| 1. |
- Cheng, Fang, et al.
(author)
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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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In: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:11, s. 1480-1485
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Journal article (peer-reviewed)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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| 2. |
- Cheng, Fang, et al.
(author)
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Suppression of amyloid beta a11-immunoreactivity by vitamin C: possible role of heparan sulfate oligosaccharides derived from glypican-1 by ascorbate-induced, no-catalyzed degradation.
- 2011
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In: Journal of Biological Chemistry. - 1083-351X. ; 286:31, s. 27559-27572
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Journal article (peer-reviewed)abstract
- Amyloid beta is generated from the copper- and heparan sulfate (HS)-binding amyloid precursor protein (APP) by proteolytic processing. APP supports S-nitrosylation of the HS-proteoglycan glypican-1 (Gpc-1). In the presence of ascorbate there is NO-catalyzed release of anhydromannose (anMan)-containing oligosaccharides from Gpc-1-SNO. We have investigated whether these oligosaccharides interact with amyolid beta during APP processing and plaque formation. anMan-Immunoreactivity was detected in amyloid plaques of Alzheimer (AD) and APP transgenic (Tg2576) mouse brains by immunofluorescence microscopy. APP/APP degradation products detected by antibodies to the C-terminus of APP, but not amyolid beta oligomers detected by the anti-amyloid beta A11 antibody, colocalized with anMan-immunoreactivity in Tg2576 fibroblasts. A 50-55-kDa anionic, SDS-stable, anMan- and amyloid beta-immunoreactive species was obtained from Tg2576 fibroblasts using immunoprecipitation with anti-APP (C-terminal). anMan-Containing HS oligo- and disaccharide preparations modulated or suppressed A11-immunoreactivity and oligomerization of amyloid beta 42 peptide in an in vitro assay. A11 immunoreactivity increased in Tg2576 fibroblasts when Gpc-1 autoprocessing was inhibited by U18666A, and decreased when Gpc-1 autoprocessing was stimulated by ascorbate. Neither overexpression of Gpc-1 in Tg2576 fibroblasts nor addition of copper ion and NO-donor to hippocampal slices from 3xTg-AD mice affected A11 immunoreactivity levels. However, A11 immunoreactivity was greatly suppressed by the subsequent addition of ascorbate. We speculate that temporary interaction between the amyolid beta domain and small, anMan-containing oligosaccharides may preclude formation of toxic amyloid beta oligomers. A portion of the oligosaccharides co-secrete with the amyloid beta peptides and are deposited in plaques. These results support the notion that inadequate supply of vitamin C could contribute to late onset AD in humans.
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