Browse
Submit Data
Databases
API
Help

Dataset Information

13 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Proteoglycan synthesis in human and murine haematopoietic progenitor cell lines: isolation and characterization of a heparan sulphate proteoglycan as a major proteoglycan from the human haematopoietic cell line TF-1.

ABSTRACT: Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the microenvironment of haematopoietic tissues. Proteoglycans might also be involved in the interaction of haematopoietic stem and stromal cells. Recently, several studies have been reported on the proteoglycan synthesis of stromal cells, but little is known about the proteoglycan synthesis of haematopoietic stem or progenitor cells. Here we report on the isolation and characterization of proteoglycans from two haematopoietic progenitor cell lines, the murine FDCP-Mix A4 and the human TF-1 cell line. Proteoglycans were isolated from metabolically labelled cells and purified by several chromatographic steps, including anion-exchange and size-exclusion chromatography. Biochemical characterization was performed by electrophoresis or gel-filtration chromatography before and after digestion with glycosaminoglycan-specific enzymes or HNO2 treatment. Whereas FDCP-Mix A4 cells synthesize a homogeneous chondroitin 4-sulphate proteoglycan, isolation and characterization of proteoglycans from the human cell line TF-1 revealed, that TF-1 cells synthesize, in addition to a chondroitin sulphate proteoglycan, a heparan sulphate proteoglycan as major proteoglycan. For this heparan sulphate proteoglycan a core protein size of approx. 59 kDa was determined. Immunochemical analysis of this heparan sulphate proteoglycan revealed that it is not related to the syndecan family nor to glypican.

SUBMITTER: Stocker G

PROVIDER: S-EPMC1217464 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Cell surface chondroitin sulphate proteoglycan 4 (CSPG4) binds to the basement membrane heparan sulphate proteoglycan, perlecan, and is involved in cell adhesion.

Project description:Chondroitin sulphate proteoglycan 4 (CSPG4) is a cell surface proteoglycan highly expressed by tumour, perivascular and oligodendrocyte cells and known to be involved cell adhesion and migration. This study showed that CSPG4 was present as a proteoglycan on the cell surface of two melanoma cell lines, MM200 and Me1007, as well as shed into the conditioned medium. CSPG4 from the two melanoma cell lines differed in the amount of chondroitin sulphate (CS) decoration, as well as the way the protein core was fragmented. In contrast, the CSPG4 expressed by a colon carcinoma cell line, WiDr, was predominantly as a protein core on the cell surface lacking glycosaminoglycan (GAG) chains. This study demonstrated that CSPG4 immunopurified from the melanoma cell lines formed a complex with perlecan synthesized by the same cultured cells. Mechanistic studies showed that CSPG4 bound to perlecan via hydrophobic protein-protein interactions involving multiple sites on perlecan including the C-terminal region. Furthermore, this study revealed that CSPG4 interacted with perlecan to support cell adhesion and actin polymerization. Together these data suggest a novel mechanism by which CSPG4 expressing cells might attach to perlecan-rich matrices so as those found in connective tissues and basement membranes.

| S-EPMC5905647 | biostudies-literature

Proteoglycan synthesis in haematopoietic cells: isolation and characterization of heparan sulphate proteoglycans expressed by the bone-marrow stromal cell line MS-5.

Project description:Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the haematopoietic microenvironment. Recently, several studies have indicated that they are involved in the interaction of haematopoietic stem and stromal cells. However, a detailed characterization of the heparan sulphate proteoglycans synthesized by bone-marrow stromal cells is still lacking. Here we report on the isolation and characterization of proteoglycans from the haematopoietic stromal cell line MS-5, that efficiently supports the growth and differentiation of human and murine haematopoietic progenitor cells. Biochemical characterization of purified proteoglycans revealed that the haematopoietic stromal cell line MS-5 synthesizes, in addition to chondroitin sulphate proteoglycans, several different heparan sulphate proteoglycans. Immunochemical analysis, using specific antibodies against the different members of the syndecan family, glypican, betaglycan and perlecan, showed that MS-5 cells synthesize all these different heparan sulphate proteoglycans. These data were further supported by reverse-transcriptase PCR and confirmed by sequence and Northern blot analysis. The relative abundance of the different heparan sulphate proteoglycans was estimated on the protein and mRNA levels.

| S-EPMC1218818 | biostudies-other

A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line.

Project description:We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.

| S-EPMC1132996 | biostudies-other

Identification of a new membrane-bound heparan sulphate proteoglycan.

