scholarly journals Identification of a 64 kDa heparan sulphate proteoglycan core protein from human lung fibroblast plasma membranes with a monoclonal antibody

1987 ◽  
Vol 247 (3) ◽  
pp. 765-771 ◽  
Author(s):  
H de Boeck ◽  
V Lories ◽  
G David ◽  
J J Cassiman ◽  
H van den Berghe
Keyword(s):  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Human Lung ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Mass ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts ◽  
The Core

Human lung fibroblasts produce heparan sulphate proteoglycans (HSPG) that are associated with the plasma membrane. A monoclonal-antibody (Mab)-secreting hybridoma, S1, was produced by fusion of SP 2/0-AG 14 mouse myeloma cells with spleen cells from mice immunized with partially purified cellular HSPG fractions. The HSPG character of the material carrying the epitope recognized by Mab S1 was demonstrated by: (i) the co-purification of the S1 epitope with the membrane HSPG of human lung fibroblasts; (ii) the decrease in size of the material carrying the S1 epitope upon treatment with heparinase or heparitinase, and the resistance of this material to heparinase treatment after N-desulphation. The S1 epitope appears to be part of the core protein, since it was destroyed by proteinase treatment and by disulphide-bond reduction, but not by treatments that depolymerize the glycosaminoglycan chains and N-linked oligosaccharide chains. Polyacrylamide-gel electrophoresis of non-reduced heparitinase-digested membrane HSPG followed by Western blotting and immunostaining with Mab S1 revealed a single band with apparent molecular mass of 64 kDa. Membrane proteoglycans isolated from detergent extracts or from 4 M-guanidinium chloride extracts of the cells yielded similar results. Additional digestion with N-glycanase lowered the apparent molecular mass of the immunoreactive material to 56 kDa, suggesting that the core protein also carries N-linked oligosaccharides. Fractionation of 125I-labelled membrane HSPG by immuno-affinity chromatography on immobilized Mab S1, followed by heparitinase digestion and polyacrylamide-gel electrophoresis of the bound material, yielded a single labelled band with apparent molecular mass 64 kDa. Treatment with dithiothreitol caused a slight increase in apparent molecular mass, suggesting that the core protein of this membrane proteoglycan of a single subunit containing (an) intrachain disulphide bond(s).

scholarly journals Structural studies on heparan sulphate from human lung fibroblasts. Characterization of oligosaccharides obtained by selective periodate oxidation of d-glucuronic acid residues followed by scission in alkali

1980 ◽  
Vol 191 (1) ◽  
pp. 103-110 ◽  
Author(s):  
Ingrid Sjöberg ◽  
Lars-Ȧke Fransson
Keyword(s):  
Uronic Acid ◽  
Glucuronic Acid ◽  
Human Lung ◽  
General Structure ◽  
Heparan Sulphate ◽  
Periodate Oxidation ◽  
Exchange Chromatography ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts ◽  
Iduronic Acid

1. 3H- and 35S-labelled heparan sulphate was isolated from monolayers of human lung fibroblasts and subjected to degradations by (a) deaminative cleavage and (b) periodate oxidation/alkaline elimination. Fragments were resolved by gel- and ion-exchange-chromatography. 2. Deaminative cleavage of the radioactive glycan afforded mainly disaccharides with a low content of ester-sulphate and free sulphate, indicating that a large part (approx. 80%) of the repeating units consisted of uronosyl-glucosamine-N-sulphate. Blocks of non-sulphated [glucuronosyl-N-acetyl glucosamine] repeats (3–4 consecutive units) accounted for the remainder of the chains. 3. By selective oxidation of glucuronic acid residues associated with N-acetylglucosamine, followed by scission in alkali, the radioactive glycan was degraded into a series of fragments. The glucuronosyl-N-acetylglucosamine-containing block regions yielded a compound N-acetylglucosamine–R, where R is the remnant of an oxidized and degraded glucuronic acid. Periodate-insensitive uronic acid residues were recovered in saccharides of the general structure glucosamine–(uronic acid–glucosamine)n–R. 4. Further degradations of these saccharides via deaminative cleavage and re-oxidations with periodate revealed that iduronic acid may be located in sequences such as glucosamine-N-sulphate→iduronic acid→N-acetylglucosamine. Occasionally the iduronic acid was sulphated. Blocks of iduronic acid-containing repeats may contain up to five consecutive units. Alternating arrangements of iduronic acid- and glucuronic acid-containing repeats were also observed. 5. 3H- and 35S-labelled heparan sulphates from sequential extracts of fibroblasts (medium, EDTA, trypsin digest, dithiothreitol extract, cell-soluble and cell-insoluble material) afforded similar profiles after both periodate oxidation/alkaline elimination and deaminative cleavage.

