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Sökning: WFRF:(Larson Göran 1953)

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1.
  • Noborn, Fredrik, et al. (författare)
  • Subtyping of cardiac amyloidosis by mass spectrometry-based proteomics of endomyocardial biopsies.
  • 2023
  • Ingår i: Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis. - : Informa UK Limited. - 1350-6129 .- 1744-2818. ; 30:1, s. 96-108
  • Tidskriftsartikel (refereegranskat)abstract
    • Cardiac amyloidosis is a severe condition leading to restrictive cardiomyopathy and heart failure. Mass spectrometry-based methods for cardiac amyloid subtyping have become important diagnostic tools but are currently used only in a few reference laboratories. Such methods include laser-capture microdissection to ensure the specific analysis of amyloid deposits. Here we introduce a direct proteomics-based method for subtyping of cardiac amyloidosis.Endomyocardial biopsies were retrospectively analysed from fresh frozen material of 78 patients with cardiac amyloidosis and from 12 biopsies of unused donor heart explants. Cryostat sections were digested with trypsin and analysed with liquid chromatography - mass spectrometry, and data were evaluated by proteomic software.With a diagnostic threshold set to 70% for each of the four most common amyloid proteins affecting the heart (LC κ, LC λ, TTR and SAA), 65 of the cases (87%) could be diagnosed, and of these, 61 cases (94%) were in concordance with the original diagnoses. The specimens were also analysed for the summed intensities of the amyloid signature proteins (ApoE, ApoA-IV and SAP). The intensities were significantly higher (p<0.001) for all assigned cases compared with controls.Cardiac amyloidosis can be successfully subtyped without the prior enrichment of amyloid deposits with laser microdissection.
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2.
  • Arnaud, Julie, et al. (författare)
  • Reduction of lectin valency drastically changes glycolipid dynamics in membranes but not surface avidity.
  • 2013
  • Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 8:9, s. 1918-24
  • Tidskriftsartikel (refereegranskat)abstract
    • Multivalency is proposed to play a role in the strong avidity of lectins for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Lectins with modified valency were designed from the β-propeller fold of Ralstonia solanacearum lectin (RSL) that presents six fucose binding sites. After identification of key amino acids by molecular dynamics calculations, two mutants with reduced valency were produced. Isothermal titration calorimetry confirmed the loss of three high affinity binding sites for both mutants. Crystal structures indicated that residual low affinity binding occurred in W76A but not in R17A. The trivalent R17A mutant presented unchanged avidity toward fucosylated surfaces, when compared to hexavalent RSL. However, R17A is not able anymore to induce formation of membrane invaginations on giant unilamellar vesicules, indicating the crucial role of number of binding sites for clustering of glycolipids. In the human lung epithelial cell line H1299, wt-RSL is internalized within seconds whereas the kinetics of R17A uptake is largely delayed. Neolectins with tailored valency are promising tools to study membrane dynamics.
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3.
  • Bally, Marta, 1981, et al. (författare)
  • A virus biosensor with single virus-particle sensitivity based on fluorescent vesicle labels and equilibrium fluctuation analysis
  • 2013
  • Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 8
  • Tidskriftsartikel (refereegranskat)abstract
    • Biosensors allowing for the rapid and sensitive detection of viral pathogens in environmental or clinical samples are urgently needed to prevent disease outbreaks and spreading. We present a bioanalytical assay for the detection of whole viral particles with single virus sensitivity. Specifically, we focus on the detection of human norovirus, a highly infectious virus causing gastroenteritis. In our assay configuration, virus-like particles are captured onto a supported lipid bilayer containing a virus-specific glycolipid and detected after recognition by a glycolipid-containing fluorescent vesicle. Read-out is performed after illumination of the vesicle labels by total internal reflection fluorescence microscopy. This allows for visualization of individual vesicles and for recording of their binding kinetics under equilibrium conditions (equilibrium fluctuation analysis), as demonstrated previously. In this work we extend the concept and demonstrate that this simple assay setup can be used as a bioanalytical assay for the detection of virus particles at a limit of detection of 16 fM. Furthermore, we demonstrate how the analysis of the single vesicle-virus-like particle interaction dynamics can contribute to increase the accuracy and sensitivity of the assay by discriminating specific from non-specific binding events. This method is suggested to be generally applicable, provided that these events display different interaction kinetics.
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4.
  • Bally, Marta, 1981, et al. (författare)
  • Interaction of Single Viruslike Particles with Vesicles Containing Glycosphingolipids
  • 2011
  • Ingår i: Physical Review Letters. - : American Physical Society. - 0031-9007 .- 1079-7114. ; 107:18
  • Tidskriftsartikel (refereegranskat)abstract
    • Glycosphingolipids are involved in the first steps of virus-cell interaction, where they mediate specific recognition of the host cell membrane. We have employed total-internal-reflection fluorescence microscopy to explore the interaction kinetics between individual unlabeled noroviruslike particles, which are attached to a glycosphingolipid-containing lipid bilayer, and fluorescent vesicles containing different types and concentrations of glycosphingolipids. Under association equilibrium, the vesicle-binding rate is found to be kinetically limited, yielding information on the corresponding activation energy. The dissociation kinetics are logarithmic over a wide range of time. The latter is explained by the vesicle-size-related distribution of the dissociation activation energy. The biological, pharmaceutical, and diagnostic relevance of the study is briefly discussed.
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5.
  • Bally, Marta, 1981, et al. (författare)
  • Interaction of virions with membrane glycolipids
  • 2012
  • Ingår i: Physical Biology. - : IOP Publishing. - 1478-3967 .- 1478-3975. ; 9:2
  • Tidskriftsartikel (refereegranskat)abstract
    • Cellular membranes contain various lipids including glycolipids (GLs). The hydrophilic head groups of GLs extend from the membrane into the aqueous environment outside the cell where they act as recognition sites for specific interactions. The first steps of interaction of virions with cells often include contacts with GLs. To clarify the details of such contacts, we have used the total internal reflection fluorescence microscopy to explore the interaction of individual unlabelled virus-like particles (or, more specifically, norovirus protein capsids), which are firmly bound to a lipid bilayer, and fluorescent vesicles containing glycosphingolipids (these lipids form a subclass of GLs). The corresponding binding kinetics were earlier found to be kinetically limited, while the detachment kinetics were logarithmic over a wide range of time. Here, the detachment rate is observed to dramatically decrease with increasing concentration of glycosphingolipids from 1% to 8%. This effect has been analytically explained by using a generic model describing the statistics of bonds in the contact area between a virion and a lipid membrane. Among other factors, the model takes the formation of GL domains into account. Our analysis indicates that in the system under consideration, such domains, if present, have a characteristic size smaller than the contact area between the vesicle and the virus-like particle.
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6.
  • Bally, Marta, 1981, et al. (författare)
  • Norovirus GII.4 Virus-like Particles Recognize Galactosylceramides in Domains of Planar Supported Lipid Bilayers.
  • 2012
  • Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 51:48, s. 12020-4
  • Tidskriftsartikel (refereegranskat)abstract
    • A sticky situation: Domain-dependent recognition of the glycosphingolipid galactosylceramide by norovirus-like particles (see picture; red/yellow) is shown using supported lipid bilayers (purple) as model membranes. Optimal ligand presentation is found to promote strong binding to GalCer. This presentation can be found at the edges of the glycosphingolipid-enriched domains (green) and binding is repressed in the absence of these domains.
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7.
  • Bally, Marta, 1981, et al. (författare)
  • Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context
  • 2021
  • Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 413, s. 7157-7178
  • Tidskriftsartikel (refereegranskat)abstract
    • The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.
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8.
