Molecular and Biochemical Parameters Related to Plasma Mannose Levels in Coronary Artery Disease Among Nondiabetic Patients.

Aims: Nondiabetic patients were studied to determine whether modest elevations in plasma mannose may be associated with a greater incidence of coronary artery disease (CAD). Materials and Methods: Plasma insulin, mannose, glucose, hexokinase 1-2, GLUT1-GLUT4 levels, and serum mannose phosphate isomerase enzyme levels were evaluated with respect to subsequent CAD using records from 120 nondiabetic CAD patients and 120 healthy volunteers. CAD was identified from myocardial infarction and new diagnoses of angina. Results: Of 120 nondiabetic CAD patients studied, their plasma GLUT4 and HK1 levels were significantly lower than those of the control group. In addition, a significant increase in plasma mannose levels was found in the patient group compared to the control group. Conclusion: Our findings showed that elevated baseline mannose levels in plasma are associated with an increased risk of CAD over time.

Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies.

Starch/cellulose has become the major feedstock for manufacturing biofuels and biochemicals because of their abundance and sustainability. In this study, we presented an artificially designed "starch-mannose-fermentation" biotransformation process through coupling the advantages of in vivo and in vitro metabolic engineering strategies together. Starch was initially converted into mannose via an in vitro metabolic engineering biosystem, and then mannose was fermented by engineered microorganisms for biomanufacturing valuable mannosyl compounds. The in vitro metabolic engineering biosystem based on phosphorylation/dephosphorylation reactions was thermodynamically favorable and the conversion rate reached 81%. The mannose production using whole-cell biocatalysts reached 75.4 g/L in a 30-L reactor, indicating the potential industrial application. Furthermore, the produced mannose in the reactor was directly served as feedstock for the fermentation process to bottom-up produced 19.2 g/L mannosyl-oligosaccharides (MOS) and 7.2 g/L mannosylglycerate (MG) using recombinant Corynebacterium glutamicum strains. Notably, such a mannose fermentation process facilitated the synthesis of MOS, which has not been achieved under glucose fermentation and improved MG production by 2.6-fold than that using the same C-mole of glucose. This approach also allowed access to produce other kinds of mannosyl derivatives from starch.

Streptomyces acidicola sp. nov., isolated from a peat swamp forest in Thailand.

A novel actinobacterium, designated strain K1PN6T, was isolated from soil sample collected in Kantulee peat swamp forest, Surat Thani province, Thailand. The morphological, chemotaxonomic, and phylogenetic characteristics were consistent with its classification in the genus Streptomyces. Based on 16S rRNA gene sequence analysis, strain K1PN6T showed highest similarity to Streptomyces phyllanthi PA1-07T (98.6 %), Streptomyces spongiae Sp080513SC-24T (98.3%) and Streptomyces adustus WH-9T (98.3%). The G + C content of the genomic DNA was 70.3 mol%. Digital DNA-DNA hybridization and average nucleotide identity values between the genome sequence of strain K1PN6T with S. phyllanthi TISTR 2346T (33.7 and 89.1%), S. spongiae NBRC 106415T (38.6 and 90.6%) and S. adustus NBRC 109810T (26.0 and 86.2%) were below the thresholds of 70 and 95-96% for prokaryotic conspecific assignation. Chemotaxonomic data revealed that strain K1PN6T possessed MK-9(H8) (45%) and MK-9(H6) (34%) as the predominant menaquinones. It contained LL-diaminopimelic acid as the diagnostic diamino acid and galactose, glucose, mannose, and ribose as whole-cell sugars. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, two unidentified aminolipids, an unidentified phospholipid, and glycophospholipid. The predominant cellular fatty acids (>10%) were iso-C16:0, C16:0, anteiso-C15:0, and iso-C14:0. On the basis of these genotypic and phenotypic data, strain K1PN6T should be designated as a representative of a novel species of the genus Streptomyces, for which the name Streptomyces acidicola sp. nov. is proposed with the type strain K1PN6T (=TBRC 11341T=NBRC 114304T).

