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Sökning: WFRF:(Nilsson Jonas 1970)

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1.
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2.
  • Johansson, Petra, 1974, et al. (författare)
  • Interaction of Helicobacter pylori with sialylated carbohydrates: the dependence on different parts of the binding trisaccharide Neu5Ac{alpha}3Gal{beta}4GlcNAc.
  • 2005
  • Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 15:6, s. 625-36
  • Tidskriftsartikel (refereegranskat)abstract
    • We have recently shown that binding of Helicobacter pylori to sialylated carbohydrates is dependent on the presence of the carboxyl group and the glycerol chain of Neu5Ac. In this work we investigated the importance of GlcNAc in the binding trisaccharide Neu5Acalpha3Galbeta4GlcNAc and the role of the N-acetamido groups of both Neu5Ac and GlcNAc. An important part of the project was epitope dissection, that is chemical derivatizations of the active carbohydrate followed by binding studies. In addition we used a panel of various unmodified carbohydrate structures in the form of free oligosaccharides or glycolipids. These were tested for binding by hemagglutination inhibition assay, TLC overlay tests, and a new quantitative approach using radiolabeled neoglycoproteins. The studies showed that the N-acetamido group of Neu5Ac is important for binding by H. pylori, whereas the same group of GlcNAc is not. In addition, Fuc attached to GlcNAc, as tested with sialyl-Lewis x, did not affect the binding. Free Neu5Ac was inactive as inhibitor, and Neu5Acalpha3Gal turned out to be active. The binding preference for neolacto structures was confirmed, although one strain also was inhibited by lacto chains. The combined results revealed that an intact Neu5Ac is crucial for the interactions with H. pylori. Parts of Gal also seem to be necessary, whereas the role of the GlcNAc is secondary. GlcNAc does influence binding, however, primarily serving as a guiding carrier for the binding epitope rather than being a part of the binding structure.
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3.
  • 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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4.
  • Nilsson, Johanna, et al. (författare)
  • LC-MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences.
  • 2014
  • Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1844:2, s. 398-405
  • Tidskriftsartikel (refereegranskat)abstract
    • Glycogen synthesis is initiated by self-glucosylation of the glycosyltransferases glycogenin-1 and -2 that, in the presence of UDP-glucose, form both the first glucose-O-tyrosine linkage, and then stepwise add a series of α1,4-linked glucoses to a growing chain of variable length. Glycogen-1 and -2 coexist in liver glycogen preparations where the proteins are known to form homodimers, and they also have been shown to interact with each other. In order to study how glycogenin-1 and -2 interactions may influence each other's glucosylations we setup a cell-free expression system for in vitro production and glucosylation of glycogenin-1 and -2 in various combinations, and used a mass spectrometry based workflow for the characterization and quantitation of tryptic glycopeptides originating from glycogenin-1 and -2. The analysis revealed that the self-glucosylation endpoint was the incorporation of 4-8 glucose units on Tyr 195 of glycogenin-1, but only 0-4 glucose units on Tyr-228 of glycogenin-2. The glucosylation of glycogenin-2 was enhanced to 2-4 glucose units by the co-presence of enzymatically active glycogenin-1. Glycogenin-2 was, however, unable to glucosylate inactive glycogenin-1, at least not an enzymatically inactivated Thr83Met glycogenin-1 mutant, recently identified in a patient with severe glycogen depletion.
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5.
  • Nilsson, Johanna, et al. (författare)
  • Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation.
  • 2012
  • Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1822:4, s. 493-9
  • Tidskriftsartikel (refereegranskat)abstract
    • Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short oligosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glucosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with glycogen depletion in skeletal muscle as a result of a non-functional glycogenin-1. The patient carried a Thr83Met substitution in glycogenin-1. In this study we have investigated the importance of threonine 83 for the catalytic activity of glycogenin-1. Non-glucosylated glycogenin-1 constructs, with various amino acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosylated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled UDP-(14)C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We demonstrate that glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated glycogenin-1.
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6.
  • Alaei-Mahabadi, Babak, et al. (författare)
  • Global analysis of somatic structural genomic alterations and their impact on gene expression in diverse human cancers.
  • 2016
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 113:48, s. 13768-13773
  • Tidskriftsartikel (refereegranskat)abstract
    • Tumor genomes are mosaics of somatic structural variants (SVs) that may contribute to the activation of oncogenes or inactivation of tumor suppressors, for example, by altering gene copy number amplitude. However, there are multiple other ways in which SVs can modulate transcription, but the general impact of such events on tumor transcriptional output has not been systematically determined. Here we use whole-genome sequencing data to map SVs across 600 tumors and 18 cancers, and investigate the relationship between SVs, copy number alterations (CNAs), and mRNA expression. We find that 34% of CNA breakpoints can be clarified structurally and that most amplifications are due to tandem duplications. We observe frequent swapping of strong and weak promoters in the context of gene fusions, and find that this has a measurable global impact on mRNA levels. Interestingly, several long noncoding RNAs were strongly activated by this mechanism. Additionally, SVs were confirmed in telomere reverse transcriptase (TERT) upstream regions in several cancers, associated with elevated TERT mRNA levels. We also highlight high-confidence gene fusions supported by both genomic and transcriptomic evidence, including a previously undescribed paired box 8 (PAX8)-nuclear factor, erythroid 2 like 2 (NFE2L2) fusion in thyroid carcinoma. In summary, we combine SV, CNA, and expression data to provide insights into the structural basis of CNAs as well as the impact of SVs on gene expression in tumors.
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7.
  • 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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8.
  • 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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9.
