aniline (Δmass = 6 Da). Additional linkage‐specific derivatization of sialic acids using 4‐(4,6‐dimethoxy‐1,3,5‐trizain‐2‐yl)‐4‐methylmorpholinium chloride offered simultaneous and advanced in‐depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N‐glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex ¹²C₆/¹³C₆ aniline 2D LC‐MS platform is ideally suited for differential glycomic analysis of structurally complex N‐glycan pools due to combination and analysis of samples in a single LC‐MS injection and the associated minimization in technical variation.     

MALDI Mass Spectrometry Imaging of Early‐ and Late‐Stage Serous Ovarian Cancer Tissue Reveals Stage‐Specific N‐Glycans

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N‐glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor‐specific N‐glycan alterations in ovarian cancer development and progression. matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N‐glycan distribution on formalin‐fixed paraffin‐embedded ovarian cancer tissue sections from early‐ and late‐stage patients. Tumor‐specific N‐glycans are identified and structurally characterized by porous graphitized carbon‐liquid chromatography‐electrospray ionization‐tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS), and then assigned to high‐resolution images obtained from MALDI‐MSI. Spatial distribution of 14 N‐glycans is obtained by MALDI‐MSI and 42 N‐glycans (including structural and compositional isomers) identified and structurally characterized by LC‐MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N‐glycan families are localized to the tumor regions of late‐stage ovarian cancer patients relative to early‐stage patients. Potential N‐glycan diagnostic markers that emerge include the oligomannose structure, (Hex)₆ + (Man)₃(GlcNAc)₂, and the complex neutral structure, (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + (Man)₃(GlcNAc)₂. The distribution of these markers is evaluated using a tissue microarray of early‐ and late‐stage patients.     

Stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG)

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of ¹³C₆/¹²C₆‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R² > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (～82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:840–848, 2015     

Epitope detection chromatography: a method to dissect the structural heterogeneity and inter‐connections of plant cell‐wall matrix glycans

Plant cell walls are complex, multi‐macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell‐wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell‐wall matrix glycan sub‐populations and inter‐connections. The method combines chromatographic separations with use of glycan‐directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell‐wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonan I sub‐populations and their modulation in A. thaliana organs.     

Localization of plant N‐glycan processing enzymes along the secretory pathway

Abstract

N‐glycosylation is an abundant covalent protein modification in all eukaryotic cells. The biosynthesis and processing of protein N‐linked glycans results from a series of highly co‐ordinated step‐by‐step enzymatic conversions occurring mainly in the endoplasmic reticulum (ER) and Golgi apparatus. N‐glycan processing enzymes are thought to act on cargo glycoproteins in a highly ordered fashion in an assembly line. Thus, the subcellular localization of these enzymes together with their in vivo substrate specificity determines the carbohydrate structures of glycoproteins transported through the secretory pathway. While the substrate specificities of many plant N‐glycan processing enzymes are fairly well characterized, the molecular mechanisms underlying enzyme localization to the ER and Golgi have remained largely elusive so far. This review discusses current data on ER and Golgi localization of plant N‐glycan processing enzymes.     

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion: Analysis of N‐ and O‐linked glycans and identification and elimination of a xylose‐based O‐linked tetrasaccharide core in the linker region

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion (huLCAT‐Fc) is a chimeric protein produced by fusing human Fc to the C‐terminus of the human enzyme via a linker sequence. The huLCAT‐Fc homodimer contains five N‐linked glycosylation sites per monomer. The heterogeneity and site‐specific distribution of the various glycans were examined using enzymatic digestion and LC‐MS/MS, followed by automatic processing. Almost all of the N‐linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N‐linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N‐linked site exclusively contain typical asialobiantennary structures. HuLCAT‐Fc was also confirmed to have mucin‐type glycans attached at T₄₀₇ and S₄₀₉. When LCAT‐Fc fusions were constructed using a G‐S‐G‐G‐G‐G linker, an unexpected +632 Da xylose‐based glycosaminoglycan (GAG) tetrasaccharide core of Xyl‐Gal‐Gal‐GlcA was attached to S₄₁₈. Several minor intermediate species including Xyl, Xyl‐Gal, Xyl‐Gal‐Gal, and a phosphorylated GAG core were also present. The mucin‐type O‐linked glycans can be effectively released by sialidase and O‐glycanase; however, the GAG could only be removed and localized using chemical alkaline β‐elimination and targeted LC‐MS/MS. E₄₁₆ (the C‐terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O‐xylosyltransferase. HuLCAT‐Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPP E₄₁₆GS₄₁₈G G G GDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.     