RMSD analysis of structures of the bacterial protein FimH identifies five conformations of its lectin domain.

FimH is a bacterial adhesin protein located at the tip of Escherichia coli fimbria that functions to adhere bacteria to host cells. Thus, FimH is a critical factor in bacterial infections such as urinary tract infections and is of interest in drug development. It is also involved in vaccine development and as a model for understanding shear-enhanced catch bond cell adhesion. To date, over 60 structures have been deposited in the Protein Data Bank showing interactions between FimH and mannose ligands, potential inhibitors, and other fimbrial proteins. In addition to providing insights about ligand recognition and fimbrial assembly, these structures provide insights into conformational changes in the two domains of FimH that are critical for its function. To gain further insights into these structural changes, we have superposed FimH's mannose binding lectin domain in all these structures and categorized the structures into five groups of lectin domain conformers using RMSD as a metric. Many structures also include the pilin domain, which anchors FimH to the fimbriae and regulates the conformation and function of the lectin domain. For these structures, we have also compared the relative orientations of the two domains. These structural analyses enhance our understanding of the conformational changes associated with FimH ligand binding and domain-domain interactions, including its catch bond behavior through allosteric action of force in bacterial adhesion.

C-mannosylation supports folding and enhances stability of thrombospondin repeats.

Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.

Mannose glycosylation is an integral step for NIS localization and function in human breast cancer cells.

Chasing an intriguing biological question on the disparity of sodium iodide symporter (NIS, officially known as SLC5A5) expression and function in the clinical scenario of breast cancer, this study addresses key molecular defects involved. NIS in cancer patients has primarily been recorded to be a cytoplasmic protein, thus limiting the scope for targeted radio-iodine therapy. We developed NIS transgene-overexpressing MCF-7 breast cancer cells, and found a few clonal derivatives that show predominant expression of NIS in the plasma membrane. The majority of clones, however, showed cytosolic NIS expression over long passages. Cells expressing membranous NIS show unperturbed dynamic trafficking of NIS through secretory pathway organelles when compared to cells expressing cytoplasmic NIS or to parental cells. Further, treatment of cells expressing membranous NIS with specific glycosylation inhibitors highlighted the importance of inherent glycosylation processing and an 84 gene signature glycosylation RT-Profiler array revealed that clones expressing NIS in their membrane cluster separately compared to the other cells. We further confirm a role of three differentially expressed genes, i.e. MAN1B1, MAN1A1 and MAN2A1, in regulating NIS localization by RNA interference. Thus, this study shows the important role of mannosidase in N-glycosylation processing in order to correctly traffic NIS to the plasma membrane in breast cancer cells.This article has an associated First Person interview with the first author of the paper.

Glycoengineering of HEK293 cells to produce high-mannose-type N-glycan structures.

Therapeutic proteins are a developing part of the modern biopharmaceutical industry, providing novel therapies to intractable diseases including cancers and autoimmune diseases. The human embryonic kidney 293 (HEK293) cell line has been widely used to produce recombinant proteins in both basic science and industry. The heterogeneity of glycan structures is one of the most challenging issues in the production of therapeutic proteins. Previously, we knocked out genes encoding α1,2-mannosidase-Is, MAN1A1, MAN1A2 and MAN1B1, in HEK293 cells, establishing a triple-knockout (T-KO) cell line, which produced recombinant protein with mainly high-mannose-type N-glycans. Here, we further knocked out MAN1C1 and MGAT1 encoding another Golgi α1,2-mannosidase-I and N-acetylglucosaminyltransferase-I, respectively, based on the T-KO cells. Two recombinant proteins, lysosomal acid lipase (LIPA) and immunoglobulin G1 (IgG1), were expressed in the quadruple-KO (QD-KO) and quintuple-KO (QT-KO) cell lines. Glycan structural analysis revealed that all the hybrid-type and complex-type N-glycans were eliminated, and only the high-mannose-type N-glycans were detected among the recombinant proteins prepared from the QD-KO and QT-KO cells. Overexpression of the oncogenes MYC and MYCN recovered the slow growth in QD-KO and QT-KO without changing the glycan structures. Our results suggest that these cell lines could be suitable platforms to produce homogeneous therapeutic proteins.

