Oligomannose Glycans Research Articles

A single glycan on IgE is indispensable for initiation of anaphylaxis.

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell-bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology.

Improving the large scale purification of the HIV microbicide, griffithsin

BackgroundGriffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery.ResultsWe achieved >99% pure griffithsin API by generating the initial green juice extract in pH 4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl2 mixture, and final purification with Capto™ multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained.ConclusionsThe methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of griffithsin. The methodology can be readily scaled to the bench top or industry and process components can be used for purification of additional proteins based on biophysical characteristics.

High Temperature SELMA: Evolution of DNA-Supported Oligomannose Clusters Which Are Tightly Recognized by HIV bnAb 2G12

SELMA (SELection with Modified Aptamers) is a directed evolution method which can be used to develop DNA-supported clusters of carbohydrates in which the geometry of clustering is optimized for strong recognition by a lectin of interest. Herein, we report a modification of SELMA which results in glycoclusters which achieve dramatically stronger target recognition (100-fold) with dramatically fewer glycans (2–3-fold). Our first applications of SELMA yielded clusters of 5–10 oligomannose glycans which were recognized by broadly neutralizing HIV antibody 2G12 with moderate affinities (150–500 nM Kd’s). In the present manuscript, we report glycoclusters containing just 3–4 glycans, which are recognized by 2G12 with Kd’s as low as 1.7 nM. These glycoclusters are recognized by 2G12 as tightly as is the HIV envelope protein gp120, and they are the first constructs to achieve this tight recognition with the minimal number of Man9units (3–4) necessary to occupy the binding sites on 2G12. They are thus of great interest as immunogens which might elicit broadly neutralizing antibodies against HIV.

Hypo-glycosylated human follicle-stimulating hormone (hFSH21/18) is much more active in vitro than fully-glycosylated hFSH (hFSH24)

Hypo-glycosylated hFSH21/18 (possesses FSHβ21 and FSHβ18bands) was isolated from hLH preparations by immunoaffinity chromatography followed by gel filtration. Fully-glycosylated hFSH24 was prepared by combining the fully-glycosylated FSHβ24 variant with hCGα and isolating the heterodimer. The hFSH21/18 glycoform preparation was significantly smaller than the hFSH24 preparation and possessed 60% oligomannose glycans, which is unusual for hFSH. Hypo-glycosylated hFSH21/18 was 9- to 26-fold more active than fully-glycosylated hFSH24 in FSH radioligand assays. Significantly greater binding of 125I-hFSH21/18 tracer than hFSH24 tracer was observed in all competitive binding assays. In addition, higher binding of hFSH21/18 was noted in association and saturation binding assays, in which twice as much hFSH21/18 was bound as hFSH24. This suggests that more ligand binding sites are available to hFSH21/18 in FSHR than to hFSH24. Hypo-glycosylated hFSH21/18 also bound rat FSHRs more rapidly, exhibiting almost no lag in binding, whereas hFSH24 specific binding proceeded very slowly for almost the first hour of incubation.

N-linked glycosylation of cortical N-methyl-D-aspartate and kainate receptor subunits in schizophrenia.

Dysfunctional glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia. Abnormal expressions in schizophrenia of ionotropic glutamate receptors (iGluRs) and the proteins that regulate their trafficking have been found to be region and subunit specific in brain, suggesting that abnormal trafficking of iGluRs may contribute toward altered glutamatergic neurotransmission. The post-translational modification N-glycosylation of iGluR subunits can be used as a proxy for their intracellular localization. Receptor complexes assemble in the lumen of the endoplasmic reticulum, where N-glycosylation begins with the addition of N-linked oligomannose glycans, and is subsequently trimmed and replaced by more elaborate glycans while trafficking through the Golgi apparatus. Previously, we found abnormalities in N-glycosylation of the GluR2 AMPA receptor subunit in schizophrenia. Here, we investigated N-glycosylation of N-methyl-D-aspartate and kainate (KA) receptor subunits in the dorsolateral prefrontal cortex from patients with schizophrenia and a comparison group. We used enzymatic deglycosylation with two glycosidases: endoglycosidase H (Endo H), which removes immature high mannose-containing sugars, and peptide-N-glycosidase F (PNGase F), which removes all N-linked sugars. The NR1, NR2A, NR2B, GluR6, and KA2 subunits were all sensitive to treatment with Endo H and PNGase F. The GluR6 KA receptor subunit was significantly more sensitive to Endo H-mediated deglycosylation in schizophrenia, suggesting a larger molecular mass of N-linked high mannose and/or hybrid sugars on GluR6. This finding, taken with our previous work, suggests that a cellular mechanism underlying abnormal glutamate neurotransmission in schizophrenia may involve abnormal trafficking of both AMPA and KA receptors.