  • De Marco Verissimo, Carolina, et al. (författare)
  • Glycan Complexity and Heterogeneity of Glycoproteins in Somatic Extracts and Secretome of the Infective Stage of the Helminth Fasciola hepatica
  • 2023
  • Ingår i: Molecular and cellular proteomics : MCP. - 1535-9484. ; 22:12
  • Tidskriftsartikel (refereegranskat)abstract
    • Fasciola hepatica is a global helminth parasite of humans and their livestock. The invasive stage of the parasite, the newly excysted juvenile (NEJs), relies on glycosylated excreted-secreted (ES) products and surface/somatic molecules to interact with host cells and tissues and to evade the host's immune responses, such as disarming complement and shedding bound antibody. While -omics technologies have generated extensive databases of NEJs' proteins and their expression, detailed knowledge of the glycosylation of proteins is still lacking. Here, we employed glycan, glycopeptide, and proteomic analyses to determine the glycan profile of proteins within the NEJs' somatic (Som) and ES extracts. These analyses characterized 123 NEJ glycoproteins, 71 of which are secreted proteins, and allowed us to map 356 glycopeptides and their associated 1690 N-glycan and 37 O-glycan forms to their respective proteins. We discovered abundant micro-heterogeneity in the glycosylation of individual glycosites and between different sites of multi-glycosylated proteins. The global heterogeneity across NEJs' glycoproteome was refined to 53 N-glycan and 16 O-glycan structures, ranging from highly truncated paucimannosidic structures to complex glycans carrying multiple phosphorylcholine (PC) residues, and included various unassigned structures due to unique linkages, particularly in pentosylated O-glycans. Such exclusive glycans decorate some well-known secreted molecules involved in host invasion, including cathepsin B and L peptidases, and a variety of membrane-bound glycoproteins, suggesting that they participate in host interactions. Our findings show that F.hepatica NEJs generate exceptional protein variability via glycosylation, suggesting that their molecular portfolio that communicates with the host is far more complex than previously anticipated by transcriptomic and proteomic analyses. This study opens many avenues to understand the glycan biology of F.hepatica throughout its life-stages, as well as other helminth parasites, and allows us to probe the glycosylation of individual NEJs proteins in the search for innovative diagnostics and vaccines against fascioliasis.
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10.
  • DuttaRoy, Smita, 1971, et al. (författare)
  • The effects of age on circulating vascular markers and cardiac prognostic markers, before and after 2 months home-based high-frequency exercise training in patients with stable coronary artery disease
  • 2014
  • Ingår i: European Heart Journal. European Society of Cardiology, 30 August - 3 September 2014, Barcelona. - 0195-668X .- 1522-9645. ; 35:Supplement: 1
  • Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
    • Purpose: Vascular endothelial growth factor (VEGF) and stromal derived factor (SDF-1) play an important role in angiogenesis. Relaxin-2 (Rlx-2) has both angiogenic and vasodilatory properties, while endothelin-1 (ET-1) is a potent vasocontrictor.VEGF, SDF-1 and Rlx-2-levels have shown to be positively modulated by exercise training, while the effect of exercise on (Rlx-2) is not known. Age is a known risk factor for morbidity and mortality in coronary artery disease (CAD). We wanted to investigate how age affects levels of these vascular factors and known prognostic cardiac markers before and after high frequency exercise training (HFE), in patients with CAD. Methods: Patients with stable CAD (age 48-80 years) were randomized to HFE (aerobic exercise 70% of max, 30 minutes, 5 times/week and resistance exercise 3 times/week), performed at home for 8 weeks, or usual lifestyle (ctrl). Serum and plasma was collected from 21 controls and 24 HFE-patients and analyzed at baseline and after 8 weeks. VEGF, SDF-1, Rlx-2 and ET-1were analyzed with enzymelinked immunoadsorbent assay (ELISA). TnT and NT-pro-BNP were analyzed on Cobas e602 (Roche). Correlation was calculated using the statistical software Graph Pad Prism 6. Pearson’s r was calculated to determine correlation between the factors prior to exercise, while Spearman’s r was used for the analysis on the exercise induced effects of the HFE-group. The exercise-induced effect on cardiac biomarkers was determined by comparing % change (from baseline to 8 weeks) between HFE and Ctrl using Mann-Whitney U-test. Results: At baseline, there was a significant positive correlation between age and TnT (r=0.38, p<0.05) and a non-significant positive correlation between age and NT-proBNP (r=0.36, p=0.06), while no correlation was found between age and levels of vascular markers (VEGF r=-0,14, SDF-1 r=-0,13, ET-1 r=0,08, Rlx-2 r=0,06, p=ns for all). As we have previously shown, home-based HFE decreased VEGF (2,6+29% (ctrl) and -3,9 +13% (HFE), p<0,05), but the other studied factors were not significantly affected. We found no correlation between age and changes in cardiac markers after exercise. Conclusions: Elderly patients with stable CAD have higher levels of TnT and NT-proBNP, indicating a higher degree of underlying CAD. This may also reflect their higher mortality in CAD. HFE-training may lower VEGF in patients with stable CAD. Interestingly, there seems to be no difference in the respone response to exercise in cardiac biomarkers, between younger and older CAD patients
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11.
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12.
  • Eriksson, Carl, 1981-, et al. (författare)
  • Clinical effectiveness of golimumab in ulcerative colitis : a prospective multicentre study based on the Swedish IBD Quality Register, SWIBREG
  • 2021
  • Ingår i: Scandinavian Journal of Gastroenterology. - : Informa UK Limited. - 0036-5521 .- 1502-7708. ; 56:11, s. 1304-1311
  • Tidskriftsartikel (refereegranskat)abstract
    • Objectives: Clinical trials demonstrated that golimumab is effective in anti-TNF naïve patients with ulcerative colitis. We aimed to assess the clinical effectiveness of golimumab in a real-world setting. Materials and methods: This was a prospective cohort study, conducted at 16 Swedish hospitals. Data were collected using an electronic case report form. Patients with active ulcerative colitis, defined as Mayo endoscopic subscore ≥2 were eligible for inclusion. The primary outcomes were clinical effectiveness at 12 weeks and 52 weeks, i.e. response (defined as a decrease in Mayo score by ≥3 points or 30% from baseline) and remission (defined as a Mayo score of ≤2 with no individual subscores >1). Results: Fifty patients were included. At study entry, 70% were previously exposed to anti-TNF, 16% to vedolizumab, and 96% to immunomodulators. The 12 and 52-week drug continuation rates were 37/50 (74%) and 23/50 (46%), respectively. The 12-week response rate was 14/50 (28%), the remission rate, 8/50 (16%) and the corresponding figures at week 52 were 13/50 (26%) and 10/50 (20%). Among patients who continued golimumab, the median Mayo score decreased from 7 (6–9) at baseline to 1 (0–5) at 52 weeks (p <.01) and the faecal calprotectin decreased from 862 (335–1759) µg/g to 90 (34–169) µg/g (p <.01). Clinical response at week 12 was highly predictive of clinical remission at week 52 (adjusted OR: 73.1; 95% CI: 4.5‒1188.9). Conclusions: The majority of golimumab treated patients represented a treatment refractory patient-group. Despite this, our results confirm that golimumab is an effective therapy in ulcerative colitis.
