The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans' capsular polysaccharide and its shed exopolysaccharide function both as key virulence factors and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this study, NOE and J-coupling values from NMR experiments were consistent with a converged structure of the synthetic decasaccharide, GXM10-Ac3, calculated from MD simulations. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is a hexasaccharide with a three-residue α-mannan backbone, modified by ß-(1→2)-xyloses (Xyl) on the first two mannoses (Man) and a ß-(1→2)-glucuronic acid (GlcA) on the third Man. Combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with Xyl/GlcA branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This derived GXM model displays high flexibility while maintaining a structural identity, yielding insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

Glycosylation States on Intact Proteins Determined by NMR Spectroscopy.

Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.

Defining HIV-1 Envelope N-Glycan Microdomains through Site-Specific Heterogeneity Profiles.

The HIV-1 envelope (Env) glycans shield the surface of Env from the immune system and form integral interactions important for a functional Env. To understand how individual N-glycosylation sites (NGS) coordinate to form a dynamic shield and evade the immune system through mutations, we tracked 20 NGS in Env from HIV-transmitted/founder (T/F) and immune escape variants and their mutants involving the N262 glycan. NGS were profiled in a site-specific manner using a high-resolution mass spectrometry (MS)-based workflow. Using this site-specific quantitative heterogeneity profiling, we empirically characterized the interdependent NGS of a microdomain in the high-mannose patch (HMP). The changes (shifts) in NGS heterogeneity between the T/F and immune escape variants defined a range of NGS that we further probed for exclusive combinations of sequons in the HMP microdomain using the Los Alamos National Laboratory HIV sequence database. The resultant sequon combinations, including the highly conserved NGS N262, N448, and N301, created an immune escape map of the conserved and variable sequons in the HMP microdomain. This report provides details on how some clustered NGS form microdomains that can be identified and tracked across Env variants. These microdomains have a limited number of N-glycan-sequon combinations that may allow the anticipation of immune escape variants.IMPORTANCE The Env protein of HIV is highly glycosylated, and the sites of glycosylation can change as the virus mutates during immune evasion. Due to these changes, the glycan location and heterogeneity of surrounding N-glycosylation sites can be altered, resulting in exposure of different glycan or proteoglycan surfaces while still producing a viable HIV variant. These changes present a need for vaccine developers to identify Env variants with epitopes most likely to induce durable protective responses. Here we describe a means of anticipating HIV-1 immune evasion by dividing Env into N-glycan microdomains that have a limited number of N-glycan sequon combinations.

Somatic Mutations Modulate Autoantibodies against Galactose-Deficient IgA1 in IgA Nephropathy.

Autoantibodies against galactose-deficient IgA1 drive formation of pathogenic immune complexes in IgA nephropathy. IgG autoantibodies against galactose-deficient IgA1 in patients with IgA nephropathy have a specific amino-acid sequence, Y1CS3, in the complementarity-determining region 3 of the heavy chain variable region compared with a Y1CA3 sequence in similar isotype-matched IgG from healthy controls. We previously found that the S3 residue is critical for binding galactose-deficient IgA1. To determine whether this difference is due to a rare germline sequence, we amplified and sequenced the corresponding germline variable region genes from peripheral blood mononuclear cells of seven patients with IgA nephropathy and six healthy controls from whom we had cloned single-cell lines secreting monoclonal IgG specific for galactose-deficient IgA1. Sanger DNA sequencing revealed that complementarity-determining region 3 in the variable region of the germline genes encoded the Y1C(A/V)3 amino-acid sequence. Thus, the A/V>S substitution in the complementarity-determining region 3 of anti-galactose-deficient-IgA1 autoantibodies of the patients with IgA nephropathy is not a rare germline gene variant. Modeling analyses indicated that the S3 hydroxyl group spans the complementarity-determining region 3 loop stem, stabilizing the adjacent ß-sheet and stem structure, important features for effective binding to galactose-deficient IgA1. Understanding processes leading to production of the autoantibodies may offer new approaches to treat IgA nephropathy.

The structure of a C. neoformans polysaccharide motif recognized by protective antibodies: A combined NMR and MD study.