Glycosylation Profile of the Transferrin Receptor in Gestational Iron Deficiency and Early-Onset Severe Preeclampsia.

OBJECTIVE: To examine the expression of hypoxia-inducible factor-1α (HIF-1α), TfR1, and TfR1-attached terminal monosaccharides in placentas of women with IDAP and severe preeclampsia. METHODS: TfR1 and HIF-1α were detected by western blot. Immunoadsorption of TfR1 was performed to characterize the terminal monosaccharides by specific lectin binding. RESULTS: There was no difference in the expression of TfR1 and HIF-1α between groups. Lectin blot analysis pointed out an overexpression of galactose ß1-4 N-acetylglucosamine (Gal-GlcNAc) and mannose in severe preeclampsia. CONCLUSION: The increase in Gal-GlcNAc may be due to the increased presence of antennary structures and the mannose glycans of TfR1 may indicate the presence of misfolded or incomplete proteins. These findings may be associated with the low expression of placental TfR1 in women with preeclampsia.

Generation of site-distinct N-glycan variants for in vitro bioactivity testing.

Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed-batch cell culture batches yielded consistent glycoprofiles of a Fc-fusion antibody comprizing three different N-glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N-glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site-specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell-based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N-glycan structures, a higher degree of antennarity, and a site-specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc-site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N-glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

Structural characterization of the Man5 glycoform of human IgG3 Fc.

Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8Šresolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.

Nucleolin is a nuclear target of heparan sulfate derived from glypican-1.

The recycling, S-nitrosylated heparan sulfate (HS) proteoglycan glypican-1 releases anhydromannose (anMan)-containing HS chains by a nitrosothiol-catalyzed cleavage in endosomes that can be constitutive or induced by ascorbate. The HS-anMan chains are then transported to the nucleus. A specific nuclear target for HS-anMan has not been identified. We have monitored endosome-to-nucleus trafficking of HS-anMan by deconvolution and confocal immunofluorescence microscopy using an anMan-specific monoclonal antibody in non-growing, ascorbate-treated, and growing, untreated, wild-type mouse embryonic fibroblasts and hypoxia-exposed Alzheimer mouse Tg2576 fibroblasts and human U87 glioblastoma cells. In all cells, nuclear HS-anMan targeted a limited number of sites of variable size where it colocalized with DNA and nucleolin, an established marker for nucleoli. HS-anMan also colocalized with ethynyl uridine-tagged nascent RNA and two acetylated forms of histone H3. Acute hypoxia increased the formation of HS-anMan in both Tg2576 and U87 cells. A portion of HS-anMan colocalized with nucleolin at small discrete sites, while most of the nucleolin and nascent RNA was dispersed. In U87 cells, HS-anMan, nucleolin and nascent RNA reassembled after prolonged hypoxia. Nucleolar HS may modulate synthesis and/or release of rRNA.

Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes.

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.

Understanding and Controlling Sialylation in a CHO Fc-Fusion Process.

A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability.

A mouse model of a human congenital disorder of glycosylation caused by loss of PMM2.

The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.

Engineering the Pichia pastoris N-Glycosylation Pathway Using the GlycoSwitch Technology.

Pichia pastoris is an important host for recombinant protein production. As a protein production platform, further development for therapeutic glycoproteins has been hindered by the high-mannose-type N-glycosylation common to yeast and fungi. Such N-glycans can complicate downstream processing, might be immunogenic or cause the rapid clearance of the glycoprotein from circulation. In recent years, much effort has gone to engineering the N-glycosylation pathway of Pichia pastoris to mimic the human N-glycosylation pathway. This can be of pivotal importance to generate the appropriate glycoforms of therapeutically relevant glycoproteins or to gain a better understanding of structure-function relationships.This chapter describes the methodology to create such glyco-engineered Pichia pastoris strains using the GlycoSwitch(®). This strategy consists of the disruption of an endogenous glycosyltransferase and the heterologous expression of a glycosidase or glycosyltransferase targeted to the Endoplasmic Reticulum or the Golgi of the host. For each step in the process, we describe the transformation procedure, small-scale screening and we also describe how to perform DNA-Sequencer-Aided Fluorophore-Assisted Capillary Electrophoresis (DSA-FACE) to select for clones with the appropriate N-glycosylation profile. The steps described in this chapter can be followed in an iterative fashion in order to generate clones of Pichia pastoris expressing heterologous proteins with humanized N-glycans.