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13.
  • Eriksson, C., et al. (författare)
  • Golimumab är effektivt vid ulcerös kolit under svenska förhållanden. Interimsanalys av en svensk prospektiv multi-centerstudie, GO-SWIBREG
  • 2018
  • Konferensbidrag (refereegranskat)abstract
    • Bakgrund: Randomiserade kontrollerade prövningar har visat effekt av golimumab vid ulcerös kolit men studiedeltagare och förhållanden i kliniska prövningar motsvarar inte alltid svensk klinisk vardag. Syftet med denna studie var att utvärdera säkerhet och effekt av behandling med golimumab vid ulcerös kolit under svenska förhållanden.Metod: Detta är en prospektiv kohortstudie med inklusion av patienter från svenska sjukhus. Patienter med måttlig till svår aktiv ulcerös kolit, definierad som endoskopiskt Mayo score ≥2 och som påbörjade golimumab fr.o.m. 1/6-2014 inkluderades efter att informerat samtycke inhämtats. Kliniska karakteristika, behandling, klinisk-, biokemisk- och endoskopisk aktivitet liksom skattning av livskvalité samlades in vid inklusion samt prospektivt med hjälp av ett elektroniskt studieformulär, integrerat i svenska kvalitetsregistret för IBD (SWIBREG). Primärt effektmått var klinisk effekt vid 3 samt 12 månader (definierat som minskat Mayo score med ≥3 poäng eller 30 % från inklusion), samt klinisk remission (definierad som Mayo score ≤ 2 utan några enskilda poäng >1). Kontinuerliga data presenteras som median och kvartilavstånd. För statistisk jämförelse mellan inklusion och uppföljning användes Wilcoxon-signed rank test. Data från induktionsbehandling samt 3-månadersuppföljning presenteras här.Resultat: 50 patienter inkluderades t.o.m. 15/9-2017. Vid studiestart var 24/50 (48 %) samtidigt behandlade med immunmodulerare, 16/50 (32 %) med perorala kortikosteroider samt 27/50 (54 %) med 5-ASA. Totalt hade 35/50 (70 %) tidigare fått behandling med minst en TNF-hämmare (tabell 1). Efter 12 veckor hade 37/50 (74 %), fortfarande behandling med golimumab. Av de patienter som fortsatte med golimumab till vecka 12 var 8 (22 %) i klinisk remission och 13 (35 %) uppvisade klinisk respons. Totalt Mayo score minskade i median från 7 (6-10) vid inklusion till 5 (1-8) vid 12 veckor (p<0.01). Fekalt calprotektin minskade från 710 (275-1850) µg/g till 390 (45-870) µg/g (p=0.02). Livskvalitet hos golimumab-behandlade patienter förbättrades, uppmätt som en signifikant minskning av poäng på short health scale (p=0.04).Slutsats: Golimumab-behandlade patienter i Sverige utgör en svårbehandlad grupp. Trots det kan förbättring av kliniska parametrar, inflammatorisk aktivitet och upplevd livskvalité uppnås redan efter 12 veckors golimumab-behandling.
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14.
  • 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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15.
  • Gritli Linde, Amel, 1959, et al. (författare)
  • Nuclear translocation of antizyme and expression of ornithine decarboxylase and antizyme are developmentally regulated
  • 2001
  • Ingår i: Developmental Dynamics. ; 220:3, s. 259-275
  • Tidskriftsartikel (refereegranskat)abstract
    • The polyamines are important regulators of cell growth and differentiation. Cells acquire polyamines by energy-dependent transport and by synthesis where the highly regulated ornithine decarboxylase (ODC) catalyzes the first and rate-controlling step. Inactivation of ODC is mainly exerted by antizyme (AZ), a 20--25 kDa polyamine-induced protein that binds to ODC, inactivates it, and targets it for degradation by the 26S proteasome without ubiquitination. In the present study, we have performed a systematic analysis of the expression of ODC and AZ, at the mRNA and protein levels, during mouse development. The expression patterns for ODC and AZ were found to be developmentally regulated, suggesting important functions for the polyamines in early embryogenesis, axonogenesis, epithelial-mesenchymal interaction, and in apoptosis. In addition, AZ protein was found to translocate to the nucleus in a developmentally regulated manner. The nuclear localization is consistent with the fact that the amino acid sequence of AZ exhibits features that characterize nuclear proteins. Interestingly, we found that cultivation of mandibular components of the first branchial arch in the presence of a selective proteasome inhibitor caused ODC accumulation in the nucleus of a subset of cells, suggesting that the observed nuclear translocation of AZ is linked to proteasome-mediated ODC degradation in the nucleus. The presence of AZ in the nucleus may suggest that nuclear ODC activity is under tight control, and that polyamine production can be rapidly interrupted when those developmental events, which depend on access to nuclear polyamines, have been completed.
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16.
  • Gupta, Priti, et al. (författare)
  • Impact of estrogen on IgG glycosylation and serum protein glycosylation in a murine model of healthy postmenopause.
