| 21. |
- Cheng, Fang, et al.
(författare)
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Non-conserved, S-nitrosylated cysteines in glypican-1 react with N-unsubstituted glucosamines in heparan sulfate and catalyze deaminative cleavage.
- 2012
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:11, s. 1480-1485
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Tidskriftsartikel (refereegranskat)abstract
- The membrane lipid-anchored glypicans (heparan sulfate proteoglycans) are present in both vertebrates and invertebrates and serve as important modulators of growth factors and morphogens during development. Their core proteins are similar and consist of a large N-terminal domain comprising 14 evolutionary conserved cysteines and a C-terminal stalk carrying the heparan sulfate side-chains and the lipid anchor. Cysteines in glypican-1 can be S-nitrosylated but their positions have not been identified. The recently determined crystal structure of the N-terminal domain of glypican-1 has revealed that all the evolutionary conserved cysteines form intramolecular disulfide bonds. However, glypican-1 contains two more, non-conserved cysteines in the C-terminal stalk, located near the heparan sulfate attachment sites. We show here that the non-conserved cysteines are free thiols as a glypican-1 core protein containing the C-terminal stalk could be biotinylated by biotin-BMCC. After S-nitrosylation by using an NO-donor and copper ions, the glypican-1 core protein was retained on an affinity matrix substituted with heparan sulfate oligosaccharides containing N-unsubstituted glucosamines. The protein was displaced with 0.2 M glucosamine but also by 2 mM ascorbate. In the latter case, the heparan sulfate of the affinity matrix was simultaneously cleaved into fragments containing anhydromannose. We propose that the S-nitrosocysteine residues interact with closely located N-unsubstituted glucosamines in the heparan sulfate side-chains of the glypican-1 proteoglycan. Addition of ascorbate induces a series of reactions that eventually releases heparan sulfate fragments with reducing terminal anhydromannose, presumably without the formation of free nitric oxide.
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| 22. |
- Cheng, Fang, et al.
(författare)
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Non-toxic amyloid beta formed in the presence of glypican-1 or its deaminatively generated heparan sulfate degradation products
- 2013
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 23:12, s. 1510-1519
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Tidskriftsartikel (refereegranskat)abstract
- The amyloid beta (A beta) peptides (mainly A beta 40 and A beta 42), which are derived from the amyloid precursor protein (APP), can oligomerize into antibody A11-positive, neurotoxic species, believed to be involved in Alzheimer's disease. Interestingly, APP binds strongly to the heparan sulfate (HS) proteoglycan (PG) glypican-1 (Gpc-1) in vitro and both proteins are colocalized inside cells. In endosomes, APP is proteolytically processed to yield A beta peptides. The HS chains of S-nitrosylated (SNO) Gpc-1 PG are cleaved into anhydromannose (anMan)-containing di- and oligosaccharides by an NO-dependent reaction in the same compartments. Here, we have studied the toxicity of oligomers/aggregates of A beta 40 and A beta 42, as well as A beta 40/42 mixtures that were formed in the presence of immobilized Gpc-1 PG or immobilized HS oligosaccharides. Afterwards, A beta was displaced from the matrices, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for A11 immunoreactivity, for effects on growth of mouse N2a neuroblastoma cells and for membrane leakage in rat cortical neurons. HS generally promoted and accelerated A beta multimerization into oligomers as well as larger aggregates that were mostly A11 positive and showed toxic effects. However, non-toxic A beta was formed in the presence of Gpc-1 PG or when anMan-containing HS degradation products were simultaneously generated. Both toxic and non-toxic A beta peptides were taken up by the cells but toxic forms appeared to enter the nuclei to a larger extent. The protection afforded by the presence of HS degradation products may reflect a normal intracellular function for the A beta peptides.
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| 23. |
- Cheng, Fang, et al.
(författare)
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Rapid nuclear transit and impaired degradation of amyloid beta and glypican-1-derived heparan sulfate in Tg2576 mouse fibroblasts.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:5, s. 548-556
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Tidskriftsartikel (refereegranskat)abstract
- Anhydromannose (anMan)-containing heparan sulfate (HS) derived from S-nitrosylated glypican-1 is generated in endosomes by an endogenously or ascorbate induced S-nitrosothiol-catalyzed reaction. Expression and processing of amyloid precursor protein (APP) is required to initiate formation and endosome-to-nucleus translocation of anMan-containing HS in wild-type mouse embryonic fibroblasts (WT MEF). HS is then transported to autophagosomes and finally degraded in lysosomes. To investigate how APP-derived amyloid beta peptide (Aβ) affects intracellular trafficking of HS we have studied nuclear transit as well as autophagosome/lysosome targeting and degradation in Tg2576 MEF which produce increased amounts of Aβ. Deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody showed anMan-staining in the nuclei of Tg2576 MEF after 5 min of ascorbate treatment and after 15 min in WT MEF. There was also greater nuclear accumulation of HS in Tg2576 MEF as determined by (35)S-sulfate labeling experiments. Tg2576 MEF was less sensitive to inhibition of NO production and copper-chelation than WT MEF. By using APP- and Aβ-recognizing antibodies we observed nuclear translocation of Aβ peptide in Tg2576 MEF but not in WT MEF. HS remained in the nucleus of WT MEF for at least 8 h and was then transported to autophagosomes. By 8 h HS had disappeared from the nuclei of Tg2576 MEF but colocalized poorly with the autophagosome marker LC3. Aβ also disappeared rapidly from the nuclei of Tg2576 MEF. Initially it appeared in acidic vesicles and later it accumulated extracellularly. Thus, in Tg2576 MEF there is nuclear accumulation as well as secretion of Aβ and impaired degradation of HS.