  • Bengtson, Per, 1971-, et al. (författare)
  • Characterization of EBV-transformed B-cells established from an individual homozygously mutated (G329A) in the FUT7 alpha1,3-fucosyltransferase gene
  • 2005
  • Ingår i: Scand J Immunol. - : Wiley. ; 62:3, s. 251-8
  • Tidskriftsartikel (refereegranskat)abstract
    • The alpha1,3-fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis x (SLe(x)) on human leukocytes. Recently, individuals were characterized carrying a missense mutation (G329A; Arg110-Gln) in the FUT7 gene encoding this enzyme. The mutated FUT7 construct produced a Fuc-TVII enzyme with impaired activity compared with the wildtype enzyme. Polymorphonuclear granulocytes from an individual carrying this mutation homozygously also showed a reduced expression of SLe(x). In the present study, we have established Epstein-Barr virus-transformed B-cell lines from this individual (SIGN) and from an individual not carrying the mutation (IWO). The cell lines were confirmed to be of B-cell origin by flow cytometry analysis. IWO cells interacted with E-selectin in an in vitro flow chamber analysis whereas SIGN cell did not. However, when SIGN cell was transiently transfected with wildtype FUT7 cDNA, interaction with E-selectin could be restored. Cell surface expression of the SLe(x)-related epitopes recognized by antibodies CSLEX-1, KM-93 and HECA-452 was elevated on IWO cells compared with that on SIGN cells, consistent with a role of these antigens in E-selectin recognition. These cell lines will be useful in further characterization of E-selectin ligands and encourage further studies on the consequences of the FUT7-G329A mutation in vivo.
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9.
  • Bernasconi, Valentina, 1989, et al. (författare)
  • A vaccine combination of lipid nanoparticles and a cholera toxin adjuvant derivative greatly improves lung protection against influenza virus infection
  • 2021
  • Ingår i: Mucosal Immunology. - : Elsevier BV. - 1933-0219 .- 1935-3456. ; 14:2, s. 523-536
  • Tidskriftsartikel (refereegranskat)abstract
    • This is a proof-of-principle study demonstrating that the combination of a cholera toxin derived adjuvant, CTA1-DD, and lipid nanoparticles (LNP) can significantly improve the immunogenicity and protective capacity of an intranasal vaccine. We explored the self-adjuvanted universal influenza vaccine candidate, CTA1-3M2e-DD (FPM2e), linked to LNPs. We found that the combined vector greatly enhanced survival against a highly virulent PR8 strain of influenza virus as compared to when mice were immunized with FPM2e alone. The combined vaccine vector enhanced early endosomal processing and peptide presentation in dendritic cells and upregulated co-stimulation. The augmenting effect was CTA1-enzyme dependent. Whereas systemic anti-M2e antibody and CD4(+)T-cell responses were comparable to those of the soluble protein, the local respiratory tract IgA and the specific Th1 and Th17 responses were strongly enhanced. Surprisingly, the lung tissue did not exhibit gross pathology upon recovery from infection and M2e-specific lung resident CD4(+)T cells were threefold higher than in FPM2e-immunized mice. This study conveys optimism as to the protective ability of a combination vaccine based on LNPs and various forms of the CTA1-DD adjuvant platform, in general, and, more specifically, an important way forward to develop a universal vaccine against influenza.
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10.
  • Breimer, Michael, 1951, et al. (författare)
  • Glycosphingolipid composition of epithelial cells isolated along the villus axis of small intestine of a single human individual
  • 2012
  • Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:12, s. 1721-1730
  • Tidskriftsartikel (refereegranskat)abstract
    • A 6-cm fresh proximal ileum surgical specimen from a blood group A(1)Le(a-b+) secretor individual was used for stepwise isolation of epithelial cells from villus tip to crypt bottom by gentle washing with ethylenediaminetetraacetic acid-containing buffer. Acid and non-acid sphingolipids were prepared from the epithelial cell fractions and the non-epithelial intestinal residue. Molecular information on the sphingolipid composition was obtained without further isolation of individual species by applying thin-layer chromatography using chemical and biological (monoclonal antibodies, cholera toxin, Escherichia coli) detection reagents, mass spectrometry and proton NMR spectroscopy of derivatized glycolipids. In this way, the structure of major and minor saccharides, ceramide components and their relative amounts were obtained. Epithelial cells and non-epithelial residue were distinctly different in their sphingolipid composition. Sphingomyelin was the major single component in both compartments. Characteristic for epithelial cells was the dominance of monoglycosylceramides, sulphatides and blood group fucolipids (mainly Le(b) hexaglycosylceramides and ALe(b) heptaglycosylceramides). The non-epithelial residue had about five times less glycolipids mainly mono-, di-, tri- and tetra-glycosylceramides and gangliosides, including the GM1 ganglioside. The ceramides were more hydroxylated (1-2 additional hydroxyls) in epithelial cell glycolipids compared with the non-epithelial residue. Combined with a separate detailed study on the glycoproteins of the same epithelial cell preparation, this human intestinal sample is the only epithelial cell preparation where both protein- and lipid-linked saccharides are characterized in detail.
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11.
  • Brinkmalm, Gunnar, et al. (författare)
  • An online nano-LC-ESI-FTICR-MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid.
  • 2012
  • Ingår i: Journal of mass spectrometry : JMS. - : Wiley. - 1096-9888 .- 1076-5174. ; 47:5, s. 591-603
  • Tidskriftsartikel (refereegranskat)abstract
    • Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top-down MS-based method.
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12.
  • Bucardo, Filemon, et al. (författare)
  • Genetic susceptibility to symptomatic norovirus infection in Nicaragua. : norovirus susceptibility in Nicaragua
  • 2009
  • Ingår i: Journal of medical virology. - : Wiley. - 1096-9071 .- 0146-6615. ; 81:4, s. 728-35
  • Tidskriftsartikel (refereegranskat)abstract
    • Host genetic resistance to Norovirus (NoV) has been observed in challenge and outbreak studies in populations from Europe, Asia, and USA. In this study, we have investigated if histo-blood group antigens can predict susceptibility to diarrhea caused by NoV in Nicaragua, Central America, and if this can be reflected in antibody-prevalence and titer to NoV among individuals with different histo-blood group antigen phenotypes. Investigation of 28 individuals infected with NoV and 131 population controls revealed 6% of non-secretors in the population and nil non-secretors among patients infected with NoV, suggesting that non-secretors may be protected against NoV disease in Nicaragua. Surprisingly, 25% of the population was Lewis negative (Le(a-b-)). NoV infections with genogroup I (GI) and GII occurred irrespective of Lewis genotype, but none of the Lewis a positive (Le(a + b-)) were infected. The globally dominating GII.4 virus infected individuals of all blood groups except AB (n = 5), while the GI viruses (n = 4) infected only blood type O individuals. Furthermore, O blood types were susceptible to infections with GI.4, GII.4, GII.7, GII.17, and GII.18-Nica viruses, suggesting that secretors with blood type O are susceptible (OR = 1.52) and non-secretors resistant. The overall antibody-prevalence to NoV GII.3 VLP was 62% with the highest prevalence among blood type B carriers (70%) followed by A (68%) and O (62%). All four investigated individuals carrying blood type AB were antibody-negative. Among secretors, 63% were antibody-positive compared to 33% among non-secretors (P = 0.151). This study extends previous knowledge about the histo-blood group antigens role in NoV disease in a population with different genetic background than North American and European.
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13.
  • Bucardo, Filemon, et al. (författare)
  • Susceptibility of Children to Sapovirus Infections, Nicaragua, 2005–2006
  • 2012
  • Ingår i: Emerging Infectious Diseases. - Atlanta, GA, USA : U.S. Department of Health and Human Services * Centers for Disease Control and Prevention. - 1080-6040 .- 1080-6059. ; 18:11, s. 1875-1878
  • Tidskriftsartikel (refereegranskat)abstract
    • We describe the genetic diversity of sapovirus (SaV) in children in Nicaragua and investigate the role of host genetic factors and susceptibility to SaV infections. Our results indicate that neither ABO blood group, Lewis phenotype, nor secretor status affects susceptibility to SaV infection in Nicaragua.