  • 2023
  • Ingår i: Frontiers in endocrinology. - 1664-2392. ; 14
  • Tidskriftsartikel (refereegranskat)abstract
    • The glycosylation of immunoglobulin (Ig) G regulates IgG interaction capability with Fc gamma receptors found in all immune cells. In pathogenic conditions, estrogen can impact IgG levels and glycosylation. Following menopause, when estrogen levels decline affecting the immune system and potentially leading to a heightened susceptibility of immune activation.In this study, we aim to determine if estrogen levels can regulate IgG glycosylation in postmenopausal healthy situations.Mice were ovariectomized to simulate an estrogen-deficient postmenopausal status and then treated with 17-beta-estradiol (E2) at different doses and different administration strategies.Using a highly sensitive liquid chromatography-tandem mass spectrometry (MS/MS) glycoproteomic method, we demonstrated that E2 treatment increased the degree of glycosylation on IgG-Fc with both galactosylation and sialylation in the position required for interaction with Fc gamma receptors. We also observed that only long-term estrogen deficiency reduces IgG levels and that estrogen status had no impact on total IgG sialylation on both Fab and Fc domains or general glycoprotein sialylation evaluated by ELISA. Furthermore, E2 status did not affect the total sialic acid content of total cells in lymphoid organs and neither B cells nor plasma cells.The study concluded that E2 treatment does not affect total serum glycoprotein sialylation but alters IgG glycosylation, including IgG sialylation, implying that estrogen functions as an intrinsic modulator of IgG sialylation and could thereby be one pathway by which estrogen modulates immunity.
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17.
  • Göransson, Jenny, 1978-, et al. (författare)
  • A single molecule array for digital targeted molecular analyses
  • 2009
  • Ingår i: Nucleic Acids Research. - England : Oxford University Press. - 0305-1048 .- 1362-4962. ; 37:1, s. e7-
  • Tidskriftsartikel (refereegranskat)abstract
    • We present a new random array format together with a decoding scheme for targeted multiplex digital molecular analyses. DNA samples are analyzed using multiplex sets of padlock or selector probes that create circular DNA molecules upon target recognition. The circularized DNA molecules are amplified through rolling-circle amplification (RCA) to generate amplified single molecules (ASMs). A random array is generated by immobilizing all ASMs on a microscopy glass slide. The ASMs are identified and counted through serial hybridizations of small sets of tag probes, according to a combinatorial decoding scheme. We show that random array format permits at least 10 iterations of hybridization, imaging and dehybridization, a process required for the combinatorial decoding scheme. We further investigated the quantitative dynamic range and precision of the random array format. Finally, as a demonstration, the decoding scheme was applied for multiplex quantitative analysis of genomic loci in samples having verified copy-number variations. Of 31 analyzed loci, all but one were correctly identified and responded according to the known copy-number variations. The decoding strategy is generic in that the target can be any biomolecule which has been encoded into a DNA circle via a molecular probing reaction.
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18.
  • 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.
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19.
  • 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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20.
  • 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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21.
  • 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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22.
  • Hedsten, Karin, 1964, et al. (författare)
  • MEMS-based VCSEL beam steering using replicated polymer diffractive lens
  • 2008
  • Ingår i: Sensors and Actuators, A: Physical. - : Elsevier BV. - 0924-4247 .- 1873-3069. ; 142:1, s. 336-345
  • Tidskriftsartikel (refereegranskat)abstract
    • Abstract. This paper describes a fully integrated micro-optical system, in which dynamic angular control of the beam from a VCSEL (vertical cavity surface emitting laser) is realized by laterally moving a collimat¬ing diffractive lens in the light path. The lens is mounted on a translatable silicon stage, which consists of a frame with an opening for the light to traverse the lens and electro-statically driven comb actuators, by which the lateral movement is achieved. Devices implementing both 1D and 2D scanning have been fabricated and evaluated. Integration of the lens onto the translatable silicon stage is done using a newly developed fabrication process based on hot embossing of an amorphous fluorocarbon polymer. This fabrication process relies on a reversed-order protocol, where the structuring of the optical element precedes the silicon microstructuring. Assembly and packaging of the VCSEL-MOEMS system, using LTCC (low temperature cofired ceramic) technique, is also demonstrated. Optical evaluation of the system and beam steering function shows significant beam deflection for a relatively low driving voltage (~70 V).
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23.
  • Jang, Su Chul, 1984, et al. (författare)
  • Bioinspired Exosome-Mimetic Nanovesicles for Targeted Delivery of Chemotherapeutics to Malignant Tumors.
  • 2013
  • Ingår i: ACS nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 7:9, s. 7698-7710
  • Tidskriftsartikel (refereegranskat)abstract
    • Exosomes, the endogenous nanocarriers that can deliver biological information between cells, were recently introduced as new kind of drug delivery system. However, mammalian cells release relatively low quantities of exosomes, and purification of exosomes is difficult. Here, we developed bioinspired exosome-mimetic nanovesicles that deliver chemotherapeutics to the tumor tissue after systemic administration. The chemotherapeutics-loaded nanovesicles were produced by the breakdown of monocytes or macrophages using a serial extrusion through filters with diminishing pore sizes (10, 5, and 1 μm). These cell-derived nanovesicles have similar characteristics with the exosomes but have 100-fold higher production yield. Furthermore, the nanovesicles have natural targeting ability of cells by maintaining the topology of plasma membrane proteins. In vitro, chemotherapeutic drug-loaded nanovesicles induced TNF-α-stimulated endothelial cell death in a dose-dependent manner. In vivo, experiments in mice showed that the chemotherapeutic drug-loaded nanovesicles traffic to tumor tissue and reduce tumor growth without the adverse effects observed with equipotent free drug. Furthermore, compared with doxorubicin-loaded exosomes, doxorubicin-loaded nanovesicles showed similar in vivo antitumor activity. However, doxorubicin-loaded liposomes that did not carry targeting proteins were inefficient in reducing tumor growth. Importantly, removal of the plasma membrane proteins by trypsinization eliminated the therapeutic effects of the nanovesicles both in vitro and in vivo. Taken together, these studies suggest that the bioengineered nanovesicles can serve as novel exosome-mimetics to effectively deliver chemotherapeutics to treat malignant tumors.
