| 1. |
- Persson, Andrea, et al.
(författare)
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Glycosaminoglycan Domain Mapping of Cellular Chondroitin/Dermatan Sulfates
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Glycosaminoglycans (GAGs) are polysaccharides produced by most mammalian cells and involved in a variety of biological processes. However, due to the size and complexity of GAGs, detailed knowledge about the structure and expression of GAGs by cells, the glycosaminoglycome, is lacking. Here we report a straightforward and versatile approach for structural domain mapping of complex mixtures of GAGs, GAGDoMa. The approach is based on orthogonal enzymatic depolymerization of the GAGs to generate internal, terminating, and initiating domains, and nanoflow reversed-phase ion-pairing chromatography with negative mode higher-energy collision dissociation (HCD) tandem mass spectrometry (MS/MS) for structural characterization of the individual domains. GAGDoMa provides a detailed structural insight into the glycosaminoglycome, and offers an important tool for deciphering the complexity of GAGs in cellular physiology and pathology. © 2020, The Author(s).
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| 2. |
- Syx, Delfien, et al.
(författare)
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Alterations in glycosaminoglycan biosynthesis associated with the Ehlers-Danlos syndromes.
- 2022
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Ingår i: American journal of physiology. Cell physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 323:6, s. C1843-C1859
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans consist of a core protein substituted with one or more glycosaminoglycan (GAG) chains and execute versatile functions during many physiological and pathological processes. The biosynthesis of GAG chains is a complex process that depends on the concerted action of a variety of enzymes. Central to the biosynthesis of heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) GAG chains is the formation of a tetrasaccharide linker region followed by biosynthesis of HS or CS/DS-specific repeating disaccharide units, which then undergo modifications and epimerization. The importance of these biosynthetic enzymes is illustrated by several severe pleiotropic disorders that arise upon their deficiency. The Ehlers-Danlos syndromes (EDS) constitute a special group among these disorders. Although most EDS types are caused by defects in fibrillar types I, III, or V collagen, or their modifying enzymes, a few rare EDS types have recently been linked to defects in GAG biosynthesis. Spondylodysplastic EDS (spEDS) is caused by defective formation of the tetrasaccharide linker region, either due to β4GalT7 or β3GalT6 deficiency, whereas musculocontractural EDS (mcEDS) results from deficiency of D4ST1 or DS-epi1, impairing DS formation. This narrative review highlights the consequences of GAG deficiency in these specific EDS types, summarizes the associated phenotypic features and the molecular spectrum of reported pathogenic variants, and defines the current knowledge on the underlying pathophysiological mechanisms based on studies in patient-derived material, in vitro analyses, and animal models.
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| 3. |
- De Leoz, M. L. A., et al.
(författare)
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NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods
- 2020
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476. ; 19:1, s. 11-30
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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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| 4. |
- Haga, K., et al.
(författare)
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Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection
- 2020
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Ingår i: mBio. - 2150-7511. ; 11:2, s. e00251-20
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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.
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| 5. |
- Nikpour, Mahnaz, et al.
(författare)
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Glycosaminoglycan linkage region of urinary bikunin as a potentially useful biomarker for β3GalT6-deficient spondylodysplastic Ehlers-Danlos syndrome.
- 2022
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Ingår i: JIMD reports. - : Wiley. - 2192-8304 .- 2192-8312. ; 63:5, s. 462-467
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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.
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| 6. |
- Noborn, Fredrik, et al.
(författare)
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Expanding the Chondroitin Sulfate Glycoproteome - But How Far?
- 2021
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Ingår i: Frontiers in cell and developmental biology. - : Frontiers Media SA. - 2296-634X. ; 9
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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.
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| 7. |
- Noborn, Fredrik, et al.
(författare)
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Mapping the Human Chondroitin Sulfate Glycoproteome Reveals an Unexpected Correlation Between Glycan Sulfation and Attachment Site Characteristics. : Chondroitin Sulfation and Attachment Site Characteristics
- 2023
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Ingår i: Molecular & cellular proteomics : MCP. - : Elsevier. - 1535-9484 .- 1535-9476. ; 22:8
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Tidskriftsartikel (refereegranskat)abstract
- Chondroitin sulfate proteoglycans (CSPGs) control key events in human health and disease and are composed of chondroitin sulfate (CS) polysaccharide(s) attached to different core proteins. Detailed information on the biological effects of site-specific CS structures is scarce as the polysaccharides are typically released from their core proteins prior to analysis. Here we present a novel glycoproteomic approach for site-specific sequencing of CS modifications from human urine. Software-assisted and manual analysis revealed that certain core proteins carried CS with abundant sulfate modifications, while others carried CS with lower levels of sulfation. Inspection of the amino acid sequences surrounding the attachment sites indicated that the acidity of the attachment site motifs increased the levels of CS sulfation, and statistical analysis confirmed this relationship. However, not only the acidity but also the sequence and characteristics of specific amino acids in the proximity of the serine glycosylation site correlated with the degree of sulfation. These results demonstrate attachment site-specific characteristics of CS polysaccharides of CSPGs in human urine and indicate that this novel method may assist in elucidating the biosynthesis and functional roles of CSPGs in cellular physiology.
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| 8. |
- Takemura, M., et al.
(författare)
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Chondroitin sulfate proteoglycan Windpipe modulates Hedgehog signaling in Drosophila
- 2020
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Ingår i: Molecular Biology of the Cell. - 1059-1524. ; 31:8, s. 813-824
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans, a class of carbohydrate-modified proteins, often modulate growth factor signaling on the cell surface. However, the molecular mechanism by which proteoglycans regulate signal transduction is largely unknown. In this study, using a recently developed glycoproteomic method, we found that Windpipe (Wdp) is a novel chondroitin sulfate proteoglycan (CSPG) in Drosophila. Wdp is a single-pass transmembrane protein with leucin-rich repeat (LRR) motifs and bears three CS sugar chain attachment sites in the extracellular domain. Here we show that Wdp modulates the Hedgehog (Hh) pathway. In the wing disc, overexpression of wdp inhibits Hh signaling, which is dependent on its CS chains and the LRR motifs. The wdp null mutant flies show a specific defect (supernumerary scutellar bristles) known to be caused by Hh overexpression. RNA interference knockdown and mutant clone analyses showed that loss of wdp leads to the up-regulation of Hh signaling. Altogether, our study demonstrates a novel role of CSPGs in regulating Hh signaling.
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