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14.
  • Carlsson, Beatrice, et al. (författare)
  • The G428A nonsense mutation in FUT2 provides strong but not absolute protection against symptomatic GII.4 Norovirus infection. : Novel GII.4 disease pattern
  • 2009
  • Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 4:5
  • Tidskriftsartikel (refereegranskat)abstract
    • In November 2004, 116 individuals in an elderly nursing home in El Grao de Castellón, Spain were symptomatically infected with genogroup II.4 (GII.4) norovirus. The global attack rate was 54.2%. Genotyping of 34 symptomatic individuals regarding the FUT2 gene revealed that one patient was, surprisingly, a non-secretor, hence indicating secretor-independent infection. Lewis genotyping revealed that Lewis-positive and negative individuals were susceptible to symptomatic norovirus infection indicating that Lewis status did not predict susceptibility. Saliva based ELISA assays were used to determine binding of the outbreak virus to saliva samples. Saliva from a secretor-negative individual bound the authentic outbreak GII.4 Valencia/2004/Es virus, but did not in contrast to secretor-positive saliva bind VLP of other strains including the GII.4 Dijon strain. Amino acid comparison of antigenic A and B sites located on the external loops of the P2 domain revealed distinct differences between the Valencia/2004/Es and Dijon strains. All three aa in each antigenic site as well as 10/11 recently identified evolutionary hot spots, were unique in the Valencia/2004/Es strain compared to the Dijon strain. To the best of our knowledge, this is the first example of symptomatic GII.4 norovirus infection of a Le(a+b-) individual homozygous for the G428A nonsense mutation in FUT2. Taken together, our study provides new insights into the host genetic susceptibility to norovirus infections and evolution of the globally dominating GII.4 viruses.
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15.
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16.
  • De Leoz, M. L. A., et al. (författare)
  • NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods
  • 2020
  • Ingår i: Molecular & Cellular Proteomics. - 1535-9476. ; 19:1, s. 11-30
  • Tidskriftsartikel (refereegranskat)abstract
    • A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories. Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.
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17.
  • Delbaere, S., et al. (författare)
  • b3galt6 Knock-Out Zebrafish Recapitulate beta 3GalT6-Deficiency Disorders in Human and Reveal a Trisaccharide Proteoglycan Linkage Region
  • 2020
  • Ingår i: Frontiers in Cell and Developmental Biology. - : Frontiers Media SA. - 2296-634X. ; 8
  • Tidskriftsartikel (refereegranskat)abstract
    • Proteoglycans are structurally and functionally diverse biomacromolecules found abundantly on cell membranes and in the extracellular matrix. They consist of a core protein linked to glycosaminoglycan chains via a tetrasaccharide linkage region. Here, we show that CRISPR/Cas9-mediated b3galt6 knock-out zebrafish, lacking galactosyltransferase II, which adds the third sugar in the linkage region, largely recapitulate the phenotypic abnormalities seen in human beta 3GalT6-deficiency disorders. These comprise craniofacial dysmorphism, generalized skeletal dysplasia, skin involvement and indications for muscle hypotonia. In-depth TEM analysis revealed disturbed collagen fibril organization as the most consistent ultrastructural characteristic throughout different affected tissues. Strikingly, despite a strong reduction in glycosaminoglycan content, as demonstrated by anion-exchange HPLC, subsequent LC-MS/MS analysis revealed a small amount of proteoglycans containing a unique linkage region consisting of only three sugars. This implies that formation of glycosaminoglycans with an immature linkage region is possible in a pathogenic context. Our study, therefore unveils a novel rescue mechanism for proteoglycan production in the absence of galactosyltransferase II, hereby opening new avenues for therapeutic intervention.
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18.
  • El Masri, R., et al. (författare)
  • Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity
  • 2022
  • Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 38:11
  • Tidskriftsartikel (refereegranskat)abstract
    • Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human SuIf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs.
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19.
  • Elmgren, A, et al. (författare)
  • DNA sequencing and screening for point mutations in the human Lewis (FUT3) gene enables molecular genotyping of the human Lewis blood group system.
  • 1996
  • Ingår i: Vox sanguinis. - 0042-9007. ; 70:2, s. 97-103
  • Tidskriftsartikel (refereegranskat)abstract
    • The human Lewis gene encodes an alpha(1,3/1,4)-fucosyltransferase responsible for synthesis of the Le(a) and a Le(b) antigens. To define the molecular background for non-functional Lewis genes we have sequenced PCR-amplified DNA fragments from two Le(a-b-) individuals. One was homozygously mutated at nucleotides 202(T --> C) and 314 (C --> T), altering Trp68 to Arg and Thr105 to Met, and the other was homozygously mutated at nucleotides 59 (T --> G) and 1067 (T --> A), altering Leu20 to Arg and Ile356 to Lys. Using PCR we screened for these and additionally one other mutation at nucleotide 508 (G --> A) among 40 Caucasians. Of 15 Le(a-b-) individuals, 7 typed as le59/1067le202/314, 4 as le202/314le202/314 and 1 as le59/1067le59/1067. Of 21 Le(a-b+) and 4 Le(a+b-), 17 typed as LeLe and 7 as Lele202/314. A pedigree study of 8 Lewis-positive individuals showed that the mutations at nucleotides 202 and 314 were located on the same allele.
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20.
  • Fernandez-Mateos, P, et al. (författare)
  • Point mutations and deletion responsible for the Bombay H null and the Reunion H weak blood groups.
  • 1998
  • Ingår i: Vox sanguinis. - 0042-9007. ; 75:1, s. 37-46
  • Tidskriftsartikel (refereegranskat)abstract
    • OBJECTIVE: Definition of the molecular basis of the Reunion and the Bombay red cell and salivary H-deficient phenotypes. METHODS: Sequence and expression of FUT1 and FUT2 genes from H-deficient individuals. Family segregation analysis of the mutations responsible for the fucosyltransferase defects of H, secretor and Lewis systems. RESULTS: The Indian red cell H null Bombay phenotype depends on a new mutation of the FUT1 gene. T725-->G changing Leu242-->Arg. Their salivary nonsecretor phenotype is secondary to a complete deletion of the FUT2 gene. The red cell H weak Reunion phenotype depends on another new mutation of FUT1, C349-->T which induces a change of His117-->Tyr. Their salivary nonsecretor phenotype is due to the known Caucasian inactivating mutation G428-->A. CONCLUSION: Single prevalent FUT1 and FUT2 point mutations and a deletion are responsible for the Indian Bombay H null and the Reunion H weak phenotypes found on Reunion island. This is in contrast with other H-deficient phenotypes where sporadic nonprevalent inactivating mutations are the rule.
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21.
  • Grahn, Ammi, 1961, et al. (författare)
  • Determination of Lewis FUT3 gene mutations by PCR using sequence-specific primers enables efficient genotyping of clinical samples.
  • 2001
  • Ingår i: Human mutation. - : Hindawi Limited. - 1098-1004 .- 1059-7794. ; 18:4, s. 358-9
  • Tidskriftsartikel (refereegranskat)abstract
    • We have developed a polymerase chain reaction method using sequence-specific primers (PCR-SSP) for rapid and correct genotyping of the common Lewis (FUT3) gene mutations 59T>G, 202T>C, 314C>T, 508G>A, and 1067T>A. The PCR-SSP method was validated on 20 healthy blood donors and 16 non-insulin-dependent diabetic patients. All individuals were in parallel genotyped by our established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The FUT3 genotypes, determined with the PCR-SSP method, were in complete accordance with the results of the PCR-RFLP reference method. The PCR-SSP method could also be adapted to assign the presence of a specific mutation to the respective FUT3 alleles. We found the method to be reliable, rapid and cheap with no requirements for restriction enzyme processing.