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24.
  • 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.
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25.
  • Le Gal, Kristell, et al. (författare)
  • Antioxidants can increase melanoma metastasis in mice.
  • 2015
  • Ingår i: Science Translational Medicine. - : American Association for the Advancement of Science (AAAS). - 1946-6242 .- 1946-6234. ; 7:308
  • Tidskriftsartikel (refereegranskat)abstract
    • Antioxidants in the diet and supplements are widely used to protect against cancer, but clinical trials with antioxidants do not support this concept. Some trials show that antioxidants actually increase cancer risk and a study in mice showed that antioxidants accelerate the progression of primary lung tumors. However, little is known about the impact of antioxidant supplementation on the progression of other types of cancer, including malignant melanoma. We show that administration of N-acetylcysteine (NAC) increases lymph node metastases in an endogenous mouse model of malignant melanoma but has no impact on the number and size of primary tumors. Similarly, NAC and the soluble vitamin E analog Trolox markedly increased the migration and invasive properties of human malignant melanoma cells but did not affect their proliferation. Both antioxidants increased the ratio between reduced and oxidized glutathione in melanoma cells and in lymph node metastases, and the increased migration depended on new glutathione synthesis. Furthermore, both NAC and Trolox increased the activation of the small guanosine triphosphatase (GTPase) RHOA, and blocking downstream RHOA signaling abolished antioxidant-induced migration. These results demonstrate that antioxidants and the glutathione system play a previously unappreciated role in malignant melanoma progression.
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26.
  • Leo, Fredrik, et al. (författare)
  • Characterization of MdpS: an in-depth analysis of a MUC5B-degrading protease from Streptococcus oralis
  • 2024
  • Ingår i: FRONTIERS IN MICROBIOLOGY. - : Frontiers Media S.A.. - 1664-302X. ; 15
  • Tidskriftsartikel (refereegranskat)abstract
    • Oral biofilms, comprising hundreds of bacteria and other microorganisms on oral mucosal and dental surfaces, play a central role in oral health and disease dynamics. Streptococcus oralis, a key constituent of these biofilms, contributes significantly to the formation of which, serving as an early colonizer and microcolony scaffold. The interaction between S. oralis and the orally predominant mucin, MUC5B, is pivotal in biofilm development, yet the mechanism underlying MUC5B degradation remains poorly understood. This study introduces MdpS (Mucin Degrading Protease from Streptococcus oralis), a protease that extensively hydrolyses MUC5B and offers an insight into its evolutionary conservation, physicochemical properties, and substrate- and amino acid specificity. MdpS exhibits high sequence conservation within the species and also explicitly among early biofilm colonizing streptococci. It is a calcium or magnesium dependent serine protease with strict physicochemical preferences, including narrow pH and temperature tolerance, and high sensitivity to increasing concentrations of sodium chloride and reducing agents. Furthermore, MdpS primarily hydrolyzes proteins with O-glycans, but also shows activity toward immunoglobulins IgA1/2 and IgM, suggesting potential immunomodulatory effects. Significantly, MdpS extensively degrades MUC5B in the N- and C-terminal domains, emphasizing its role in mucin degradation, with implications for carbon and nitrogen sequestration for S. oralis or oral biofilm cross-feeding. Moreover, depending on substrate glycosylation, the amino acids serine, threonine or cysteine triggers the enzymatic action. Understanding the interplay between S. oralis and MUC5B, facilitated by MdpS, has significant implications for the management of a healthy eubiotic oral microenvironment, offering potential targets for interventions aimed at modulating oral biofilm composition and succession. Additionally, since MdpS does not rely on O-glycan removal prior to extensive peptide backbone hydrolysis, the MdpS data challenges the current model of MUC5B degradation. These findings emphasize the necessity for further research in this field.
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27.
  • 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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28.
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29.
  • 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.
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30.
  • Nasir, Waqas, et al. (författare)
  • Parvovirus B19 VLP recognizes globoside in supported lipid bilayers
  • 2014
  • Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 456, s. 364-369
  • Tidskriftsartikel (refereegranskat)abstract
    • Studies have suggested that the glycosphingolipid globoside (Gb4Cer) is a receptor for human parvovirus B19. Virus-like particles bind to Gb4Cer on thin-layer chromatograms, but a direct interaction between the virus and lipid membrane-associated Gb4Cer has been debated. Here, we characterized the binding of parvovirus B19 VP1/VP2 virus-like particles to glycosphingolipids (i) on thin-layer chromatograms (TLCs) and (ii) incorporated into supported lipid bilayers (SLBs) acting as cell-membrane mimics. The binding specificities of parvovirus B19 determined in the two systems were in good agreement; the VLP recognized both Gb4Cer and the Forssman glycosphingolipid on TLCs and in SLBs compatible with the role of Gb4Cer as a receptor for this virus.
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31.
  • 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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32.
  • 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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33.
  • Nilsson, Erik, 1975-, et al. (författare)
  • Pregnancy Associated Plasma Protein-A as a Cardiovascular Risk Marker in Patients with Stable Coronary Heart Disease During 10 Years Follow-Up-A CLARICOR Trial Sub-Study
  • 2020
  • Ingår i: Journal of Clinical Medicine. - : MDPI. - 2077-0383. ; 9:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Elevated pregnancy-associated plasma protein A (PAPP-A) is associated with mortality in acute coronary syndromes. Few studies have assessed PAPP-A in stable coronary artery disease (CAD) and results are conflicting. We assessed the 10-year prognostic relevance of PAPP-A levels in stable CAD. The CLARICOR trial was a randomized controlled clinical trial including outpatients with stable CAD, randomized to clarithromycin versus placebo. The placebo group constituted our discovery cohort (n = 1.996) and the clarithromycin group the replication cohort (n = 1.975). The composite primary outcome was first occurrence of cardiovascular event or death. In the discovery cohort, incidence rates (IR) for the composite outcome were higher in those with elevated PAPP-A (IR 12.72, 95% Confidence Interval (CI) 11.0-14.7 events/100 years) compared to lower PAPP-A (IR 8.78, 8.25-9.34), with comparable results in the replication cohort. Elevated PAPP-A was associated with increased risk of the composite outcome in both cohorts (discovery Hazard Ratio (HR) 1.45, 95% CI 1.24-1.70; replication HR 1.29, 95% CI 1.10-1.52). In models adjusted for established risk factors, these trends were attenuated. Elevated PAPP-A was associated with higher all-cause mortality in both cohorts. We conclude that elevated PAPP-A levels are associated with increased long-term mortality in stable CAD, but do not improve long-term prediction of death or cardiovascular events when added to established predictors.