Molecular characterization of acidic peptide:N-glycanase from the dimorphic yeast Yarrowia lipolytica.

Peptide:N-glycanase (PNGase) A is used preferentially to cleave the glycans from plant and insect glycopeptides. Although many putative PNGase A homologous genes have been found in the plant and fungus kingdoms through sequence similarity analyses, only several PNGases from plants and one from a filamentous fungus have been characterized. In this study, we identified and characterized a PNGase A-like enzyme, PNGase Yl, in the dimorphic yeast Yarrowia lipolytica. The corresponding gene was cloned and recombinantly expressed in Pichia pastoris. The purified enzyme cleaved glycans from glycopeptides with the maximum activity at pH 5. No metal ions were required for full activity, and rather it was repressed by three metal ions (Fe(3+), Cu(2+) and Zn(2+)). Using glycopeptide substrates, PNGase Yl was shown to release various types of N-glycans including high-mannose and complex-type glycans as well as glycans containing core-linked α(1,3)-fucose that are frequently found in plants and insects. Moreover, in comparison with PNGase A, PNGase Yl was able to cleave with higher efficiency the glycans from some denatured glycoproteins. Taken together, our results suggest that PNGase Yl, the first biochemically characterized yeast PNGase A homologue, can be developed through protein engineering as a useful deglycosylation tool for N-glycosylation study.

Mannosylglycerate: structural analysis of biosynthesis and evolutionary history.

Halophilic and halotolerant microorganisms adapted to thrive in hot environments accumulate compatible solutes that usually have a negative charge either associated with a carboxylic group or a phosphodiester unit. Mannosylglycerate (MG) has been detected in several members of (hyper)thermophilic bacteria and archaea, in which it responds primarily to osmotic stress. The outstanding ability of MG to stabilize protein structure in vitro as well as in vivo has been convincingly demonstrated. These findings led to an increasingly supported link between MG and microbial adaptation to high temperature. However, the accumulation of MG in many red algae has been known for a long time, and the peculiar distribution of MG in such distant lineages was intriguing. Knowledge on the biosynthetic machinery together with the rapid expansion of genome databases allowed for structural and phylogenetic analyses and provided insight into the distribution of MG. The two pathways for MG synthesis have distinct evolutionary histories and physiological roles: in red algae MG is synthesised exclusively via the single-step pathway and most probably is unrelated with stress protection. In contrast, the two-step pathway is strongly associated with osmoadaptation in (hyper)thermophilic prokaryotes. The phylogenetic analysis of the two-step pathway also reveals a second cluster composed of fungi and mesophilic bacteria, but MG has not been demonstrated in members of this cluster; we propose that the synthase is part of a more complex pathway directed at the synthesis of yet unknown molecules containing the mannosyl-glyceryl unit.

Recent advances in the understanding of biological implications and modulation methodologies of monoclonal antibody N-linked high mannose glycans.

With the prevalence of therapeutic monoclonal antibodies (mAbs) in the biopharmaceutical industry, the use of mammalian cell culture systems, particularly Chinese hamster ovary (CHO) cells, has become the main method for the production of therapeutics. Despite their similarity to human cells, one major challenge of mammalian cell based biopharmaceutical production is controlling aberrant glycosylation, especially glycans with five to nine mannose residues-high mannose glycoforms. Glycosylation plays a critical role in determining the therapeutic profile of therapeutic glycoproteins; high mannose glycoforms in particular have been shown to have a significant impact on clinical efficacy and pharmacokinetics. Thus, producing glycoform profiles with consistent levels of high mannose is necessary to reduce batch-to-batch therapeutic variability and to meet regulatory standards. Studies have shown that high mannose glycoforms can be modulated through the genetic engineering of cell lines, addition of inhibitors to key enzymes in the glycosylation pathways, and varying cell culture conditions. Focusing on these three types of techniques, this review will examine and critically assess current methods for high mannose glycosylation control and future developments in this area.