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22.
  • Grahn, Ammi, 1961, et al. (författare)
  • Glycobiology and cancer
  • 2009
  • Ingår i: Encyclopedia of Cancer. - Berlin : Springer Verlag. - 9783540476481
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)
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23.
  • Grahn, Ammi, 1961, et al. (författare)
  • Identification of seven new alpha2,3-sialyltransferase III, ST3Gal III, transcripts from human foetal brain
  • 2004
  • Ingår i: Glycoconj J. ; 20:7-8, s. 493-500
  • Tidskriftsartikel (refereegranskat)abstract
    • We have recently cloned and sequenced 19 human ST3Gal III gene isotranscripts from peripheral blood leukocytes and identified very complex patterns of isotranscripts of this gene in neuronal tissues. We have now cloned and sequenced additionally seven new isotranscripts from foetal brain. These novel isotranscripts showed losses of complete exons along the whole length of the coding sequence. None of the new isotranscripts coded for proteins with the two (L- and S-) sialylmotifs intact. One of the isotranscripts belonged to the isoform ST3Gal III -B, five to the ST3Gal III -C isoform and one to ST3Gal III -D isoform of isotranscripts, which lacks exon 3, exons 3 and 4 and exon 4 respectively. Two of the C series isotranscripts, ST3Gal III C4 and C11 had both lost exons 12 and 13 containing the S-motif but had otherwise the L- and the VS-motifs intact. Three isotranscripts, ST3Gal III C5, C12 and D5, were similar in the 3'-end coding for an identical amino acid sequence unrelated to the original enzyme. Isotranscripts ST3Gal III C9 and B10 were distinctly different from all other forms identified so far. The splice variants reported here are unlikely to express enzymatic activities but may other biological functions.
  •  
24.
  • Haga, K., et al. (författare)
  • Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection
  • 2020
  • Ingår i: mBio. - 2150-7511. ; 11:2
  • Tidskriftsartikel (refereegranskat)abstract
    • Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme, while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals, while HIEs from secretors are permissive to infection. However, whether FUT2 expression alone is critical for infection remains unproven, since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication, we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies, these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status. IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however, previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GII and several GII HuNoV strains.
  •  
25.
  • Halim, Adnan, et al. (författare)
  • Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC-MS/MS of Glycopeptides.
  • 2014
  • Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 13:12, s. 6024-32
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.
  •  
26.
  • Halim, Adnan, et al. (författare)
  • Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD.
  • 2012
  • Ingår i: Molecular & cellular proteomics : MCP. - 1535-9484. ; 11:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.
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27.
  • Halim, Adnan, et al. (författare)
  • LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins.
  • 2013
  • Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:2, s. 573-584
  • Tidskriftsartikel (refereegranskat)abstract
    • The GalNAc O-glycosylation on Ser/Thr residues of extracellular proteins has not been well characterized from a proteomics perspective. We previously reported a sialic acid capture-and-release protocol to enrich tryptic N- and O-glycopeptides from human cerebrospinal fluid glycoproteins using nanoLC-ESI-MS/MS with collision-induced dissociation (CID) for glycopeptide characterization. Here, we have introduced peptide N-glycosidase F (PNGase F) pre-treatment of CSF samples to remove the N-glycans facilitating the selective characterization of O-glycopeptides and enabling the use of an automated CID-MS2/MS3 search protocol for glycopeptide identification. We used electron capture and transfer dissociation (ECD/ETD) to pinpoint the glycosylation site(s) of the glycopeptides, identified as predominantly core 1 like HexHexNAc-O- glycans attached to 1-4 Ser/Thr residues. We characterized 108 O-glycosylations and found Pro residues preferentially in the n-1, n+1 and/or n+3 positions in relation to the Ser/Thr attachment site (n). The characterization of glycans and glycosylation sites in glycoproteins from human clinical samples provides a basis for future studies addressing the biological and diagnostic importance of specific protein glycosylations in relation to human disease.
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28.
  • Halim, Adnan, et al. (författare)
  • Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid {beta}-peptides in human cerebrospinal fluid.
  • 2011
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 108:29, s. 11848-53
  • Tidskriftsartikel (refereegranskat)abstract
    • The proteolytic processing of human amyloid precursor protein (APP) into shorter aggregating amyloid β (Aβ)-peptides, e.g., Aβ1-42, is considered a critical step in the pathogenesis of Alzheimer's disease (AD). Although APP is a well-known membrane glycoprotein carrying both N- and O-glycans, nothing is known about the occurrence of released APP/Aβ glycopeptides in cerebrospinal fluid (CSF). We used the 6E10 antibody and immunopurified Aβ peptides and glycopeptides from CSF samples and then liquid chromatography-tandem mass spectrometry for structural analysis using collision-induced dissociation and electron capture dissociation. In addition to 33 unglycosylated APP/Aβ peptides, we identified 37 APP/Aβ glycopeptides with sialylated core 1 like O-glycans attached to Thr(-39, -21, -20, and -13), in a series of APP/AβX-15 glycopeptides, where X was -63, -57, -52, and -45, in relation to Asp1 of the Aβ sequence. Unexpectedly, we also identified a series of 27 glycopeptides, the Aβ1-X series, where X was 20 (DAEFRHDSGYEVHHQKLVFF), 19, 18, 17, 16, and 15, which were all uniquely glycosylated on Tyr10. The Tyr10 linked O-glycans were (Neu5Ac)(1-2)Hex(Neu5Ac)HexNAc-O- structures with the disialylated terminals occasionally O-acetylated or lactonized, indicating a terminal Neu5Acα2,8Neu5Ac linkage. We could not detect any glycosylation of the Aβ1-38/40/42 isoforms. We observed an increase of up to 2.5 times of Tyr10 glycosylated Aβ peptides in CSF in six AD patients compared to seven non-AD patients. APP/Aβ sialylated O-glycans, including that of a Tyr residue, the first in a mammalian protein, may modulate APP processing, inhibiting the amyloidogenic pathway associated with AD.
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29.
  • Hedberg, Carola, 1969, et al. (författare)
  • Hereditary myopathy with early respiratory failure is associated with misfolding of the titin fibronectin III 119 subdomain
  • 2014
  • Ingår i: Neuromuscular Disorders. - : Elsevier BV. - 0960-8966. ; 24:5, s. 373-379
  • Tidskriftsartikel (refereegranskat)abstract
    • Hereditary myopathy with early respiratory failure is a rare disease with muscle weakness and respiratory failure as early symptoms. Muscle pathology is characterized by the presence of multiple cytoplasmic bodies and other protein aggregates in muscle fibers. The disease is associated with mutations in the titin gene (TTN). All patients harbor mutations located in exon 343 in the TTN gene that codes for the fibronectin III domain 119 (FN3 119) in the 10th motif of the 11-element motif A-band super-repeat. We investigated how such disease-causing mutations affect the biochemical behavior of this titin domain. All five disease-causing amino acid changes analyzed by us (p.P30068R, p.C30071R, p.W30088R, p.W30088C and p.P30091L) resulted in impaired FN3 119 domain solubility. In contrast, amino acid changes associated with common SNPs (p.V30076I, p.R30107C and p.S30125F) did not have this effect. In silica analyses further support the notion that disease-causing mutations impair proper folding of the FN3 119 domain. The results suggest that hereditary myopathy with early respiratory failure is caused by defective protein folding. (C) 2014 Elsevier B.V. All rights reserved.