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34.
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35.
  • Nilsson, Hanna, et al. (författare)
  • Risk of hernia formation after radical prostatectomy : a comparison between open and robot-assisted laparoscopic radical prostatectomy within the prospectively controlled LAPPRO trial
  • 2022
  • Ingår i: Hernia. - : Springer Science and Business Media LLC. - 1265-4906 .- 1248-9204. ; 26, s. 157-164
  • Tidskriftsartikel (refereegranskat)abstract
    • Purpose: In addition to incisional hernia, inguinal hernia is a recognized complication to radical retropubic prostatectomy. To compare the risk of developing inguinal and incisional hernias after open radical prostatectomy compared to robot-assisted laparoscopic prostatectomy. Method: Patients planned for prostatectomy were enrolled in the prospective, controlled LAPPRO trial between September 2008 and November 2011 at 14 hospitals in Sweden. Information regarding patient characteristics, operative techniques and occurrence of postoperative inguinal and incisional hernia were retrieved using six clinical record forms and four validated questionnaires. Results: 3447 patients operated with radical prostatectomy were analyzed. Within 24 months, 262 patients developed an inguinal hernia, 189 (7.3%) after robot-assisted laparoscopic prostatectomy and 73 (8.4%) after open radical prostatectomy. The relative risk of having an inguinal hernia after robot-assisted laparoscopic prostatectomy was 18% lower compared to open radical retropubic prostatectomy, a non-significant difference. Risk factors for developing an inguinal hernia after prostatectomy were increased age, low BMI and previous hernia repair. The incidence of incisional hernia was low regardless of surgical technique. Limitations are the non-randomised setting. Conclusions: We found no difference in incidence of inguinal hernia after open retropubic and robot-assisted laparoscopic radical prostatectomy. The low incidence of incisional hernia after both procedures did not allow for statistical analysis. Risk factors for developing an inguinal hernia after prostatectomy were increased age and BMI.
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36.
  • 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.
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37.
  • 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.
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38.
  • Nilsson, Jonas, 1970, et al. (författare)
  • Enrichment of glycopeptides for glycan structure and attachment site identification.
  • 2009
  • Ingår i: Nature methods. - : Springer Science and Business Media LLC. - 1548-7105 .- 1548-7091. ; 6:11, s. 809-11
  • Tidskriftsartikel (refereegranskat)abstract
    • We present a method to enrich for glycoproteins from proteomic samples. Sialylated glycoproteins were selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of sialic acid glycosidic bonds. Mass spectrometric fragment analysis allowed identification of glycan structures, and additional fragmentation of deglycosylated ions yielded peptide sequence information, which allowed glycan attachment site and protein identification. We identified 36 N-linked and 44 O-linked glycosylation sites on glycoproteins from human cerebrospinal fluid.
  •  
39.
  • Nilsson, Jonas, 1970, et al. (författare)
  • Expression of influenza A virus glycan receptor candidates in mallard, chicken, and tufted duck
  • 2024
  • Ingår i: GLYCOBIOLOGY. - 0959-6658 .- 1460-2423. ; 34:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate alpha 2-3- from alpha 2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including alpha 2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both alpha 2-3- and alpha 2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.
  •  
40.
  • Nilsson, Jonas, 1970- (författare)
  • Initial-Boundary-Value Problems for the Stokes and Navier–Stokes Equations on Staggered Grids
  • 2000
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • In the first part of the thesis various types of boundary conditions for the steady state Stokes equations are considered. We formulate the boundary conditions in a new way, such that the boundary value problem becomes non-singular, and derive estimates of the solution. By using a second-order finite difference approximation on a staggered grid, we are able to derive a non-singular approximation in a direct way. Furthermore, we derive the same type of estimates as for the continuous case. The estimates of the solution are derived for the case where the velocity field is prescribed at the boundary, and moreover for the case where the pressure is specified instead of one of the velocity components. We also show that the same estimates hold when the continuity condition is substituted by the Poisson equation for the pressure if we add the divergence condition as a boundary condition. Next, by using a Laplace transform in time for the analysis, we are able to derive the same type of estimates for the linearized Navier-Stokes equations as for the steady state Stokes equations. Also here, we make an analogous analysis for a difference approximation on a staggered grid, and derive the corresponding estimates. Numerical experiments confirm the theoretical results. We also show that the results are valid for more complicated geometries, by using composite overlapping grids, with local velocity components on orthogonal staggered subgrids. The analysis and derivation of the estimates here, give new insight into the behavior of the solutions and are used for developing a fourth-order method for the incompressible Navier-Stokes equations. In order to eliminate problems with non-physical parasitic solutions and extra numerical boundary conditions, we construct a finite difference scheme with compact high order difference approximations of Padé type on a staggered grid. We use the same type of boundary conditions proposed, developed and analyzed for the linearized Navier-Stokes equations. Several numerical experiments demonstrate the accuracy and efficiency of the method.
  •  
41.
