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1.
Proc Natl Acad Sci U S A ; 121(7): e2315733121, 2024 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-38330012

RESUMEN

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.


Asunto(s)
Criptococosis , Cryptococcus neoformans , Humanos , Mananos , Cryptococcus neoformans/química , Polisacáridos/química , Criptococosis/microbiología , Espectroscopía de Resonancia Magnética , Anticuerpos Monoclonales , Anticuerpos Antifúngicos
2.
Molecules ; 26(14)2021 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-34299586

RESUMEN

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.


Asunto(s)
Glicoproteínas/química , Manosa/química , Resonancia Magnética Nuclear Biomolecular , Ribonucleasas/química , Glicosilación
3.
J Virol ; 93(1)2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30305355

RESUMEN

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.


Asunto(s)
VIH-1/metabolismo , Mutación , Polisacáridos/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Glicosilación , Células HEK293 , VIH-1/química , VIH-1/genética , Células HeLa , Humanos , Evasión Inmune , Espectrometría de Masas , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética
4.
bioRxiv ; 2023 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-37732210

RESUMEN

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.

5.
Carbohydr Polym ; 291: 119547, 2022 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-35698377

RESUMEN

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.


Asunto(s)
Cryptococcus neoformans , Polisacáridos , Cryptococcus neoformans/metabolismo , Liofilización , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Transmisión , Polisacáridos/metabolismo , Polisacáridos Bacterianos/química
6.
ACS Chem Biol ; 16(10): 1951-1960, 2021 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-33769035

RESUMEN

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.


Asunto(s)
Hemaglutininas Virales/metabolismo , Polisacáridos/metabolismo , Ácidos Siálicos/metabolismo , Proteínas Virales de Fusión/metabolismo , Acetilación , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Hemaglutininas Virales/química , Estructura Molecular , Oxidación-Reducción , Ácido Peryódico/química , Fenilendiaminas/química , Polisacáridos/análisis , Polisacáridos/química , Unión Proteica , Ácidos Siálicos/análisis , Ácidos Siálicos/química , Torovirus/enzimología , Proteínas Virales de Fusión/química
7.
iScience ; 23(11): 101711, 2020 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-33205023

RESUMEN

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.

8.
Curr Opin Virol ; 36: 56-66, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31202133

RESUMEN

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.


Asunto(s)
Proteínas de la Membrana/química , Polisacáridos/química , Procesamiento Proteico-Postraduccional , Proteínas de la Matriz Viral/química , Fenómenos Fisiológicos de los Virus , Glicosilación , Espectrometría de Masas , Proteínas de la Membrana/genética , Unión Proteica , Proteínas de la Matriz Viral/genética , Virulencia , Virus/química
9.
Metallomics ; 11(4): 784-798, 2019 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-30855050

RESUMEN

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.


Asunto(s)
Antibacterianos/farmacología , Cobre/farmacología , Pirimidinonas/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Biopelículas/efectos de los fármacos , Cobre/química , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/fisiología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Pirimidinonas/química , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/fisiología
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