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30.
  • Hesse, Camilla, et al. (författare)
  • The N-terminal domain of α-dystroglycan is released as a 38kDa protein and is increased in cerebrospinal fluid in patients with Lyme neuroborreliosis.
  • 2011
  • Ingår i: Biochemical and biophysical research communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 412:3, s. 494-499
  • Tidskriftsartikel (refereegranskat)abstract
    • α-Dystroglycan is an extracellular adhesion protein that is known to interact with different ligands. The interaction is thought to stabilize the integrity of the plasma membrane. The N-terminal part of α-dystroglycan may be proteolytically processed to generate a small 38kDa protein (α-DG-N). The physiological significance of α-DG-N is unclear but has been suggested to be involved in nerve regeneration and myelination and to function as a potential biomarker for neurodegenerative and neuromuscular diseases. In this report we show that α-DG-N is released into different body fluids, such as lachrimal fluid, cerebrospinal fluid (CSF), urine and plasma. To investigate the significance of α-DG-N in CSF we examined the levels of α-DG-N and known neurodegenerative markers in CSF from patients diagnosed with Lyme neuroborreliosis (LNB) and healthy controls. In untreated acute phase LNB patients, 67% showed a significant increase of CSF α-DG-N compared to healthy controls. After treatment with antibiotics the CSF α-DG-N levels were normalized in the LNB patients.
  •  
31.
  • Kawahara, R., et al. (författare)
  • Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis
  • 2021
  • Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 18, s. 1304-1316
  • Tidskriftsartikel (refereegranskat)abstract
    • Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics. This analysis presents the results of a community-based evaluation of existing software for large-scale glycopeptide data analysis.
  •  
32.
  • Kindberg, Elin, 1981-, et al. (författare)
  • Host genetic resistance to symptomatic norovirus (GGII.4) infections in Denmark
  • 2007
  • Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 45:8, s. 2720-2
  • Tidskriftsartikel (refereegranskat)abstract
    • A total of 61 individuals involved in five norovirus outbreaks in Denmark were genotyped at nucleotides 428 and 571 of the FUT2 gene, determining secretor status, i.e., the presence of ABH antigens in secretions and on mucosa. A strong correlation (P = 0.003) was found between the secretor phenotype and symptomatic disease, extending previous knowledge and confirming that nonsense mutations in the FUT2 gene provide protection against symptomatic norovirus (GGII.4) infections. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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33.
  • Koppisetty, Ashok Krishna Chaitanya, 1982, et al. (författare)
  • Computational studies on the interaction of ABO-active saccharides with the norovirus VA387 capsid protein can explain experimental binding data
  • 2010
  • Ingår i: Journal of Computer-Aided Molecular Design. - : Springer Science and Business Media LLC. - 0920-654X .- 1573-4951. ; 24:5, s. 423-431
  • Tidskriftsartikel (refereegranskat)abstract
    • Norovirus strains are known to cause recurring epidemics of winter vomiting disease. The crystal structure of the capsid protein of VA387, a representative of the clinically important GII.4 genocluster, was recently solved in complex with histo-blood group A- and B-trisaccharides. However, the VA387 strain is known to bind also to other natural carbohydrates for which detailed structural information of the complexes is not available. In this study we have computationally explored the fit of the VA387 with a set of naturally occurring carbohydrate ligands containing a terminal alpha 1,2-linked fucose. MD simulations both with explicit and implicit solvent models indicate that type 1 and 3 extensions of the ABO-determinant including ALe(b) and BLe(b) pentasaccharides can be well accommodated in the site. Scoring with Glide XP indicates that the downstream extensions of the ABO-determinants give an increase in binding strength, although the alpha 1,2-linked fucose is the single strongest interacting residue. An error was discovered in the geometry of the GalNAc-Gal moiety of the published crystal structure of the A-trisaccharide/VA387 complex. The present modeling of the complexes with histo-blood group A-active structures shows some contacts which provide insight into mutational data, explaining the involvement of I389 and Q331. Our results can be applicable in structure-based design of adhesion inhibitors of noroviruses.
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34.
  • Kunze, Angelika, 1978, et al. (författare)
  • Equilibrium-fluctuation-analysis of single liposome binding events reveals how cholesterol and Ca2+ modulate glycosphingolipid trans-interactions
  • 2013
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 3
  • Tidskriftsartikel (refereegranskat)abstract
    • Carbohydrate-carbohydrate interactions (CCIs) are of central importance for several biological processes. However, the ultra-weak nature of CCIs generates difficulties in studying this interaction, thus only little is known about CCIs. Here we present a highly sensitive equilibrium-fluctuation-analysis of single liposome binding events to supported lipid bilayers (SLBs) based on total internal reflection fluorescence (TIRF) microscopy that allows us to determine apparent kinetic rate constants of CCIs. The liposomes and SLBs both contained natural Le(x) glycosphingolipids (Gal beta 4( Fuc alpha 3) GlcNAc beta 3Gal beta 4Glc beta 1Cer), which were employed to mimic cell-cell contacts. The kinetic parameters of the self-interaction between Le(x)-containing liposomes and SLBs were measured and found to be modulated by bivalent cations. Even more interestingly, upon addition of cholesterol, the strength of the CCIs increases, suggesting that this interaction is strongly influenced by a cholesterol-dependent presentation and/or spatial organization of glycosphingolipids in cell membranes.
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35.
  • Larson, Göran, 1953, et al. (författare)
  • Typing for the human lewis blood group system by quantitative fluorescence-activated flow cytometry: large differences in antigen presentation on erythrocytes between A(1), A(2), B, O phenotypes.
  • 1999
  • Ingår i: Vox sanguinis. - 0042-9007. ; 77:4, s. 227-36
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: Lewis phenotyping by hemagglutination is an unreliable routine method for Lewis antigen designation. Now genomic typing of the Lewis gene is available. Additionally, flow cytometry has been used for typing. We wanted to compare the results of Lewis typing in healthy individuals using the three methods. MATERIALS AND METHODS: Ninety-three randomly selected plasma donors were genotyped for inactivating Secretor (FUT2) G428A and Lewis (FUT3) T59G, T202C, C314T, G508A and T1067A point mutations. All Le(a+b-) individuals (nonsecretors) were homozygous for the FUT2 G428A mutation and all Le(a-b-) individuals had inactivating mutations on both FUT3 alleles. Fixed erythrocytes were analyzed by fluorescence-activated flow cytometry and the results were compared with hem- agglutination and genotypic data. Antigen availability was expressed as median fluorescence intensity and as percentage positive cells with fluorescence intensities > or =10(2). RESULTS: Using an anti-Le(a) reagent a mean of 99% of erythrocytes from Le(a+b-) individuals and 1% of erythrocytes from Le(a-b-) or Le(a-b+) individuals were stained positive. Using an anti-Le(b) reagent, a mean of 71% of erythrocytes from A(1), 95% from B and 99% from O and A(2) Le(a-b+) individuals and less than 10% of erythrocytes from Le(a-b-) or Le(a+b-) individuals were stained positive. After papain treatment 100% of the erythrocytes from A(1) and A(1)B Le(a-b+) individuals stained positive without increase in background staining. The flow cytometric technique revealed large differences in staining intensities, within each ABO Le(a-b+) subgroup which was not directly correlated to plasma donation frequencies nor to Secretor or Lewis genotypes. CONCLUSION: Flow cytometry may prove valuable as a Lewis blood group typing technique but also as a research tool when investigating Lewis phenotypes of human erythrocytes.
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36.