  • Nilsson, Jonas, 1970 (författare)
  • Liquid chromatography-tandem mass spectrometry-based fragmentation analysis of glycopeptides
  • 2016
  • Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 33:3, s. 261-272
  • Tidskriftsartikel (refereegranskat)abstract
    • The use of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) for the glycoproteomic characterization of glycopeptides is a growing field of research. The N- and O-glycosylated peptides (N- and O-glycopeptides) analyzed typically originate from protease-digested glycoproteins where many of them are expected to be biomedically important. Examples of LC-MS2 and MS3 fragmentation strategies used to pursue glycan structure, peptide identity and attachment-site identification analyses of glycopeptides are described in this review. MS2 spectra, using the CID and HCD fragmentation techniques of a complex biantennary N-glycopeptide and a core 1 O-glycopeptide, representing two examples of commonly studied glycopeptide types, are presented. A few practical tips for accomplishing glycopeptide analysis using reversed-phase LC-MSn shotgun proteomics settings, together with references to the latest glycoproteomic studies, are presented.
  •  
42.
  • Nilsson, Jonas, 1970, et al. (författare)
  • Norwalk virus-like particles bind specifically to A, H and difucosylated Lewis but not to B histo-blood group active glycosphingolipids.
  • 2009
  • Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080.
  • Tidskriftsartikel (refereegranskat)abstract
    • Noroviruses and norovirus virus-like particles (VLPs) exhibit strain specific patterns in their binding to ABH and Lewis histo-blood group antigens. In this study we demonstrate for the first time specific binding of Norwalk virus VLPs to type 1 and type 2 chain glycosphingolipids (GSLs) carrying ABH and Lewis antigens. N-succinimidyl-3-tributylstannyl benzoate (ATE) was precursor labeled with (125)I and then conjugated to VLPs. The (125)I-VLPs were used in GSL thin-layer chromatogram binding assays and displayed binding to H type 1, Lewis b, A type 1, A Lewis b GSLs but no binding to B type 1 or B Lewis b GSLs. For the type 2 chain GSLs the Norwalk VLPs bound to H type 2, Lewis y, A type 2 and A Lewis y. In addition, the VLPs bound to several complex GSLs from blood group O and A, but not from blood group B red blood cells.
  •  
43.
  • Nilsson, Jonas, 1970, et al. (författare)
  • Sialic acid capture-and-release and LC-MSn analysis of glycopeptides
  • 2013
  • Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 951, s. 79-100
  • Tidskriftsartikel (refereegranskat)abstract
    • Extracellular glycoproteins frequently carry terminal sialic acids on their N-linked and/or O-linked glycan structures. In this chapter a sialic acid specific capture-and-release protocol for the enrichment of N- and O-glycopeptides originating from glycoproteins in complex biological samples is described. The enriched glycopeptides are subjected to reversed phase liquid chromatography (LC) interfaced with electrospray ionization and multistage tandem mass spectrometry (MSn). The glycopeptide precursor ions are fragmented by collision-induced dissociation (CID) for analysis of the glycan parts in the MS2 spectra. Further fragmentation (i.e., MS 3) of deglycosylated peptide ions results in peptide backbone fragmentation, which is used in protein database searches to identify protein sequences. For O-glycopeptides the use of both CID and electron capture dissociation (ECD) fragmentation of the peptide backbone with intact glycans still attached are used to pinpoint the glycosylation sites of glycopeptides containing several Ser/Thr residues. The step-by-step protocols for fragmentation analyses of O- and N-glycopeptides enriched from human cerebrospinal fiuid are described. © Springer Science+Business Media, LLC 2013.
  •  
44.
  • Nilsson, Jens, 1970-, et al. (författare)
  • Swedish civil air traffic control dataset
  • 2023
  • Ingår i: Data in Brief. - : ELSEVIER. - 2352-3409. ; 48
  • Tidskriftsartikel (refereegranskat)abstract
    • The Swedish Civil Air Traffic Control (SCAT) dataset consists of 13 weeks of data collected from the area control in Sweden flight information region. The dataset consists of detailed data from almost 170,000 flights as well as airspace data and weather forecasts. The flight data includes system updated flight plans, clearances from air traffic control, surveillance data and trajectory prediction data. Each week of data is continuous but the 13 weeks are spread over one year to provide variations in weather and seasonal traffic patterns. The dataset does only include scheduled flights not involved in any incident reports. Sensitive data such as military and private flight has been removed.The SCAT dataset can be useful for any research related to air traffic control, e.g. analysis of transportation patterns, environmental impact, optimization and automation/AI.
  •  
45.
  • Nilsson, Jonas, 1970, et al. (författare)
  • Synthetic standard aided quantification and structural characterization of amyloid-beta glycopeptides enriched from cerebrospinal fluid of Alzheimer's disease patients
  • 2019
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
  • Tidskriftsartikel (refereegranskat)abstract
    • An early pathological hallmark of Alzheimer's disease (AD) is amyloid-beta (A beta) deposits in the brain, which largely consist of up to 43 amino acids long A beta peptides derived from the amyloid precursor protein (APP). We previously identified a series of sialylated Tyr-10 O-glycosylated A beta peptides, 15-20 residues long, from human cerebrospinal fluid (CSF) and observed a relative increase of those in AD vs non-AD patients. We report here on the synthesis and use of an isotopically double-labeled A beta 1-15 glycopeptide, carrying the core 1Gal beta 3GalNAc alpha 1-O-Tyr-10 structure, to (1) identify by HCD LC-MS/MS the definite glycan core 1 structure of immunopurified and desialylated A beta glycopeptides in human CSF and to (2) establish a LC-MS/MS quantification method for desialylated A beta 1-15 (and A beta-17) glycopeptides and to (3) compare the concentrations of these A beta glycopeptides in CSF from 20 AD patients and 20 healthy controls. Although we unambiguously identified the core 1 structures and Tyr-10 attachment sites of the glycopeptides, we did not observe any quantitative differences, determined through both peptide and oxonium ion fragments, of the desialylated A beta 1-15 or A beta 1-17 glycopeptides between the AD and non-AD group. The new quantitative glycoproteomic approach described, using double-labeled glycopeptide standards, will undoubtedly facilitate future studies of glycopeptides as clinical biomarkers but should also embrace sialylated A beta standards to reveal specific sialylation patterns of individual A beta glycopeptides in AD patients and controls.
  •  
46.
  • Nilsson, Jonas, 1970, et al. (författare)
  • Targeting the glycoproteome.
