137 research outputs found

    Fluctuating Nonlinear Spring Model of Mechanical Deformation of Biological Particles

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    We present a new theory for modeling forced indentation spectral lineshapes of biological particles, which considers non-linear Hertzian deformation due to an indenter-particle physical contact and bending deformations of curved beams modeling the particle structure. The bending of beams beyond the critical point triggers the particle dynamic transition to the collapsed state, an extreme event leading to the catastrophic force drop as observed in the force (F)-deformation (X) spectra. The theory interprets fine features of the spectra: the slope of the FX curves and the position of force-peak signal, in terms of mechanical characteristics --- the Young's moduli for Hertzian and bending deformations E_H and E_b, and the probability distribution of the maximum strength with the strength of the strongest beam F_b^* and the beams' failure rate m. The theory is applied to successfully characterize the FXFX curves for spherical virus particles --- CCMV, TrV, and AdV

    Examining the Heterogeneous Genome Content of Multipartite Viruses BMV and CCMV by Native Mass Spectrometry

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    Since the concept was first introduced by Brian Chait and co-workers in 1991, mass spectrometry of proteins and protein complexes under non-denaturing conditions (native MS) has strongly developed, through parallel advances in instrumentation, sample preparation, and data analysis tools. However, the success rate of native MS analysis, particularly in heterogeneous mega-Dalton (MDa) protein complexes, still strongly depends on careful instrument modification. Here, we further explore these boundaries in native mass spectrometry, analyzing two related endogenous multipartite viruses: the Brome Mosaic Virus (BMV) and the Cowpea Chlorotic Mottle Virus (CCMV). Both CCMV and BMV are approximately 4.6 megadalton (MDa) in mass, of which approximately 1 MDA originates from the genomic content of the virion. Both viruses are produced as mixtures of three particles carrying different segments of the genome, varying by approximately 0.1 MDA in mass (~2%). This mixture of particles poses a challenging analytical problem for high-resolution native MS analysis, given the large mass scales involved. We attempt to unravel the particle heterogeneity using both Q-TOF and Orbitrap mass spectrometers extensively modified for analysis of very large assemblies. We show that manipulation of the charging behavior can provide assistance in assigning the correct charge states. Despite their challenging size and heterogeneity, we obtained native mass spectra with resolved series of charge states for both BMV and CCMV, demonstrating that native MS of endogenous multipartite virions is feasible. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-016-1348-6) contains supplementary material, which is available to authorized users

    Glycan shield of the ebolavirus envelope glycoprotein GP

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    The envelope glycoprotein GP of the ebolaviruses is essential for host cell entry and the primary target of the host antibody response. GP is heavily glycosylated with up to 17 N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation is important for host cell attachment, GP stability and fusion activity, and shielding from neutralization by serum antibodies. Here, we use glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the MLD, and two O-linked sites in the receptor-binding GP1 subunit. Multiple O-linked glycans are observed within N-linked glycosylation sequons, suggesting crosstalk between the two types of modifications. We confirmed C-mannosylation of W288 in full-length trimeric GP. We find complex glycosylation at the majority of N-linked sites, while the conserved sites N257 and especially N563 are enriched in unprocessed glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. Our findings illustrate how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP, guiding future immunological studies and functional interpretation of ebolavirus GP-antibody interactions

    Frequency chasing of individual megadalton ions in an Orbitrap analyser improves precision of analysis in single-molecule mass spectrometry

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    To enhance the performance of charge-detection mass spectrometry, we investigated the behaviour of macromolecular single ions on their paths towards and within the Orbitrap analyser. Ions with a mass beyond one megadalton reach a plateau of stability and can be successfully trapped for seconds, travelling a path length of multiple kilometres, thereby enabling precise mass analysis with an effective resolution of greater than 100,000 at a mass-to-charge ratio of 35,000. Through monitoring the frequency of individual ions, we show that these high-mass ions, rather than being lost from the trap, can gradually lose residual solvent molecules and, in rare cases, a single elementary charge. We also demonstrate that the frequency drift of single ions due to desolvation and charge stripping can be corrected, which improves the effective ion sampling 23-fold and gives a twofold improvement in mass precision and resolution. [Figure not available: see fulltext.

    Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

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    Recent human H3N2 influenza A viruses have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells, which are commonly employed to propagate influenza A virus, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase 1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAcs), would result in improved A/H3N2 propagation. Stable overexpression of β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase 1 in Madin-Darby Canine Kidney and "humanized" Madin-Darby Canine Kidney cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the β-1,3-N-acetylglucosaminyltransferase and/or β-1,4-galactosyltransferase 1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on "humanized" Madin-Darby Canine Kidney-β-1,3-N-acetylglucosaminyltransferase cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 influenza A viruses require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.</p

    Reverse-engineering the anti-MUC1 antibody 139H2 by mass spectrometry–based de novo sequencing

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    Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at mucosal surfaces. MUC1 has a barrier function against bacterial invasion and is well known for its aberrant expression and glycosylation in adenocarcinomas. The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated. Monoclonal antibodies against the MUC1 VNTR are powerful research tools with applications in the diagnosis and treatment of MUC1-expressing cancers. Here, we report direct mass spectrometry–based sequencing of anti-MUC1 hybridoma-derived 139H2 IgG, enabling reverse-engineering of the functional recombinant monoclonal antibody. The crystal structure of the 139H2 Fab fragment in complex with the MUC1 epitope was solved, revealing the molecular basis of 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostic, targeting, and treatment strategies

    Contemporary human H3N2 influenza a viruses require a low threshold of suitable glycan receptors for efficient infection

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    Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells (MDCK and hCK) which are commonly employed to propagate IAV, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency

    Glycan shield of the ebolavirus envelope glycoprotein GP

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    The envelope glycoprotein GP of the ebolaviruses is essential for host cell entry and the primary target of the host antibody response. GP is heavily glycosylated with up to 17 N-linked sites, numerous O-linked glycans in its disordered mucin-like domain (MLD), and three predicted C-linked mannosylation sites. Glycosylation is important for host cell attachment, GP stability and fusion activity, and shielding from neutralization by serum antibodies. Here, we use glycoproteomics to profile the site-specific glycosylation patterns of ebolavirus GP. We detect up to 16 unique O-linked glycosylation sites in the MLD, and two O-linked sites in the receptor-binding GP1 subunit. Multiple O-linked glycans are observed within N-linked glycosylation sequons, suggesting crosstalk between the two types of modifications. We confirmed C-mannosylation of W288 in full-length trimeric GP. We find complex glycosylation at the majority of N-linked sites, while the conserved sites N257 and especially N563 are enriched in unprocessed glycans, suggesting a role in host-cell attachment via DC-SIGN/L-SIGN. Our findings illustrate how N-, O-, and C-linked glycans together build the heterogeneous glycan shield of GP, guiding future immunological studies and functional interpretation of ebolavirus GP-antibody interactions

    Reverse engineering the anti-MUC1 hybridoma antibody 139H2 by mass spectrometry-based de novo sequencing

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    Mucin 1 (MUC1) is a transmembrane mucin expressed at the apical surface of epithelial cells at different mucosal surfaces including breast and intestine. In the gastrointestinal tract, MUC1 has a barrier function against bacterial invasion, but can also serve as an entry receptor for pathogenic Salmonella bacteria. Moreover, MUC1 is well known for its aberrant expression and glycosylation in adenocarcinomas The MUC1 extracellular domain contains a variable number of tandem repeats (VNTR) of 20 amino acids, which are heavily O-linked glycosylated.. Monoclonal antibodies against the MUC1 VNTR can be powerful tools because of their multiplicity of binding and possible applications in the diagnosis and treatment of MUC1-expressing cancers. One such antibody is the hybridoma mouse monoclonal 139H2, which is also widely used as a research tool to study non-cancer MUC1. Here we report direct mass spectrometry-based sequencing of hybridoma-derived 139H2 IgG, which enabled reverse engineering of a recombinant 139H2. The performance of the reverse engineered 139H2 IgG and its Fab fragment were validated by comparison to the hybridoma-derived product in Western blot and immunofluorescence microscopy. The reverse engineering of 139H2 allowed us to characterize binding to the VNTR peptide epitope by surface plasmon resonance (SPR) and solve the crystal structure of the 139H2 Fab fragment in complex with the MUC1 VNTR peptide. These analyses reveal the molecular basis for 139H2 binding specificity to MUC1 and its tolerance to O-glycosylation of the VNTR. The available sequence of 139H2 will allow further development of MUC1-related diagnostics, targeting and treatment strategies
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