  • Le Pendu, J., et al. (författare)
  • Human Norovirus Receptors
  • 2016
  • Ingår i: Viral Gastroenteritis: Molecular Epidemiology and Pathogenesis. - : Elsevier. - 9780128026595 ; , s. 379-396
  • Bokkapitel (refereegranskat)abstract
    • Due to the lack of cell culture methods, studies of human norovirus receptors have been limited to virus challenge and outbreak studies and in vitro binding studies of virus-like particles (VLPs). Norovirus VLPs recognize glycans from the ABO(H) and Lewis histo-blood group family on glycoproteins and glycosphingolipids in a strain-dependent manner. Host genetic studies have shown that for the clinically dominating strains the binding pattern correlates with susceptibility to infection. Particularly, the so-called nonsecretors, characterized by their lack of expression of ABO(H) structures in the gastrointestinal tract, have been identified as resistant to infections by many strains. In support of a receptor status, norovirus VLPs have been shown to bind to intestinal epithelium expressing ABO(H) epitopes, to intestinal glycosphingolipids carrying ABO(H) epitopes and to induce membrane invaginations, resembling endocytosis intermediates, on model membranes carrying such glycosphingolipids. However, in addition to the ABO(H) structures, the VLPs also recognizes other glycans that could play an important role in virus entry into the host cells. © 2016 Elsevier Inc. All rights reserved.
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37.
  • Leymarie, N., et al. (författare)
  • Interlaboratory Study on Differential Analysis of Protein Glycosylation by Mass Spectrometry: The ABRF Glycoprotein Research Multi-Institutional Study 2012
  • 2013
  • Ingår i: Molecular & Cellular Proteomics. - 1535-9476. ; 12:10, s. 2935-2951
  • Tidskriftsartikel (refereegranskat)abstract
    • One of the principal goals of glycoprotein research is to correlate glycan structure and function. Such correlation is necessary in order for one to understand the mechanisms whereby glycoprotein structure elaborates the functions of myriad proteins. The accurate comparison of glycoforms and quantification of glycosites are essential steps in this direction. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information. As part of the 2012 ABRF Glycoprotein Research Group study, we explored the use of mass spectrometry and ancillary methodologies to characterize the glycoforms of two sources of human prostate specific antigen (PSA). PSA is used as a tumor marker for prostate cancer, with increasing blood levels used to distinguish between normal and cancer states. The glycans on PSA are believed to be biantennary N-linked, and it has been observed that prostate cancer tissues and cell lines contain more antennae than their benign counterparts. Thus, the ability to quantify differences in glycosylation associated with cancer has the potential to positively impact the use of PSA as a biomarker. We studied standard peptide-based proteomics/glycomics methodologies, including LC-MS/MS for peptide/glycopeptide sequencing and label-free approaches for differential quantification. We performed an interlaboratory study to determine the ability of different laboratories to correctly characterize the differences between glycoforms from two different sources using mass spectrometry methods. We used clustering analysis and ancillary statistical data treatment on the data sets submitted by participating laboratories to obtain a consensus of the glycoforms and abundances. The results demonstrate the relative strengths and weaknesses of top-down glycoproteomics, bottom-up glycoproteomics, and glycomics methods. T6G 2G2, Canada. [Cipollo, John F.; An, Yanming] US FDA, Ctr Biol Evaluat & Res, Bethesda, MD 20993 USA. [Desaire, Heather; Go, Eden P.] Univ Kansas, Lawrence, KS 66045 USA. [Goldman, Radoslav; Pompach, Petr; Sanda, Miloslav] Georgetown Univ, Dept Oncol, Washington, DC [Halim, Adnan; Larson, Goran; Nilsson, Jonas] Univ Gothenburg, Sahlgrenska Acad, Dept Clin Chem & [Hensbergen, Paul J.; Wuhrer, Manfred] Leiden Univ, Med Ctr, Biomol Mass Spectrometry Unit, NL- [Jabs, Wolfgang; Marx, Kristina; Resemann, Anja; Schweiger-Hufnagel, Ulrike; Suckau, Detlev] Bruker [Ly, Mellisa; Staples, Gregory O.] Agilent Technol, Agilent Labs, Santa Clara, CA 95051 USA. [Mechref, Yehia; Song, Ehwang] Texas Tech Univ, Dept Chem & Biochem, Lubbock, TX 79409 USA. [Nyalwidhe, Julius O.; Watson, Megan] Eastern Virginia Med Sch, Leroy T Canoles Jr Canc Res Ctr, Dept [Packer, Nicolle H.; Thaysen-Andersen, Morten] Macquarie Univ, Dept Chem & Biomol Sci, Biomol [Sihlbom, Carina] Gothenburg Univ, Prote Core Facil, Gothenburg, Sweden. [Tang, Haixu] Indiana Univ, Sch Informat, Bloomington, IN 47405 USA. [Valmuv, Leena] Finnish Red Cross Blood Serv, Helsinki 00310, Finland. [Wada, Yoshinao] Osaka Med Ctr Maternal & Child Hlth, Res Inst, Izumi Ku, Osaka 5941101, Japan.
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38.
  • Mehmedbasic, Arnela, et al. (författare)
  • SorLA CR-Domains Protect the Amyloid Precursor Protein against Processing.
  • 2015
  • Ingår i: The Journal of biological chemistry. - 1083-351X. ; 290, s. 3359-3376
  • Tidskriftsartikel (refereegranskat)abstract
    • SorLA is a neuronal sorting receptor that is genetically associated with Alzheimer's disease. SorLA interacts directly with the amyloid precursor protein (APP) and affects the processing of the precursor, leading to a decreased generation of the amyloid-β (Aβ) peptide. The sorLA complement-type repeat (CR)-domains associate in vitro with APP, but the precise molecular determinants of sorLA-APP complex formation and the mechanisms responsible for the effect of binding on APP processing have not yet been elucidated. Here, we have generated protein expression constructs for sorLA devoid of the 11 CR-domains and for two sorLA mutants harboring substitutions of the fingerprint residues in the central CR-domains. We generated SH-SY5Y cell lines that stably express these sorLA variants to study the binding and processing of APP using co-immunoprecipitation and western blotting/ELISA assays, respectively. We found that the sorLA CR-cluster is essential for interaction with APP and that deletion of the CR-cluster abolishes the protection against APP processing. Mutation of identified fingerprint residues in the sorLA CR-domains leads to changes in the O-linked glycosylation of APP when expressed in SH-SY5Y cells. Our results provide novel information on the mechanisms behind the influence of sorLA activity on APP metabolism by controlling post-translational glycosylation in the Golgi, suggesting new strategies against amyloidogenesis in Alzheimer's disease.
  •  
39.
  • Mirgorodskaya, Ekaterina, et al. (författare)
  • Cracking the Sugar Code by Mass Spectrometry
  • 2018
  • Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305. ; 29:6, s. 1065-1074
  • Tidskriftsartikel (refereegranskat)abstract
    • The structural study of glycans and glycoconjugates is essential to assign their roles in homeostasis, health, and disease. Once dominated by nuclear magnetic resonance spectroscopy, mass spectrometric methods have become the preferred toolbox for the determination of glycan structures at high sensitivity. The patterns of such structures in different cellular states now allow us to interpret the sugar codes in health and disease, based on structure-function relationships. Dr. Catherine E. Costello was the 2017 recipient of the American Society for Mass Spectrometry's Distinguished Contribution Award. In this Perspective article, we describe her seminal work in a historical and geographical context and review the impact of her research accomplishments in the field.
  •  
40.