Project description:The morphological changes that occur during intestinal development have been extensively described, but the molecular basis of these changes is largely unknown. As a result of our efforts to identify molecules that play a role in intestinal morphogenesis during development, we have previously isolated a cDNA that is developmentally regulated in the intestine. This cDNA, named OCI-5, was recently shown to have 20-25% identity at the protein-sequence level with glypican and cerebroglycan, two heparan sulphate proteoglycans (HSPG) that are attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Here we provide experimental evidence indicating that OCI-5 is also a GPI-linked HSPG. We demonstrate this by showing that OCI-5 can be labelled with radioactive sulphate and can be digested by heparitinase, but not by chondroitinase. We also show that treatment with phosphatidylinositol-specific phospholipase C releases OCI-5 from the cell surface of COS cells transfected with an OCI-5 expression vector. The identification of OCI-5 as a GPI-linked HSPG confirms that this proteoglycan belongs to the same family of HSPGs that include glypican and cerebroglycan.

| S-EPMC1136036 | biostudies-other

Chondroitin Sulphate Proteoglycan Axonal Coats in the Human Mediodorsal Thalamic Nucleus.

Project description:Mounting evidence supports a key involvement of the chondroitin sulfate proteoglycans (CSPGs) NG2 and brevican (BCAN) in the regulation of axonal functions, including axon guidance, fasciculation, conductance, and myelination. Prior work suggested the possibility that these functions may, at least in part, be carried out by specialized CSPG structures surrounding axons, termed axonal coats. However, their existence remains controversial. We tested the hypothesis that NG2 and BCAN, known to be associated with oligodendrocyte precursor cells, form axonal coats enveloping myelinated axons in the human brain. In tissue blocks containing the mediodorsal thalamic nucleus (MD) from healthy donors (n = 5), we used dual immunofluorescence, confocal microscopy, and unbiased stereology to characterize BCAN and NG2 immunoreactive (IR) axonal coats and measure the percentage of myelinated axons associated with them. In a subset of donors (n = 3), we used electron microscopy to analyze the spatial relationship between axons and NG2- and BCAN-IR axonal coats within the human MD. Our results show that a substantial percentage (∼64%) of large and medium myelinated axons in the human MD are surrounded by NG2- and BCAN-IR axonal coats. Electron microscopy studies show NG2- and BCAN-IR axonal coats are interleaved with myelin sheets, with larger axons displaying greater association with axonal coats. These findings represent the first characterization of NG2 and BCAN axonal coats in the human brain. The large percentage of axons surrounded by CSPG coats, and the role of CSPGs in axonal guidance, fasciculation, conductance, and myelination suggest that these structures may contribute to several key axonal properties.

| S-EPMC9298528 | biostudies-literature

Purification and characterization of heparan sulphate proteoglycan from bovine brain.

Project description:A heparan sulphate proteoglycan was purified from adult bovine brain tissues and its structure was characterized. The major heparan sulphate proteoglycan from whole bovine brain had a molecular mass of >200 kDa on denaturing SDS/PAGE and a core protein size of 66 kDa following the removal of glycosaminoglycan chains. Fractionation on DEAE-Sephacel showed that this proteoglycan consisted of three major forms having high, intermediate and low overall charge. All core proteins were identical in size and reacted with heparan sulphate proteoglycan-stub antibody and an antibody made to a synthetic peptide based on rat glypican. The three forms of proteoglycans had identical peptide maps and their amino acid compositional analysis did not match any of the known glypicans. The internal sequence of a major peptide showed only 37.5% sequence similarity with human glypican 5. The glycosaminoglycan chain sizes of the three forms of this proteoglycan, determined after beta-elimination by PAGE, were identical. The disaccharide compositional analysis on the heparan sulphate chains from the three forms of the proteoglycan, determined by treatment with a mixture of heparin lyases followed by high-resolution capillary electrophoresis, showed that they differed primarily by degree of sulphation. The most highly sulphated proteoglycan isolated had a disaccharide composition similar to heparan sulphate glycosaminoglycans found in brain tissue. Based on their sensitivity to low pH nitrous acid treatment, the N-sulphate groups in these proteoglycans were found to be primarily in the smaller glycosaminoglycan chains. The heparan sulphate proteoglycans were also heavily glycosylated with O-linked glycans and no glycosylphosphatidylinositol anchor could be detected.

| S-EPMC1220693 | biostudies-other

Heparan sulphate proteoglycan and the low-density lipoprotein receptor-related protein 1 constitute major pathways for neuronal amyloid-beta uptake.