  • 2013
  • Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 30:2, s. 119-36
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite numerous original publications describing the structural complexity of N- and O-linked glycans on glycoproteins, only very few answer the basic question of which particular glycans are linked to which amino acid residues along the polypeptide chain. Such structural information is of fundamental importance for understanding the biological roles of complex glycosylations as well as deciphering their non-template driven biosynthesis. This review focuses on presenting and commenting on recent strategies, specifically aimed at identifying the glycoproteome of cultured cells and biological samples, using targeted and global enrichment procedures and utilizing the high resolution power, high through-put capacity and complementary fragmentation techniques of tandem mass spectrometry. The goal is to give an update of this emerging field of protein and glyco-sciences and suggest routes to bridge the data gap between the two aspects of glycoprotein characteristics, i.e. glycan structures and their attachment sites.
  •  
47.
  • Noborn, Fredrik, et al. (författare)
  • A Glycoproteomic Approach to Identify Novel Proteoglycans
  • 2022
  • Ingår i: Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Balagurunathan K., Nakato H., Desai U., Saijoh Y. (eds). - New York, NY : Springer. - 1064-3745. - 9781071613986 ; , s. 71-85
  • Bokkapitel (refereegranskat)abstract
    • In this chapter, we describe a glycoproteomic approach for the identification of novel chondroitin sulfate proteoglycans (CSPGs) using a combination of biochemical enrichments, enzymatic digestions, and nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. The identification is achieved by trypsin digestion of CSPG-containing samples, followed by enrichment of chondroitin sulfate (CS) glycopeptides by strong anion exchange chromatography (SAX). The enriched CS glycopeptides are then digested with chondroitinase ABC to depolymerize the CS polysaccharides, generating a residual hexasaccharide structure, composed of the linkage region tetrasaccharide extended with a terminal dehydrated disaccharide, still attached to the peptide. The obtained CS glycopeptides are analyzed by nLC-MS/MS, and the generated data sets are evaluated through proteomic software with adjustment in the settings to allow for glycopeptide identification. This approach has enabled the identification of several novel core proteins in human samples and in Caenorhabditis elegans. Here we specifically describe the procedure for the enrichment and characterization of CS glycopeptides from human cerebrospinal fluid (CSF). © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
  •  
48.
  • Noborn, Fredrik, et al. (författare)
  • Expanding the chondroitin glycoproteome of Caenorhabditis elegans. : Chondroitin proteoglycans in C. elegans
  • 2018
  • Ingår i: The Journal of biological chemistry. - 1083-351X. ; 293:1, s. 379-389
  • Tidskriftsartikel (refereegranskat)abstract
    • Chondroitin sulfate proteoglycans (CSPGs) are important structural components of connective tissues in essentially all metazoan organisms. In vertebrates, CSPGs are involved also in more specialized processes such as neurogenesis and growth factor signaling. In invertebrates, however, knowledge of CSPGs core proteins and proteoglycan-related functions is relatively limited, even for Caenorhabditis elegans. This nematode produces large amounts of non-sulfated chondroitin in addition to low-sulfated chondroitin sulfate chains. So far, only nine core proteins (CPGs) have been identified, some of which have been shown to be involved in extracellular matrix formation. We recently introduced a protocol to characterize proteoglycan core proteins by identifying CS-glycopeptides with a combination of biochemical enrichment, enzymatic digestion, and nano-scale liquid chromatography MS/MS analysis. Here, we have used this protocol to map the chondroitin glycoproteome in C. elegans, resulting in the identification of 15 novel CPG proteins in addition to the nine previously established. Three of the newly identified CPGs displayed homology to vertebrate proteins. Bioinformatics analysis of the primary protein sequences revealed that the CPG proteins altogether contained 19 unique functional domains, including Kunitz and endostatin domains, suggesting direct involvement in protease inhibition and axonal migration, respectively. The analysis of the core protein domain organization revealed that all chondroitin attachment sites are located in unstructured regions. Our results suggest that CPGs display a much greater functional and structural heterogeneity than previously appreciated and indicate that specialized proteoglycan-mediated functions evolved early in metazoan evolution.
  •  
49.
  • Noborn, Fredrik, et al. (författare)
  • Expanding the Chondroitin Sulfate Glycoproteome - But How Far?
  • 2021
  • Ingår i: Frontiers in cell and developmental biology. - : Frontiers Media SA. - 2296-634X. ; 9
  • Forskningsöversikt (refereegranskat)abstract
    • Chondroitin sulfate proteoglycans (CSPGs) are found at cell surfaces and in connective tissues, where they interact with a multitude of proteins involved in various pathophysiological processes. From a methodological perspective, the identification of CSPGs is challenging, as the identification requires the combined sequencing of specific core proteins, together with the characterization of the CS polysaccharide modification(s). According to the current notion of CSPGs, they are often considered in relation to a functional role in which a given proteoglycan regulates a specific function in cellular physiology. Recent advances in glycoproteomic methods have, however, enabled the identification of numerous novel chondroitin sulfate core proteins, and their glycosaminoglycan attachment sites, in humans and in various animal models. In addition, these methods have revealed unexpected structural complexity even in the linkage regions. These findings indicate that the number and structural complexity of CSPGs are much greater than previously perceived. In light of these findings, the prospect of finding additional CSPGs, using improved methods for structural and functional characterizations, and studying novel sample matrices in humans and in animal models is discussed. Further, as many of the novel CSPGs are found in low abundance and with not yet assigned functions, these findings may challenge the traditional notion of defining proteoglycans. Therefore, the concept of proteoglycans is considered, discussing whether "a proteoglycan" should be defined mainly on the basis of an assigned function or on the structural evidence of its existence.
  •  
50.
  • Noborn, Fredrik, et al. (författare)
  • Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.
  • 2015
  • Ingår i: Molecular & cellular proteomics : MCP. - 1535-9484 .- 1535-9476. ; 14:1, s. 41-9
  • Tidskriftsartikel (refereegranskat)abstract
    • Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.
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