Cryptococcus neoformans is a fungal pathogen responsible for cryptococcosis and cryptococcal meningitis. The C. neoformans capsular polysaccharide and shed exopolysaccharide functions both as a key virulence factor and to protect the fungal cell from phagocytosis. Currently, a glycoconjugate of these polysaccharides is being explored as a vaccine to protect against C. neoformans infection. In this combined NMR and MD study, experimentally determined NOEs and J-couplings support a structure of the synthetic decasaccharide, GXM10-Ac3, obtained by MD. GXM10-Ac3 was designed as an extension of glucuronoxylomannan (GXM) polysaccharide motif (M2) which is common in the clinically predominant serotype A strains and is recognized by protective forms of GXM-specific monoclonal antibodies. The M2 motif is characterized by a 6-residue α-mannan backbone repeating unit, consisting of a triad of α-(1→3)-mannoses, modified by ß-(1→2)-xyloses on the first two mannoses and a ß-(1→2)-glucuronic acid on the third mannose. The combined NMR and MD analyses reveal that GXM10-Ac3 adopts an extended structure, with xylose/glucuronic acid branches alternating sides along the α-mannan backbone. O-acetyl esters also alternate sides and are grouped in pairs. MD analysis of a twelve M2-repeating unit polymer supports the notion that the GXM10-Ac3 structure is uniformly represented throughout the polysaccharide. This experimentally consistent GXM model displays high flexibility while maintaining a structural identity, yielding new insights to further explore intermolecular interactions between polysaccharides, interactions with anti-GXM mAbs, and the cryptococcal polysaccharide architecture.

Lyophilization induces physicochemical alterations in cryptococcal exopolysaccharide.

Microbial polysaccharide characterization requires purification that often involves detergent precipitation and lyophilization. Here we examined physicochemical changes following lyophilization of Cryptococcus neoformans exopolysaccharide (EPS). Solution 1H Nuclear Magnetic Resonance (NMR) reveals significant anomeric signal attenuation following lyophilization of native EPS while 1H solid-state Nuclear Magnetic Resonance (ssNMR) shows few changes, suggesting diminished molecular motion and consequent broadening of 1H NMR polysaccharide resonances. 13C ssNMR, dynamic light scattering, and transmission electron microscopy show that, while native EPS has rigid molecular characteristics and contains small, loosely packed polysaccharide assemblies, lyophilized and resuspended EPS is disordered and contains larger dense aggregates, suggesting that structural water molecules in the interior of the polysaccharide assemblies are removed during extensive lyophilization. Importantly, mAbs to C. neoformans polysaccharide bind native EPS more strongly than lyophilized EPS. Together, these observations argue for caution when interpreting the biological and immunological attributes of polysaccharides that have been lyophilized to dryness.

Reversible O-Acetyl Migration within the Sialic Acid Side Chain and Its Influence on Protein Recognition.

O-Acetylation is a common naturally occurring modification of carbohydrates and is especially widespread in sialic acids, a family of nine-carbon acidic monosaccharides. O-Acetyl migration within the exocyclic glycerol-like side chain of mono-O-acetylated sialic acid reported previously was from the C7- to C9-hydroxyl group with or without an 8-O-acetyl intermediate, which resulted in an equilibrium that favors the formation of the 9-O-acetyl sialic acid. Herein, we provide direct experimental evidence demonstrating that O-acetyl migration is bidirectional, and the rate of equilibration is influenced predominantly by the pH of the sample. While the O-acetyl group on sialic acids and sialoglycans is stable under mildly acidic conditions (pH < 5, the rate of O-acetyl migration is extremely low), reversible O-acetyl migration is observed readily at neutral pH and becomes more significant when the pH increases to slightly basic. Sialoglycan microarray studies showed that esterase-inactivated porcine torovirus hemagglutinin-esterase bound strongly to sialoglycans containing a more stable 9-N-acetylated sialic acid analog, but these compounds were less resistant to periodate oxidation treatment compared to their 9-O-acetyl counterparts. Together with prior studies, the results support the possible influence of sialic acid O-acetylation and O-acetyl migration to host-microbe interactions and potential application of the more stable synthetic N-acetyl mimics.

Glycan Positioning Impacts HIV-1 Env Glycan-Shield Density, Function, and Recognition by Antibodies.

HIV-1 envelope (Env) N-glycosylation impact virus-cell entry and immune evasion. How each glycan interacts to shape the Env-protein-sugar complex and affects Env function is not well understood. Here, analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation sites affected the Env structure at distant locations and had a ripple effect on Env-wide glycan processing, virus infectivity, antibody recognition, and virus neutralization. Specifically, the N262 glycan, although not in the CD4-binding site, modulated Env binding to the CD4 receptor, affected Env recognition by several glycan-dependent neutralizing antibodies, and altered site-specific glycosylation heterogeneity, with, for example, N448 displaying limited glycan processing. Molecular-dynamic simulations visualized differences in glycan density and how specific oligosaccharide positions can move to compensate for a glycan loss. This study demonstrates how changes in individual glycans can alter molecular dynamics, processing, and function of the Env-glycan shield.