HIV-1 envelope proteins and V1/V2 domain scaffolds with mannose-5 to improve the magnitude and quality of protective antibody responses to HIV-1.

Two lines of investigation have highlighted the importance of antibodies to the V1/V2 domain of gp120 in providing protection from HIV-1 infection. First, the recent RV144 HIV-1 vaccine trial documented a correlation between non-neutralizing antibodies to the V2 domain and protection. Second, multiple broadly neutralizing monoclonal antibodies to the V1/V2 domain (e.g. PG9) have been isolated from rare infected individuals, termed elite neutralizers. Interestingly, the binding of both types of antibodies appears to depend on the same cluster of amino acids (positions 167­171) adjacent to the junction of the B and C strands of the four-stranded V1/V2 domain ß-sheet structure. However, the broadly neutralizing mAb, PG9, additionally depends on mannose-5 glycans at positions 156 and 160 for binding. Because the gp120 vaccine immunogens used in previous HIV-1 vaccine trials were enriched for complex sialic acid-containing glycans, and lacked the high mannose structures required for the binding of PG9-like mAbs, we wondered if these immunogens could be improved by limiting glycosylation to mannose-5 glycans. Here, we describe the PG9 binding activity of monomeric gp120s from multiple strains of HIV-1 produced with mannose-5 glycans. We also describe the properties of glycopeptide scaffolds from the V1/V2 domain also expressed with mannose-5 glycans. The V1/V2 scaffold from the A244 isolate was able to bind the PG9, CH01, and CH03 mAbs with high affinity provided that the proper glycans were present. We further show that immunization with A244 V1/V2 fragments alone, or in a prime/boost regimen with gp120, enhanced the antibody response to sequences in the V1/V2 domain associated with protection in the RV144 trial.

Accumulation of free oligosaccharides and tissue damage in cytosolic α-mannosidase (Man2c1)-deficient mice.

Free Man(7-9)GlcNAc2 is released during the biosynthesis pathway of N-linked glycans or from misfolded glycoproteins during the endoplasmic reticulum-associated degradation process and are reduced to Man5GlcNAc in the cytosol. In this form, free oligosaccharides can be transferred into the lysosomes to be degraded completely. α-Mannosidase (MAN2C1) is the enzyme responsible for the partial demannosylation occurring in the cytosol. It has been demonstrated that the inhibition of MAN2C1 expression induces accumulation of Man(8-9)GlcNAc oligosaccharides and apoptosis in vitro. We investigated the consequences caused by the lack of cytosolic α-mannosidase activity in vivo by the generation of Man2c1-deficient mice. Increased amounts of Man(8-9)GlcNAc oligosaccharides were recognized in all analyzed KO tissues. Histological analysis of the CNS revealed neuronal and glial degeneration with formation of multiple vacuoles in deep neocortical layers and major telencephalic white matter tracts. Enterocytes of the small intestine accumulate mannose-containing saccharides and glycogen particles in their apical cytoplasm as well as large clear vacuoles in retronuclear position. Liver tissue is characterized by groups of hepatocytes with increased content of mannosyl compounds and glycogen, some of them undergoing degeneration by hydropic swelling. In addition, lectin screening showed the presence of mannose-containing saccharides in the epithelium of proximal kidney tubules, whereas scattered glomeruli appeared collapsed or featured signs of fibrosis along Bowman's capsule. Except for a moderate enrichment of mannosyl compounds and glycogen, heterozygous mice were normal, arguing against possible toxic effects of truncated Man2c1. These findings confirm the key role played by Man2c1 in the catabolism of free oligosaccharides.