| 1. |
- Nilsson, Jonas, 1970, et al.
(författare)
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Synthetic standard aided quantification and structural characterization of amyloid-beta glycopeptides enriched from cerebrospinal fluid of Alzheimer's disease patients
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
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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.
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| 2. |
- Parveen, Nagma, 1988, et al.
(författare)
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Competition for Membrane Receptors: Norovirus Detachment via Lectin Attachment
- 2019
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 141:41, s. 16303-16311
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Tidskriftsartikel (refereegranskat)abstract
- Virus internalization into the host cells occurs via multivalent interactions, in which a single virus binds to multiple receptors in parallel. Because of analytical and experimental limitations this complex type of interaction is still poorly understood and quantified. Herein, the multivalent interaction of norovirus-like particles (noroVLPs) with H or B type 1 glycosphingolipids (GSLs), embedded in a supported phospholipid bilayer, is investigated by following the competition between noroVLPs and a lectin (from Ralstonia solanacearum) upon binding to these GSLs. Changes in noroVLP and lectin coverage, caused by competition, were monitored for both GSLs and at different GSL concentrations using quartz crystal microbalance with dissipation monitoring. The study yields information about the minimum GSL concentration needed for (i) noroVLPs to achieve firm attachment to the bilayer prior to competition and to (ii) remain firmly attached to the bilayer during competition. We show that these two concentrations are almost identical for the H type 1-noroVLP interaction but differ for B type 1, indicating an accumulation of B type 1 GSLs in the noroVLP-bilayer interaction area. Furthermore, the GSL concentration required for firm attachment is significantly larger for H type 1 than for B type 1, indicating a higher affinity of noroVLP toward B type 1. This finding is supported by extracting the energy of single noroVLP-H type 1 and noroVLP-B type 1 bonds from the competition kinetics, which were estimated to be 5 and 6 kcal/mol, respectively. This demonstrates the potential of utilizing competitive binding kinetics to analyze multivalent interactions, which has remained difficult to quantify using conventional approaches.
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| 3. |
- Nordén, Rickard, 1977, et al.
(författare)
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O-linked glycosylation of the mucin domain of the herpes simplex virus type 1 specific glycoprotein gC-1 is temporally regulated in a seed-and-spread manner.
- 2015
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 290:8, s. 5078-5091
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Tidskriftsartikel (refereegranskat)abstract
- The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, participating in viral receptor interactions and immunity interference, harbors a mucin-like domain with multiple clustered O-linked glycans. Using HSV-1 infected diploid human fibroblasts, an authentic target for HSV-1 infection, and a protein immunoaffinity procedure, we enriched fully glycosylated gC-1 and a series of its biosynthetic intermediates. This fraction was subjected to trypsin digestion and a LC-MS/MS glycoproteomics approach. In parallel, we characterized the expression patterns of the 20 isoforms of human GalNAc transferases responsible for initiation of O-linked glycosylation. The gC-1 O-glycosylation was regulated in an orderly manner initiated by synchronous addition of one GalNAc unit each to T87 and T91, and one GalNAc unit to either T99 or T101, forming a core glycopeptide for subsequent additions of in all 11 GalNAc residues to selected Ser and Thr residues of the T76-L107 stretch of the mucin domain. The expression patterns of GalNAc transferases in the infected cells suggested that initial additions of GalNAc were carried out by initiating GalNAc transferases, in particular GalNAc-T2, whereas subsequent GalNAc additions were carried out by follow up transferases, in particular GalNAc-T10. Essentially all of the susceptible Ser or Thr residues had to acquire their GalNAc units before any elongation to longer O-linked glycans of the gC-1-associated GalNAc units was permitted. Since the GalNAc occupancy pattern is of relevance for receptor binding of gC-1, the data provides a model to delineate biosynthetic steps of O-linked glycosylation of the gC-1 mucin domain in HSV-1 infected target cells.
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| 4. |
- Mehmedbasic, Arnela, et al.
(författare)
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SorLA CR-Domains Protect the Amyloid Precursor Protein against Processing.
- 2015
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 290, s. 3359-3376
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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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| 5. |
- Mirgorodskaya, Ekaterina, et al.
(författare)
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Cracking the Sugar Code by Mass Spectrometry
- 2018
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305. ; 29:6, s. 1065-1074
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Tidskriftsartikel (refereegranskat)abstract
- The structural study of glycans and glycoconjugates is essential to assign their roles in homeostasis, health, and disease. Once dominated by nuclear magnetic resonance spectroscopy, mass spectrometric methods have become the preferred toolbox for the determination of glycan structures at high sensitivity. The patterns of such structures in different cellular states now allow us to interpret the sugar codes in health and disease, based on structure-function relationships. Dr. Catherine E. Costello was the 2017 recipient of the American Society for Mass Spectrometry's Distinguished Contribution Award. In this Perspective article, we describe her seminal work in a historical and geographical context and review the impact of her research accomplishments in the field.
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| 6. |
- Nilsson, Jonas, 1970, et al.
(författare)
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Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS.
- 2017
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1879-1123 .- 1044-0305. ; 28:2, s. 229-41
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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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| 7. |
- Noborn, Fredrik, et al.
(författare)
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Expanding the chondroitin glycoproteome of Caenorhabditis elegans. : Chondroitin proteoglycans in C. elegans
- 2018
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 293:1, s. 379-389
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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.
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| 8. |
- Noborn, Fredrik, et al.
(författare)
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Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.
- 2015
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Ingår i: Molecular & cellular proteomics : MCP. - 1535-9484 .- 1535-9476. ; 14:1, s. 41-9
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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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| 9. |
- Noborn, Fredrik, et al.
(författare)
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Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- © 2016 The Author(s).Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans.
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| 10. |
- Persson, Andrea, et al.
(författare)
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Identification of a non-canonical chondroitin sulfate linkage region trisaccharide
- 2019
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 29:5, s. 366-371
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Tidskriftsartikel (refereegranskat)abstract
- It is generally accepted that the biosynthesis of chondroitin sulfate and heparan sulfate is proceeding from a common linkage region tetrasaccharide comprising GlcA-Gal-Gal-Xyl-O-. The linkage region can undergo various modifications such as sulfation, phosphorylation and sialylation, and as the methods for studying glycosaminoglycan structure have been developed and refined, the number of discovered modifications has increased. Previous studies on the linkage region and the glycosyltransferases involved in the biosynthesis suggest that variants of the linkage region tetrasaccharide may also be possible. Here, using LC-MS/MS, we describe a non-canonical linkage region trisaccharide comprising GlcA-Gal-Xyl-O-. The trisaccharide was identified as a minor constituent in the proteoglycan bikunin from urine of human healthy donors present as a disulfated pentasaccharide, Delta HexA-GalNAc(S)-GlcA-Gal(S)-Xyl-O-, after chondroitinase ABC degradation. Furthermore, it was present as the corresponding disulfated pentasaccharide after chondroitinase ABC degradation in chondroitin sulfate primed on xylosides isolated from human cell lines. This linkage region trisaccharide may serve as an alternative point of entry for glycosaminoglycan biosynthesis.
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