Deficiency of α-glucosidase I alters glycoprotein glycosylation and lifespan in Caenorhabditis elegans

Endoplasmic reticulum (ER) α-glucosidase I is an enzyme that trims the distal α1,2-linked glucose (Glc) residue from the Glc3Man9GlcNAc2 oligosaccharide following its addition to nascent glycoproteins in the initial step of processing. This reaction is critical to the subsequent processing of N-glycans and thus defects in α-glucosidase I gene in human cause congenital disorder of glycosylation (CDG) type IIb. We identified the Caenorhabditis elegans α-glucosidase I gene (F13H10.4, designated agl-1) that encodes a polypeptide with 36% identity to human α-glucosidase I. The agl-1 cDNA restored the expression of complex-type N-glycans on the cell surface of α-glucosidase I-defective Chinese hamster ovary Lec23 cells. RNAi knockdown of agl-1 [agl-1(RNAi)] produced worms that were visibly similar to wild-type, but lifespan was reduced to about half of the control. Analyses of N-glycosylation in agl-1(RNAi) animals by western blotting and mass spectrometry showed reduction of paucimannose and complex-type glycans and dramatic increase of glucosylated oligomannose glycans. In addition, a significant amount of unusual terminally fucosylated N-glycans were found in agl-1(RNAi) animals. ER stress response was also provoked, leading to the accumulation of large amounts of triglucosylated free oligosaccharides (FOSs) (Glc3Man4-5GlcNAc1-2) in agl-1(RNAi) animals. Acceleration of ER-associated degradation in response to the accumulation of unfolded glycoproteins and insufficient interaction with calnexin/calreticulin in the ER lumen likely accounts for the increase of FOSs. Taken together, these studies in C. elegans demonstrate that decreased ER α-glucosidase I affects the entire N-glycan profile and induces chronic ER stress, which may contribute to the pathophysiology of CDG-IIb in humans.

Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml−1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.

Hypo‐glycosylated hFSH binds and activates FSH receptors more rapidly than fully‐glycosylated hFSH

Follitropin (FSH) heterodimers possess two potential N‐glycosylation sites on both common α and hormone‐specific β subunits. Hypo‐glycosylated hFSH isolated from lutropin preparations possessed a partially glycosylated FSHβ subunit decorated with one glycan, while there were two in the hFSHα subunit. Mass spectrometry revealed the presence of oligomannose glycans, which are normally rare in hFSH preparations. Hypo‐glycosylated hFSH was 5‐ to 26‐fold more active than fully‐glycosylated hFSH in several FSH radioligand assays. In rat testis homogenate, 125I‐hypo‐hFSH bound receptors immediately, without the 45–60 min lag observed for 125I‐fully‐glycosylated hFSH. In porcine granulosa cells, short‐term incubations revealed an earlier onset of cAMP accumulation, protein kinase A (PKA) activity and CREB phosphorylation. Hypo‐glycosylated hFSH increased intracellular cAMP accumulation within one min and PKA‐dependent CREB phosphorylation within 5 min; whereas fully‐glycosylated hFSH induced CREB phosphorylation after a 10–15 min lag. Additionally, CREB phosphorylation was sustained for greater than 60 min in response to hypo‐glycosylated hFSH, but returned to basal within 60 min in response to fully‐glycosylated hFSH. Thus, partial glycosylation of hFSH results in faster binding to its cognate receptor and this is attended by a more rapid cellular response. Supported by NIH grant 5P01 AG‐029531.