Project description:Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder in which the aggregation and deposition of amyloid-? (A?) peptides in the brain are central to its pathogenesis. In healthy brains, A? is effectively metabolized with little accumulation. Cellular uptake and subsequent degradation of A? is one of the major pathways for its clearance in the brain. Increasing evidence has demonstrated significant roles for the low-density lipoprotein receptor-related protein 1 (LRP1) in the metabolism of A? in neurons, glia cells, and along the brain vasculatures. Heparan sulfate proteoglycan (HSPG) has also been implicated in several pathogenic features of AD, including its colocalization with amyloid plaques. Here, we demonstrate that HSPG and LRP1 cooperatively mediate cellular A? uptake. Fluorescence-activated cell sorter and confocal microscopy revealed that knockdown of LRP1 suppresses A? uptake, whereas overexpression of LRP1 enhances this process in neuronal cells. Heparin, which antagonizes HSPG, significantly inhibited cellular A? uptake. Importantly, treatment with heparin or heparinase blocked LRP1-mediated cellular uptake of A?. We further showed that HSPG is more important for the binding of A? to the cell surface than LRP1. The critical roles of HSPG in cellular A? binding and uptake were confirmed in Chinese hamster ovary cells genetically deficient in HSPG. We also showed that heparin and a neutralizing antibody to LRP1 suppressed A? uptake in primary neurons. Our findings demonstrate that LRP1 and HSPG function in a cooperative manner to mediate cellular A? uptake and define a major pathway through which A? gains entry to neuronal cells.

| S-EPMC3272839 | biostudies-literature

Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver.

Project description:Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

| S-EPMC1149861 | biostudies-other

Unraveling Heparan Sulfate Proteoglycan Binding Motif for Cancer Cell Selectivity.

Project description:Membrane heparan sulfate proteoglycans (HSPG) regulate cell proliferation, migration, and differentiation and are therefore considered key players in cancer cell development processes. Here, we used the NT4 peptide to investigate how the sulfation pattern of HSPG on cells drives binding specificity. NT4 is a branched peptide that binds the glycosaminoglycan (GAG) chains of HSPG. It has already been shown to inhibit growth factor-induced migration and invasiveness of cancer cells, implying antagonist binding of HSPG. The binding affinity of NT4 with recombinant HSPG showed that NT4 bound glypican-3 and -4 and, with lower affinity, syndecan-4. NT4 binding to the cancer cell membrane was inversely correlated with sulfatase expression. NT4 binding was higher in cell lines with lower expression of SULF-1 and SULF-2, which confirms the determinant role of sulfate groups for recognition by NT4. Using 8-mer and 9-mer heparan sulfate (HS) oligosaccharides with analog disaccharide composition and different sulfation sites, a possible recognition motif was identified that includes repeated 6-O-sulfates alternating with N- and/or 2-O-sulfates. Molecular modeling provided a fully descriptive picture of binding architecture, showing that sulfate groups on opposite sides of the oligosaccharide can interact with positive residues on two peptide sequences of the branched structure, thus favoring multivalent binding and explaining the high affinity and selectivity of NT4 for highly sulfated GAGs. NT4 and possibly newly selected branched peptides will be essential probes for reconstructing and unraveling binding sites for cancer-involved ligands on GAGs and will pave the way for new cancer detection and treatment options.

| S-EPMC6759624 | biostudies-literature

Evidence for the existence of a multichain proteoglycan of heparan sulphate.

Project description:1. Glycosaminoglycans were extracted with 2m-potassium chloride from bovine aorta and purified by precipitation with cetylpyridinium chloride from 0.5m-potassium chloride. The yield amounted to 24% of the total glycosaminoglycan content of the tissue. 2. After removal of chondroitin sulphate by digestion with testicular hyaluronidase, the residual glycosaminoglycan material (11% of the extracted polysaccharide) was fractionated by gel chromatography on Sephadex G-200. Two peaks (I and II) were obtained, the more retarded of which (II) corresponded to single polysaccharide chains. 3. The macromolecular properties of fraction I were investigated by repeated gel chromatography, after treatment of the fraction with alkali or digestion with papain. In both cases the elution position of fraction I was shifted towards that of the single polysaccharide chains. 4. Analysis of fraction I showed approximately equal amounts of heparan sulphate and dermatan sulphate. It is concluded that these glycosaminoglycans both occur in the aortic wall as multichain proteoglycans.

| S-EPMC1179020 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data