Glycosylation of viral surface proteins probed by mass spectrometry.

Glycosylation is a common and biologically significant post-translational modification that is found on numerous virus surface proteins (VSPs). Many of these glycans affect virulence through modulating virus receptor binding, masking antigenic sites, or by stimulating the host immune response. Mass spectrometry (MS) has arisen as a pivotal technique for the characterization of VSP glycosylation. This review will cover how MS-based analyses, such as released glycan profiles, glycan site localization, site-occupancy, and site-specific heterogeneity, are being utilized to map VSP glycosylation. Furthermore, this review will provide information on how MS glycoprofiling data are being used in conjunction with molecular and structural experiments to provide a better understanding of the role of specific glycans in VSP function.

Human neutrophil peptide-1 inhibits thrombus formation under arterial flow via its terminal free cysteine thiols.

Essentials Biological activity of human neutrophil peptide (HNP)-1 in hemostasis under physiological conditions is not fully understood. HNP-1 inhibits the adhesion/aggregation of murine platelets on a fibrillar collagen surface or an activated endothelial cell surface under flow. The anti-adhesion activity appears to depend on the terminal free thiols of HNP-1, which may inhibit VWF-VWF lateral associations. Our results suggest a protective role and potential novel therapeutic use of HNP-1 for arterial thrombosis. SUMMARY: Background Human neutrophil peptides (HNPs), also known as α-defensins, are released from degranulated neutrophils and play an important role in innate immunity. However, their biological roles in hemostasis under flow are not fully explored. Objective This study aims to determine the role of HNP-1 on platelet adhesion and aggregation on a collagen surface or ultra large von Willebrand factor (ULVWF) on endothelium under flow and elucidate the structural elements required for its activity. Methods Anticoagulated whole blood from wild-type or Adamts13-/- mice was incubated with a fluorescein-conjugated anti-human CD41 in the presence of increasing concentrations of a synthetic HNP-1 and perfused over a collagen surface or a tumor necrosis factor (TNF)-α activated murine endothelial cell surface under arterial flow. The rate of accumulation and the final surface coverage of fluoresceinated murine platelets or the rate of forming platelet-decorated ULVWF strings were determined using the BioFlux microfluidic system. Results HNP-1 inhibited the rate and final coverage of fluorescein-labeled murine platelets on a fibrillar collagen surface under flow (100 dyne/cm2 ) in a concentration-dependent manner and the anti-adhesive activity of HNP-1 depended on its terminal free cysteine thiols. HNP-1 (20 µM) also dramatically inhibited the formation of platelets-decorated ULVWF strings on TNF-α activated murine endothelial surface under arterial flow. Conclusions Our results demonstrate for the first time an antiplatelet adhesion or antithrombotic activity of HNP-1; this activity depends on its terminal free thiols, likely affecting VWF-VWF lateral associations. These findings may suggest a potential novel therapeutic strategy for arterial thrombosis.

Pyrazolopyrimidinones, a novel class of copper-dependent bactericidal antibiotics against multi-drug resistant S. aureus.

The treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections poses a therapeutic challenge as even last resort drugs become increasingly ineffective. As the demand for antibiotics with novel modes of action is growing, new approaches are needed to probe a greater spectrum of antimicrobial activities for their potential efficacy against drug-resistant pathogens. The use of copper (Cu) by the innate immune system to mount an antimicrobial response against bacterial invaders has created an opportunity to explore a role for Cu in antimicrobial therapy. Here we describe pyrazolopyrimidinones (PZP) as novel copper-dependent inhibitors (CDI) of S. aureus. 5-Benzyl-3-(4-chlorophenyl)-2-methyl-4H,7H-pyrazolo[1,5-a]pyrimidin-7-one (PZP-915) showed potent bactericidal properties at sub-micromolar concentrations and activity against clinical MRSA isolates and biofilms cultures. This cupricidal activity is founded on the molecule's ability to coordinate Cu and induce accumulation of Cu ions inside S. aureus cells. We demonstrate that exposure to 915 + Cu led to an almost instantaneous collapse of the membrane potential which was accompanied by a complete depletion of cellular ATP, loss of cell-associated K+, a substantial gain of cell associated Na+, and an inability to control the influx of protons in slightly acidic medium, while the integrity of the cell membrane remained intact. These findings highlight PZP-915 as a novel membrane-directed metalloantibiotic against S. aureus that is likely to target a multiplicity of membrane associated protein functions rather than imposing physical damage to the membrane structure.