Human IL-34 and CSF-1 Establish Structurally Similar Extracellular Assemblies with Their Common Hematopoietic Receptor

The discovery that hematopoietic human colony stimulating factor-1 receptor (CSF-1R) can be activated by two distinct cognate cytokines, colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34), created puzzling scenarios for the two possible signaling complexes. We here employ a hybrid structural approach based on small-angle X-ray scattering (SAXS) and negative-stain EM to reveal that bivalent binding of human IL-34 to CSF-1R leads to an extracellular assembly hallmarked by striking similarities to the CSF-1:CSF-1R complex, including homotypic receptor-receptor interactions. Thus, IL-34 and CSF-1 have evolved to exploit the geometric requirements of CSF-1R activation. Our models include N-linked oligomannose glycans derived froma systematic approach resulting in the accurate fitting of glycosylated models to the SAXS data. We further show that the C-terminal region of IL-34 is heavily glycosylated and that it can be proteolytically cleaved from the IL-34:hCSF-1R complex, providing insights into its role in the functional nonredundancy of IL-34 and CSF-1.

Glycomodification and characterization of anti-colorectal cancer immunotherapeutic monoclonal antibodies in transgenic tobacco

Anti-colorectal cancer mAb CO17-1A (IgG2a) recognizes the antigen GA733, which is highly expressed on the surface membrane of human colorectal carcinoma cells. In this study, a transgenic tobacco system for the production of mAb CO17-1A was developed. The mAb construct included a KDEL sequence, an endoplasmic reticulum (ER) retention signal attached to the C-terminus of the heavy chain, to target accumulation of mAb into ER. An immunoblot showed significantly enhanced levels of expression of the plant-derived mAbK (mAbPK) CO17-1A compared to mAbP CO17-1A mAb without the KDEL sequence. An ELISA assay using human colorectal carcinoma cells confirmed that expression of mAbPK was also significantly higher than that of mAbP. Glycosylation analysis revealed that mAbP had plant-specific glycans; whereas, mAbPK primarily had oligomannose glycans. FACS showed that the Fc domains of both mAbPK and mammalian-derived mAb (mAbM) had similar binding activity to the FcγRI receptor (CD64). However, the Fc domains of the mAbP had slightly lower binding activity to the FcγRI receptor than both mAbPK and mAbM. The antibody-dependent cell cytotoxicity of mAbPK, against human colorectal cancer cells, was as efficient as mAbM; whereas mAbP was very low. These results suggest that KDEL localized and accumulated mAbP in the ER and eventually enhanced the expression of mAbP with oligomannose glycan and similar anti-cancer biological activity to the parental mAbM.

Structural characterisation of theN-glycan moiety of the barnacle settlement-inducing protein complex (SIPC)

Many barnacle species are gregarious and their cypris larvae display a remarkable ability to explore surfaces before committing to permanent attachment. The chemical cue to gregarious settlement behaviour - the settlement-inducing protein complex (SIPC) - is an α(2)-macroglobulin-like glycoprotein. This cuticular protein may also be involved in cyprid reversible adhesion if its presence is confirmed in footprints of adhesive deposited during exploratory behaviour, which increase the attractiveness of surfaces and signal other cyprids to settle. The full-length open-reading frame of the SIPC gene encodes a protein of 1547 amino acids with seven potential N-glycosylation sites. In this study on Balanus amphitrite, glycan profiling of the SIPC via hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-fluorescence) provided evidence of predominantly high mannose glycans (M2-9), with the occurrence of monofucosylated oligomannose glycans (F(6)M2-4) in lower proportions. The high mannose glycosylation found supports previous observations of an interaction with mannose-binding lectins and exogenous mannose increasing settlement in B. amphitrite cypris larvae. Transmission electron microscopy of the deglycosylated SIPC revealed a multi-lobed globular protein with a diameter of ~8 nm. Obtaining a complete structural characterisation of the SIPC remains a goal that has the potential to inspire solutions to the age-old problem of barnacle fouling.