Host-soluble galectin-1 promotes HIV-1 replication through a direct interaction with glycans of viral gp120 and host CD4

Sexual transmission of HIV-1 requires virus adsorption to a target cell, typically a CD4(+) T lymphocyte residing in the lamina propria, beneath the epithelium. To escape the mucosal clearance system and reach its target cells, HIV-1 has evolved strategies to circumvent deleterious host factors. Galectin-1, a soluble lectin found in the underlayers of the epithelium, increases HIV-1 infectivity by accelerating its binding to susceptible cells. By comparison, galectin-3, a family member expressed by epithelial cells and part of the mucosal clearance system, does not perform similarly. We show here that galectin-1 directly binds to HIV-1 in a β-galactoside-dependent fashion through recognition of clusters of N-linked glycans on the viral envelope gp120. Unexpectedly, this preferential binding of galectin-1 does not rely on the primary sequence of any particular glycans. Instead, glycan clustering arising from the tertiary structure of gp120 hinders its binding by galectin-3. Increased polyvalency of a specific ligand epitope is a common strategy for glycans to increase their avidity for lectins. In this peculiar occurrence, glycan clustering is instead exploited to prevent binding of gp120 by galectin-3, which would lead to a biological dead-end for the virus. Our data also suggest that galectin-1 binds preferentially to CD4, the host receptor for gp120. Together, these results suggest that HIV-1 exploits galectin-1 to enhance gp120-CD4 interactions, thereby promoting virus attachment and infection events. Since viral adhesion is a rate-limiting step for HIV-1 entry, modulation of the gp120 interaction with galectin-1 could thus represent a novel approach for the prevention of HIV-1 transmission.     

Preparation,characterization and immunogenicity of HIV-1 related high-mannose oligosaccharides-CRM<Subscript>197</Subscript> glycoconjugates

The dense glycan shield on the surface of human immunodeficiency virus type 1 (HIV-1) gp120 masks conserved protein epitopes and facilitates virus entry via interaction to glycan binding proteins on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope is currently being considered for the design of a synthetic vaccine. The cluster nature of the 2G12 epitope suggests that a multivalent antigen presentation is important to develop a carbohydrate-based vaccine candidate. In this work we describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates. We exploited flexible polyamidoamine (PAMAM) scaffold to generate four- and eight-valent sugar clusters of HIV-1-related oligomannose antigens Man₄, Man₆ and Man₉. The multivalent presentation of oligomannoses increased the avidity of Man₄ and Man₉ to 2G12. The synthetic glycodendrons were then covalently coupled to the protein carrier CRM₁₉₇, formulated with the adjuvant MF59, and used to immunize two animal species. Oligomannose-specific IgG antibodies were generated; however, the antisera failed to recognize recombinant HIV-1 gp120 proteins. We conclude that further structural vaccinology work is needed to identify an antigen presentation that closely matches in vivo the structure of the epitope mapped by 2G12.     

Expression-System-Dependent Modulation of HIV-1 Envelope Glycoprotein Antigenicity and Immunogenicity

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly related Env surface (gp120) antigens produced in different systems: (a) mammalian (293 FreeStyle™ cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), which confer mainly paucimannosidic glycans; (c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high-mannose, hybrid and complex glycans without sialic acid; and (d) 293F cells, which impart high-mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4 binding and CD4-induced sites compared to those expressed in the system that do, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic-acid-containing gp120 was significantly more immunogenic than the sialylated version when administered in two different adjuvants, and induced higher titers of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic-acid-imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations that should be considered when selecting expression systems for glycosylated antigens to be used for structure-function studies and for vaccine use.     

A first view on the unsuspected intragenus diversity of N‐glycans in Chlorella microalgae

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health‐promoting food supplements. A mass spectrometry‐based survey of N‐glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N‐glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N‐acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan‐type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4‐O‐methyl‐N‐acetylglucosamine), and substitution of the second N‐acetylglucosamine. Analysis of barcode ITS1–5.8S–ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N‐glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella‐like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N‐glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.