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| 24. |
- Cherian, Reeja Maria, et al.
(författare)
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Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins
- 2014
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 24:1, s. 26-38
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Tidskriftsartikel (refereegranskat)abstract
- The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG(2b) (PSGL-1/mIgG(2b)), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG(2b) fusion protein, the pigeon alpha 1,4-galactosyltransferase (alpha 4Gal-T) and the core 2 beta 1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG(2b) and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG(2b). In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG(2b) and a P-k-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG(2b) and P-k-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon alpha 4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG(2b) bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.
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| 25. |
- Conze, Tim, 1977-, et al.
(författare)
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MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas
- 2010
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 20:2, s. 199-206
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Tidskriftsartikel (refereegranskat)abstract
- Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.
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| 26. |
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| 27. |
- Cui, Hao, et al.
(författare)
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Glucuronyl C5-epimerase is crucial for epithelial cell maturation during embryonic lung development
- 2021
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Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 31:3, s. 223-230
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Tidskriftsartikel (refereegranskat)abstract
- Glucuronyl C5-epimerase (Hsepi) is a key enzyme in the biosynthesis of heparan sulfate that is a sulfated polysaccharide expressed on the cell surface and in the extracellular matrix of alveolar walls and blood vessels. Targeted interruption of the Hsepi gene, Glce, in mice resulted in neonatal lethality, which is most likely due to lung atelectasis. In this study, we examined the potential mechanisms behind the defect in lung development. Histological analysis of the lungs from embryos revealed no difference in the morphology between wild-type and mutant animals up to E16.5. This suggests that the initial events leading to formation of the lung primordium and branching morphogenesis are not disturbed. However, the distal lung of E17.5-18.5 mutants is still populated by epithelial tubules, lacking the typical saccular structural characteristic of a normal E17.5 lung. Immunostaining revealed strong signals of surfactant protein-C, but a weaker signal of T1 alpha in the mutant lungs in comparison to WT littermates, suggesting differentiation of type I alveolar epithelial cells (AT1) is impaired. One of the parameters contributed to the failure of AT1 maturation is reduced vascularization in the developing lungs.
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| 28. |
- Cui, Hao, et al.
(författare)
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Re-expression of glucuronyl C5-epimerase in the mutant MEF cells increases heparan sulfate epimerization but has no influence on the Golgi localization and enzymatic activity of 2-O-sulfotransferase
- 2021
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Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 31:8, s. 1018-1025
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) is a linear and complex polysaccharide that modulates the biological activities through protein recognition and interaction. Evidence indicates that protein-binding properties of HS are largely dependent on distinctive sulfation and epimerization patterns that are modified by a series of Golgi-localized enzymes. In particular, the glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) residues to L-iduronic acid (IdoA) and 2-O-sulfotransferase (2OST) catalyzes sulfation at C2 position of IdoA and rarely GlcA residues. Mice lacking both Hsepi and 2OST display multiple development defects, indicating the importance of IdoA in HS. Here, to gain greater insights of HS structure-function relationships, as well as a better understanding of the regulatory mechanisms of Hsepi and 2OST, the fine structure and cellular signaling functions of HS were investigated after restoration of Hsepi in the mutant mouse embryonic fibroblast (MEF) cells. Introduction of Hsepi into the Hsepi mutant MEF cells led to robustly increased proportion of IdoA residues, which rescued the cell signaling in response to fibroblast growth factor 2. However, we found that Hsepi knockout had no influence on either cellular transport or enzymatic activity of 2OST in the MEF cells, which is not in accord with the findings suggesting that the enzymatic activity and cellular transport of 2OST and Hsepi might be differently regulated.
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| 29. |
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| 30. |
- Derba-Maceluch, Marta, et al.
(författare)
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O-Acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants
- 2014
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 24, s. 494-506
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Tidskriftsartikel (refereegranskat)abstract
- O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 -> 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.
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