Engineering novel Lec1 glycosylation mutants in CHO–DUKX cells: Molecular insights and effector modulation of N‐acetylglucosaminyltransferase I

Many secreted or cell surface proteins are post-translationally modified by carbohydrate chains which are a primary source of heterogeneity. The Lec1 mutant, which is defective in Golgi N-acetylglucosaminyltransferase I (GnTI) activity, produces relatively homogeneous Man(5) GlcNAc(2) glycan modifications, and is widely used for various applications. To facilitate the investigation of GnTI, its Man5 glycan endproduct, and the impact of Man5 on effector function, the present study has established several novel Lec1 mutants in dhfr(-) CHO-DUKX cells through chemical mutagenesis and lectin selection. A total of nine clonal lines exhibiting the Lec1-phenotype are characterized, six of which harbor non-sense mutations leading to a truncated GnTI, and three (R415K, D291N, and P138L) of which are novel loss-of-function sense mutations. Analysis of the rabbit GnTI structure (Unligil et al., 2000) indicates that D291 is the proposed catalytic base and R415 is a crucial residue in forming the substrate binding pocket, whereas P138 is key to maintaining two β strands in proximity to the substrate binding pocket. Computational modeling reveals that the oligomannose glycan backbone of a glycoprotein (the acceptor substrate) fits nicely into the unoccupied channel of the substrate binding pocket partly through hydrogen bonding with R415 and D291. This finding is consistent with the ordered sequential Bi Bi kinetic mechanism suggested for GnTI, in which binding of UDP-GlcNAc (the donor substrate)/Mn(2+) induces conformational changes that promote acceptor binding. When an anti-human CD20 antibody protein is stably expressed in one CHO-DUKX-Lec1 line, it is confirmed that N-glycans are predominantly Man(5) GlcNAc(2) and they do not contain an α1,6-fucose linked to the innermost GlcNAc. Furthermore, this Man(5) GlcNAc(2) modified antibody exhibits a significantly increased ADCC activity than the wild-type protein, while displaying a lower CDC activity. The data support the hypothesis that modulating GnTI activity can influence antibody effector functions for proteins with an IgG1 immunoglobulin Fc domain.

Expression of GA733-Fc Fusion Protein as a Vaccine Candidate for Colorectal Cancer in Transgenic Plants

The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K), GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), and GA733-Fc fused to the ER retention sequence (GA733-FcK). Agrobacterium-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins.

Expression of Anti-breast Cancer Monoclonal Antibody in Transgenic Plant

BACKGROUND: Plant expression system for mass production of recombinant proteins has several advantages over other existing expression systems with economical and safety issues. Breast cancer is a cancer originating from breast tissue and comprises almost 25% of all cancers in women world widely. Lewis-Y antigen is difucosylated oligosaccharide and is carried by glycoconjugates at cancer cell surface. In this study, the anti-breast cancer mAb BR55, which recognizes the epitope Lewis-Y, was expressed in the plant expression system. METHODS AND RESULTS: We have developed plant system for production of mAb BR55 with or without KDEL (the ER retention signal). This ER retention signal was attached to C-terminus of protein to help retain the recombinant glycoprotein carrying oligomannose glycans and enhance glycoprotein accumulation. PCR analysis was performed and confirmed the presence of recombinant genes. Western blot validated that the recombinant proteins mAb BR55 with or without KDEL were expressed in transgenic plants, moreover, the expression level of the mAb BR55 with KDEL was higher compared to the mAb BR55 without KDEL. CONCLUSION: These results indicate that KDEL fusion is a good way to produce proteins and plant can be an ideal expression system to obtain proteins and enhance accumulation of proteins.

The Glycan Shield of HIV Is Predominantly Oligomannose Independently of Production System or Viral Clade

The N-linked oligomannose glycans of HIV gp120 are a target for both microbicide and vaccine design. The extent of cross-clade conservation of HIV oligomannose glycans is therefore a critical consideration for the development of HIV prophylaxes. We measured the oligomannose content of virion-associated gp120 from primary virus from PBMCs for a range of viral isolates and showed cross-clade elevation (62–79%) of these glycans relative to recombinant, monomeric gp120 (∼30%). We also confirmed that pseudoviral production systems can give rise to notably elevated gp120 oligomannose levels (∼98%), compared to gp120 derived from a single-plasmid viral system using the HIVLAI backbone (56%). This study highlights differences in glycosylation between virion-associated and recombinant gp120.

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the "core" trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewis(x), Lewis(y), Lewis(a) and Lewis(b) epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.

Human Plasma Glycome in Attention-Deficit Hyperactivity Disorder and Autism Spectrum Disorders

Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.