  • Modin Larsson, Malin, 1980-, et al. (författare)
  • Antibody prevalence and titer to norovirus (genogroup II) correlate with secretor (FUT2) but not with ABO phenotype or Lewis (FUT3) genotype
  • 2006
  • Ingår i: J Infect Dis. - : Oxford University Press. ; 194:10, s. 1422-1427
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: Histo-blood group antigens and secretor status have been associated with susceptibility to Norovirus infections, which suggests that antibody prevalence and titer might correlate with these phenotypes. METHODS: Plasma samples (n = 105) from Swedish blood donors that had been genotyped for secretor (FUT2) and Lewis (Le; FUT3) genotypes and phenotyped for ABO and Le blood groups were analyzed for immunoglobulin G antibody prevalence and titers to norovirus genogroup (GG) II.4. RESULTS: The results showed that nonsecretors (se4128se428) and Lea+b- individuals not only had significantly lower antibody titers than did secretors (P < .0001) and Lea-b+ individuals (P < .0002) but were also significantly more often antibody negative (P < .05). Antibody titers in secretors were not significantly different between individuals of different Le (FUT3) genotypes or different ABO phenotypes. CONCLUSIONS: Nonsecretors and Lea+b- individuals are significantly less prone to be infected with GGII noroviruses. This new information extends previous knowledge and supports the hypothesis that nonsecretors are relatively but not absolutely resistant to norovirus infections.
  •  
41.
  • Mottram, Lynda, et al. (författare)
  • FUT2 non-secretor status is associated with altered susceptibility to symptomatic enterotoxigenic Escherichia coli infection in Bangladeshiw
  • 2017
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Polymorphisms of the FUT2 gene alters glycan ABO(H) blood group and Lewis antigen expression (commonly known as non-secretor status) in the small intestinal mucosa. Whilst non-secretor status affects 20% of the population worldwide, it has been reported to be present in up to 40% of all Bangladeshis. Furthermore, Bangladeshi children are reportedly more susceptible to symptomatic enterotoxigenic Escherichia coli (ETEC) infection if they are non-secretors. Therefore, in an attempt to identify a non-secretor status genotypic biomarker of altered susceptibility to ETEC infection, we used the 1000 Genomes Project to identify three population related non-synonymous FUT2 single nucleotide polymorphisms (SNPs). We then assessed the genotypic frequency of these SNPs in Bangladeshi children who had been clinically monitored for ETEC infection. One novel missense FUT2 SNP, rs200157007-TT and the earlier established rs601338-AA SNP were shown to be causing non-secretor status, with these SNPs being associated with symptomatic but not asymptomatic ETEC infection. Moreover, rs200157007-TT and rs601338-AA were associated with symptomatic but not asymptomatic ETEC infection irrespective of the child's Lewis secretor status, suggesting FUT2, the regulator of Lewis and ABO(H) antigens in the intestinal mucosa, could be a host genotypic feature affecting susceptibility to ETEC infection.
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42.
  • Naguib, Mahmoud, et al. (författare)
  • A Comparison of Host Responses to Infection with Wild-Type Avian Influenza Viruses in Chickens and Tufted Ducks
  • 2023
  • Ingår i: Microbiology Spectrum. - : American Society for Microbiology. - 2165-0497. ; 11:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV.IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks show a limited immune response to infection with low pathogenic avian influenza viruses. These findings and future studies in different IAV hosts are important for our understanding of barriers to IAV transmission between species and ultimately from the wild reservoir to humans. Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species.
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43.
  • Nasir, Waqas, et al. (författare)
  • Histo-Blood Group Antigen Presentation Is Critical for Binding of Norovirus VLP to Glycosphingolipids in Model Membranes
  • 2017
  • Ingår i: Acs Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 12:5, s. 1288-1296
  • Tidskriftsartikel (refereegranskat)abstract
    • Virus entry depends on biomolecular recognition at the surface of cell membranes. In the case of glycolipid receptors, these events are expected to be influenced by how the glycan epitope close to the membrane is presented to the virus. This presentation of membrane associated glycans is more restricted than that of glycans in solution, particularly because of orientational constraints imposed on the glycolipid through its lateral interactions with other membrane lipids and proteins. We have developed and employed a total internal reflection fluorescence microscopy-based binding assay and. a scheme for molecular dynamics (MD) membrane simulations to investigate the consequences of various glycan presentation effects. The system studied was histo-blood group antigen (HBGA) epitopes of membrane-bound glycosphingolipids (GSLs) derived from small intestinal epithelium of humans (type 1 chain) and dogs (type 2 chain) interacting with GII.4 norovirus-like particles. Our experimental results showed strong binding to all lipid-linked type 1 chain HBGAs but no or only weak binding to the corresponding type 2 chain HBGAs. This is in contrast to results derived from STD experiments with free HBGAs in solution where binding was observed for Lewis x. The MD data suggest that the strong binding to type 1 chain glycolipids was due to the well-exposed (1,2)-linked alpha-L-Fucp and (1,4)- linked alpha-L-Fucp residues, while the weaker binding or lack of binding to type 2 chain HBGAs was due to the very restricted accessibility of the (1,3) -linked alpha-L-Fucp residue when the glycolipid is embedded in a phospholipid membrane. Our results not only contribute to a general understanding of protein carbohydrate interactions on model membrane surfaces, particularly in the context of virus binding, but also suggest a possible role of human intestinal GSLs as potential receptors for norovirus uptake.
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44.
  • Nasir, Waqas, et al. (författare)
  • Interaction of Virus-Like Particles with Vesicles Containing Glycolipids: Kinetics of Detachment
  • 2015
  • Ingår i: The Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 119:35, s. 11466-11472
  • Tidskriftsartikel (refereegranskat)abstract
    • Many viruses interact with their host cells via glycosphingolipids (GSLs) and/or glycoproteins present on the outer cell membrane. This highly specific interaction includes virion attachment and detachment. The residence time determined by the detachment is particularly interesting, since it is directly related to internalization and infection as well as to virion egress and spreading. In an attempt to deepen the understanding of virion detachment kinetics, we have used total internal reflection fluorescence (TIRF) microscopy to probe the interaction between individual fluorescently labeled GSL-containing lipid vesicles and surface-bound virus-like particles (VLPs) of a norovirus genotype II.4 strain. The distribution of the VLP-vesicle residence time was investigated for seven naturally occurring GSLs, all of which are candidates for the not yet identified receptor(s) mediating norovirus entry into host cells. As expected for interactions involving multiple GSL binding sites at a viral capsid, the detachment kinetics displayed features typical for a broad activation-energy distribution for all GSLs. Detailed inspection of these distributions revealed significant differences among the different GSLs. The results are discussed in terms of strength of the interaction, vesicle size, as well as spatial distribution and clustering of GSLs in the vesicle membrane. (Figure Presented).
  •  
45.
  • Nasir, Waqas, et al. (författare)
  • SweetNET: A Bioinformatics Workflow for Glycopeptide MS/MS Spectral Analysis
  • 2016
  • Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 15:8, s. 2826-2840
  • Tidskriftsartikel (refereegranskat)abstract
    • Glycoproteomics has rapidly become an independent analytical platform bridging the fields of glycomics and proteomics to address site-specific protein glycosylation and its impact in biology. Current glycopeptide characterization relies on time-consuming manual interpretations and demands high levels of personal expertise. Efficient data interpretation constitutes one of the major challenges to be overcome before true high throughput glycopeptide analysis can be achieved. The development of new glyco-related bioinformatics tools is thus of crucial importance to fulfill this goal. Here we present SweetNET: a data-oriented bioinformatics workflow for efficient analysis of hundreds of thousands of glycopeptide MS/MS-spectra. We have analyzed MS data sets from two separate glycopeptide enrichment protocols targeting sialylated glycopeptides and chondroitin sulfate linkage region glycopeptides, respectively. Molecular networking was performed to organize the glycopeptide MS/MS data based on spectral similarities. The combination of spectral clustering, oxonium ion intensity profiles, and precursor ion m/z shift distributions provided typical signatures for the initial assignment of different N-, O- and CS-glycopeptide classes and their respective glycoforms. These signatures were further used to guide database searches leading to the identification and validation of a large number of glycopeptide variants including novel deoxyhexose (fucose) modifications in the linkage region of chondroitin sulfate proteoglycans.
