Structural domain analysis of heparin and heparan sulfate combined with non-labeled quantitative proteomics for elucidating their functional diversity in liver cancer and APAP-induced liver injury
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Lianli Chi
PROVIDER: PXD064406 | iProX | Thu May 29 00:00:00 BST 2025
REPOSITORIES: iProX
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Project description: Not available
| S-EPMC12639849 | biostudies-literature
Project description:Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan-sulfate binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC–MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and, using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire glycan-binding proteins for further study.
2020-01-09 | MSV000084782 | MassIVE
Project description:Heparan sulfate (HS) in the vascular endothelial glycocalyx (eGC) is a critical regulator of blood vessel homeostasis. Trauma results in HS shedding from the eGC, but the impact of HS shedding on mechanisms of vascular injury and repair has not been evaluated. The objective of this work was to characterize the effect of eGC HS shedding on the transcriptional landscape of vascular endothelial cells. In vitro work was performed using flow conditioned primary human lung microvascular ECs treated with vehicle or heparin lyase III to simulate human heparanase activity. Bulk RNA sequencing was performed to determine differentially expressed gene-enriched pathways following heparin lyase III treatment. Pathway analysis demonstrated downregulation of genes that support cell junction integrity, EC polarity, and EC senescence while upregulating genes that promote cell differentiation and proliferation following HS shedding.
2024-08-14 | GSE260628 | GEO
Project description:Transforming growth factor-ß1 (TGF-ß1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-ß1’s activity on hMSCs. Using a proteomics “Protect and Label” approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin. Competition assays in an optical biosensor demonstrated that TGF-ß1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-ß1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14–24 saccharides (dp14–24) in length all increased the phosphorylation of SMAD2 after 6 h of stimulation with TGF-ß1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-ß1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-ß1 in hMSCs.
2015-09-01 | PXD002772 | Pride
Project description:We report a novel proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind to heparin. The technique is based on Limited Proteolysis of live cells in the absence of denaturation and fixation, Heparin-Affinity chromatography, and high-resolution LC-MS/MS, which we designate as LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells led to the identification of 64 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitates the mapping of the heparin-binding domains, making it possible to make predictions on the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly discovered heparin-binding proteins, CLEC14A, a member of the C-type lectin family that modulates angiogenesis. The C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity and the heparin-binding site was mapped by molecular modeling and mutagenesis. CLEC14A can physically interact with other glycosaminoglycans including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The combination of limited proteolysis and mass spectrometry led to the identification of previously undocumented glycosaminoglycan-binding proteins and mapping of their ligand binding sites. The technique should be applicable to other cells and glycans and provides a way to expand the repertoire glycan-binding proteins for further study.
2019-10-27 | MSV000084510 | MassIVE
Project description:The aim of this study was to identify the biological mechanisms involved in the effects of natural compounds such as heparin analogues obtained from marine invertebrates in wound healing We performed the obtaining, purifying and chemical characterization of the dermatan sulfate extracted from Styela plicata, an ascidian species. DS was used in wound healing in vitro assay and was analyzed for its effect in the toxicity, proliferation and cellular migration of murine fibroblasts. In addition, a gene expression test was performed to determine which genes responsible for the healing process were being modulated.
2020-11-30 | GSE160142 | GEO
Project description:Transforming growth factor-Ã?1 (TGF-Ã?1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-Ã?1â??s activity on hMSCs. Using a proteomics â??Protect and Labelâ?? approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin. Competition assays in an optical biosensor demonstrated that TGF-Ã?1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-Ã?1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14â??24 saccharides (dp14â??24) in length all increased the phosphorylation of SMAD2 after 6 h of stimulation with TGF-Ã?1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-Ã?1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-Ã?1 in hMSCs.
2016-07-19 | MSV000079921 | MassIVE
Project description:In order to obtain a global view of energy metabolism pathways of the sulfate-reducer Desulfovibrio vulgaris Hildenborough and the proteins involved therein whole-genome microarrays were used to compare the transcriptional response of cells grown with hydrogen/sulfate, pyruvate/sulfate, lactate/thiosulfate, and pyruvate with limiting sulfate, relative to growth in standard lactate/sulfate condition. Growth with hydrogen/sulfate showed the largest number of differently expressed genes and the largest changes in expression levels. The most up-regulated energy metabolism genes were those coding for the periplasmic [NiFeSe] hydrogenase, followed by the Ech hydrogenase, and the most down-regulated were genes coding for the Coo hydrogenase. The results point to the involvement of formate cycling and the ethanol pathway during growth on hydrogen, whereas there is evidence for CO cycling during growth on lactate and pyruvate, but not on H2. Growth with thiosulfate showed the hallmarks of a reduced energy status of the cells with down regulation of the ATP synthase and the Qmo and Dsr complexes., whereas growth with pyruvate showed the smallest differences but an increased role for the Ech hydrogenase.that in this case functions in reverse from the case of growth with H2. The multiple periplasmic hydrogenases and formate dehydrogenases, do not display the same regulation pattern showing that their metabolic roles are not totally interchangeable. This result together with the observation that several genes coding for proteins that have not been biochemically characterised were considerably affected in this study, reveals a more complex energy metabolism than previously considered and provides guidance for further studies. Keywords: Growth protocol
2008-05-31 | GSE8072 | GEO
Project description:In order to obtain a global view of energy metabolism pathways of the sulfate-reducer Desulfovibrio vulgaris Hildenborough and the proteins involved therein whole-genome microarrays were used to compare the transcriptional response of cells grown with hydrogen/sulfate, pyruvate/sulfate, lactate/thiosulfate, and pyruvate with limiting sulfate, relative to growth in standard lactate/sulfate condition. Growth with hydrogen/sulfate showed the largest number of differently expressed genes and the largest changes in expression levels. The most up-regulated energy metabolism genes were those coding for the periplasmic [NiFeSe] hydrogenase, followed by the Ech hydrogenase, and the most down-regulated were genes coding for the Coo hydrogenase. The results point to the involvement of formate cycling and the ethanol pathway during growth on hydrogen, whereas there is evidence for CO cycling during growth on lactate and pyruvate, but not on H2. Growth with thiosulfate showed the hallmarks of a reduced energy status of the cells with down regulation of the ATP synthase and the Qmo and Dsr complexes., whereas growth with pyruvate showed the smallest differences but an increased role for the Ech hydrogenase.that in this case functions in reverse from the case of growth with H2. The multiple periplasmic hydrogenases and formate dehydrogenases, do not display the same regulation pattern showing that their metabolic roles are not totally interchangeable. This result together with the observation that several genes coding for proteins that have not been biochemically characterised were considerably affected in this study, reveals a more complex energy metabolism than previously considered and provides guidance for further studies. Keywords: Growth protocol Desulfovibrio vulgaris Hildenborough was cultured at 37°C with thiosulfate as the only electron acceptor (with lactate as the electron donor) to mid-log phase. Cultures were also cultivated similarly using sulfate as the sole electron acceptor to mid-log phase. Gene expression profiles of cultures grown with thiosulfate as the electron acceptor were compared with those of the cultures grown with sulfate as the electron acceptor for dissimilatory sulfate reductiontion. Total RNA was harvested from four replicate cultures for microarray analysis. RNA extraction, purification, and labeling were performed independently on each cell sample. Two samples of each total RNA preparation were labeled, one with Cy3-dUTP and another with Cy5-dUTP for microarray hybridization (dye swap).
2008-05-31 | E-GEOD-8072 | biostudies-arrayexpress
Project description:sulfate reducing bacteria Metagenome
| PRJNA421399 | ENA