Lack of complex N-glycans on HIV-1 envelope glycoproteins preserves protein conformation and entry function

The HIV-1 envelope glycoprotein complex (Env) is the focus of vaccine development aimed at eliciting humoral immunity. Env's extensive and heterogeneous N-linked glycosylation affects folding, binding to lectin receptors, antigenicity and immunogenicity. We characterized recombinant Env proteins and virus particles produced in mammalian cells that lack N-acetylglucosaminyltransferase I (GnTI), an enzyme necessary for the conversion of oligomannose N-glycans to complex N-glycans. Carbohydrate analyses revealed that trimeric Env produced in GnTI(-/-) cells contained exclusively oligomannose N-glycans, with incompletely trimmed oligomannose glycans predominating. The folding and conformation of Env proteins was little affected by the manipulation of the glycosylation. Viruses produced in GnTI(-/-) cells were infectious, indicating that the conversion to complex glycans is not necessary for Env entry function, although virus binding to the C-type lectin DC-SIGN was enhanced. Manipulating Env's N-glycosylation may be useful for structural and functional studies and for vaccine design.

Polysaccharide mimicry of the epitope of the broadly neutralizing anti-HIV antibody, 2G12, induces enhanced antibody responses to self oligomannose glycans

Immunologically, "self" carbohydrates protect the HIV-1 surface glycoprotein, gp120, from antibody recognition. However, one broadly neutralizing antibody, 2G12, neutralizes primary viral isolates by direct recognition of Manalpha1-->2Man motifs formed by the host-derived oligomannose glycans of the viral envelope. Immunogens, capable of eliciting antibodies of similar specificity to 2G12, are therefore candidates for HIV/AIDS vaccine development. In this context, it is known that the yeast mannan polysaccharides exhibit significant antigenic mimicry with the glycans of HIV-1. Here, we report that modulation of yeast polysaccharide biosynthesis directly controls the molecular specificity of cross-reactive antibodies to self oligomannose glycans. Saccharomyces cerevisiae mannans are typically terminated by alpha1-->3-linked mannoses that cap a Manalpha1-->2Man motif that otherwise closely resembles the part of the oligomannose epitope recognized by 2G12. Immunization with S. cerevisiae deficient for the alpha1-->3 mannosyltransferase gene (DeltaMnn1), but not with wild-type S. cerevisiae, reproducibly elicited antibodies to the self oligomannose glycans. Carbohydrate microarray analysis of DeltaMnn1 immune sera revealed fine carbohydrate specificity to Manalpha1-->2Man units, closely matching that of 2G12. These specificities were further corroborated by enzyme-linked immunosorbent assay with chemically defined glycoforms of gp120. These antibodies exhibited remarkable similarity in the carbohydrate specificity to 2G12 and displayed statistically significant, albeit extremely weak, neutralization of HIV-1 compared to control immune sera. These data confirm the Manalpha1-->2Man motif as the primary carbohydrate neutralization determinant of HIV-1 and show that the genetic modulation of microbial polysaccharides is a route towards immunogens capable of eliciting antibody responses to the glycans of HIV-1.

Cryo-electron tomography of homophilic adhesion mediated by the neural cell adhesion molecule L1.

The neural cell adhesion molecule L1 participates in homophilic interactions important for axon guidance and neuronal development. The structural details of homophilic adhesion mediated by L1 and other immunoglobulin superfamily members containing an N-terminal horseshoe arrangement of four immunoglobulin-like domains are unknown. Here we used cryo-electron tomography to study liposomes to which intact or truncated forms of the L1 ectodomain were attached. Tomographic reconstructions revealed an adhesion interface with a regular and repeating pattern consistent with interactions between paired horseshoes contributed by L1 proteins from neighboring liposomes. The characteristics of the pattern changed when N-linked carbohydrates were altered by removing sialic acids or converting from complex to high mannose or oligomannose glycans, suggesting a regulatory role for carbohydrates in L1-mediated homophilic adhesion. Using the results from tomograms and crystal structures of L1-related molecules, we present a structural model for L1-mediated homophilic adhesion that depends on protein-protein, protein-carbohydrate, and carbohydrate-carbohydrate interactions.