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46.
  • Nikpour, Mahnaz, 1980, et al. (författare)
  • Glycosaminoglycan linkage region of urinary bikunin as a potentially useful biomarker for β3GalT6-deficient spondylodysplastic Ehlers-Danlos syndrome.
  • 2022
  • Ingår i: JIMD reports. - : Wiley. - 2192-8304 .- 2192-8312. ; 63:5, s. 462-467
  • Tidskriftsartikel (refereegranskat)abstract
    • The spondylodysplastic type of Ehlers-Danlos syndrome (spEDS) is caused by genetic defects in the B4GALT7 or B3GALT6 genes both deranging the biosynthesis of the glycosaminoglycan linkage region of chondroitin/dermatan sulfate and heparan sulfate proteoglycans. In this study, we have analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of three siblings, diagnosed with spEDS and carrying biallelic pathogenic variants of the B3GALT6 gene. Proteoglycans were digested with trypsin, glycopeptides enriched on anion-exchange columns, depolymerized with chondroitinase ABC, and analyzed by nLC-MS/MS. In urine of the unaffected mother, the dominating glycopeptide of bikunin/protein AMBP appeared as only one dominating (99.9%) peak with the canonical tetrasaccharide linkage region modification. In contrast, the samples of the three affected siblings contained two different glycopeptide peaks, corresponding to the canonical tetrasaccharide and to the non-canonical trisaccharide linkage region modifications in individual ratios of 61/38, 73/27, and 59/41. We propose that the relative distribution of glycosaminoglycan linkage regions of urinary bikunin glycopeptides may serve as a phenotypic biomarker in a diagnostic test but also as a biomarker to follow the effect of future therapies in affected individuals.
  •  
47.
  • Nikpour, Mahnaz, 1980, et al. (författare)
  • Proteoglycan profiling of human, rat and mouse insulin-secreting cells
  • 2021
  • Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 31:8, s. 916-930
  • Tidskriftsartikel (refereegranskat)abstract
    • Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography tandem mass spectrometry. We identified CS modifications of chromogranin-A (CgA), islet amyloid polypeptide, secretogranin-1 and secretogranin-2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (CgA and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For CgA, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.
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48.
  • Nilsson, Jonas, 1970, et al. (författare)
  • A glycomic workflow for LC-MS/MS analysis of urine glycosaminoglycan biomarkers in mucopolysaccharidoses
  • 2023
  • Ingår i: Glycoconjugate Journal. - 0282-0080. ; 40:5, s. 523-40
  • Tidskriftsartikel (refereegranskat)abstract
    • In recent years, several rational designed therapies have been developed for treatment of mucopolysaccharidoses (MPS), a group of inherited metabolic disorders in which glycosaminoglycans (GAGs) are accumulated in various tissues and organs. Thus, improved disease-specific biomarkers for diagnosis and monitoring treatment efficacy are of paramount importance. Specific non-reducing end GAG structures (GAG-NREs) have become promising biomarkers for MPS, as the compositions of the GAG-NREs depend on the nature of the lysosomal enzyme deficiency, thereby creating a specific pattern for each subgroup. However, there is yet no straightforward clinical laboratory platform which can assay all MPS-related GAG-NREs in one single analysis. Here, we developed and applied a GAG domain mapping approach for analyses of urine samples of ten MPS patients with various MPS diagnoses and corresponding aged-matched controls. We describe a nano-LC-MS/MS method of GAG-NRE profiling, utilizing 2-aminobenzamide reductive amination labeling to improve the sensitivity and the chromatographic resolution. Diagnostic urinary GAG-NREs were identified for MPS types IH/IS, II, IIIc, IVa and VI, corroborating GAG-NRE as biomarkers for these known enzyme deficiencies. Furthermore, a significant reduction of diagnostic urinary GAG-NREs in MPS IH (n = 2) and MPS VI (n = 1) patients under treatment was demonstrated. We argue that this straightforward glycomic workflow, designed for the clinical analysis of MPS-related GAG-NREs in one single analysis, will be of value for expanding the use of GAG-NREs as biomarkers for MPS diagnosis and treatment monitoring.
  •  
49.
  • Nilsson, Jonas, 1970, et al. (författare)
  • Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS.
  • 2017
  • Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1879-1123 .- 1044-0305. ; 28:2, s. 229-41
  • Tidskriftsartikel (refereegranskat)abstract
    • Purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of glycopeptides, originating from protease digests of glycoproteins, enables site-specific analysis of protein N- and O-glycosylations. We have described a protocol to enrich, hydrolyze by chondroitinase ABC, and characterize chondroitin sulfate-containing glycopeptides (CS-glycopeptides) using positive mode LC-MS/MS. The CS-glycopeptides, originating from the Bikunin proteoglycan of human urine samples, had ΔHexAGalNAcGlcAGalGalXyl-O-Ser hexasaccharide structure and were further substituted with 0-3 sulfate and 0-1 phosphate groups. However, it was not possible to exactly pinpoint sulfate attachment residues, for protonated precursors, due to extensive fragmentation of sulfate groups using high-energy collision induced dissociation (HCD). To circumvent the well-recognized sulfate instability, we now introduced Na(+) ions to form sodiated precursors, which protected sulfate groups from decomposition and facilitated the assignment of sulfate modifications. Sulfate groups were pinpointed to both Gal residues and to the GalNAc of the hexasaccharide structure. The intensities of protonated and sodiated saccharide oxonium ions were very prominent in the HCD-MS2 spectra, which provided complementary structural analysis of sulfate substituents of CS-glycopeptides. We have demonstrated a considerable heterogeneity of the bikunin CS linkage region. The realization of these structural variants should be beneficial in studies aimed at investigating the importance of the CS linkage region with regards to the biosynthesis of CS and potential interactions to CS binding proteins. Also, the combined use of protonated and sodiated precursors for positive mode HCD fragmentation analysis will likely become useful for additional classes of sulfated glycopeptides. Graphical Abstract ᅟ.
  •  
50.
  • Nilsson, Johanna, et al. (författare)
  • Characterization of site-specific O-glycan structures within the mucin-like domain of alpha-dystroglycan from human skeletal muscle.
  • 2010
  • Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 20:9, s. 1160-9
  • Tidskriftsartikel (refereegranskat)abstract
    • The glycosylation of the extracellular protein alpha-dystroglycan is important for its ligand-binding activity, and altered or blocked glycosylation is associated with several forms of congenital muscular dystrophies. By immunoprecipitation and sialic acid capture-and-release enrichment strategies, we isolated tryptic glycopeptides of alpha-dystroglycan from human skeletal muscle. Nano-liquid chromatography tandem mass spectrometry was used to identify both glycopeptides and peptides corresponding to the mucin-like and C-terminal domain of alpha-dystroglycan. The O-glycans found had either Hex-O-Thr or HexNAc-O-Ser/Thr anchored structures, which were often elongated and frequently, but not always, terminated with sialic acid. The HexNAc-O-Ser/Thr, but not Hex-O-Thr glycopeptides, displayed heterogeneity regarding glycan core structures and level of glycosylation site occupancy. We demonstrate for the first time glycan attachment sites of the NeuAcHexHexNAcHex-O structure corresponding to the anticipated Neu5Acalpha3Galbeta4GlcNAcbeta2Man-O-glycan (sLacNAc-Man), within the mucin-like domain of human alpha-dystroglycan from human skeletal muscle. Twenty-five glycopeptides were characterized from human alpha-dystroglycan, which provide insight to the complex in vivo O-glycosylation of alpha-dystroglycan.
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