scholarly journals The small dermatan sulphate proteoglycans synthesized by fibroblasts derived from skin, synovium and gingiva show tissue-related heterogeneity

1988 ◽  
Vol 256 (1) ◽  
pp. 35-40 ◽  
Author(s):  
H Larjava ◽  
J Heino ◽  
T Krusius ◽  
E Vuorio ◽  
M Tammi
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Core Protein ◽  
Culture Media ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gingival Fibroblasts ◽  
Dermatan Sulphate ◽  
Different Tissues

Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix.

scholarly journals Effect of transforming growth factor-β1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican

1995 ◽  
Vol 310 (1) ◽  
pp. 73-81 ◽  
Author(s):  
M Romarís ◽  
A Bassols ◽  
G David
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Fibroblast Growth Factor ◽  
Transforming Growth Factor ◽  
Human Lung ◽  
Core Protein ◽  
Lung Fibroblasts ◽  
Fibroblast Growth ◽  
Tgf Beta ◽  
Human Lung Fibroblasts

We have tested the effects of transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF) and TGF-beta 1 + bFGF on the expression of the cell surface proteoglycans (CD44, syndecans and glypican) in cultures of human lung fibroblasts (HLF). Cell surface proteoglycan expression was monitored by quantitative immunoprecipitation from metabolically labelled cells. Western and Northern blotting and evaluation of the glycanation of the proteoglycans. Stimulation of the cells with TGF-beta 1 increased the length of the chondroitin sulphate (CS) chains on CD44 (approximately 1.6-fold). bFGF, administered solely, also increased the length of the CS chains on CD44 (approximately 1.4-fold), whereas the combination of TGF-beta 1 + bFGF nearly doubled both the length and the number of the CS chains on CD44. None of these treatments lead to changes in CD44 message or core-protein expression. This enhanced glycanation of CD44 after the TGF-beta 1, bFGF and combined treatments correlated with a 2-fold increase in the affinity of the proteoglycan for fibronectin but had no influence on the binding to type I collagen. TGF-beta 1, alone or in combination with bFGF, also stimulated the CS content of syndecan-1, but none of the other syndecans was significantly affected by any of the factors or combinations tested. The expression of glypican however was significantly decreased (nearly halved) by the combination of TGF-beta 1 + bFGF, less so by TGF-beta 1 and not at all by bFGF. This decrease occurred both at the level of the message and of the core protein. These data demonstrate specific and differential effects of TGF-beta 1 and bFGF on the structure, expression and interactions of the cell surface proteoglycans of HLF.

scholarly journals Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts.

1989 ◽  
Vol 108 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
G David ◽  
V Lories ◽  
A Heremans ◽  
B Van der Schueren ◽  
J J Cassiman ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Human Lung ◽  
Core Protein ◽  
Human Fibroblasts ◽  
Chondroitin Sulfate Proteoglycans ◽  
Lung Fibroblasts ◽  
Hydrophobic Membrane ◽  
Human Lung Fibroblasts ◽  
Core Proteins

Cultured human fetal lung fibroblasts produce some chondroitin sulfate proteoglycans that are extracted as an aggregate in chaotropic buffers containing 4 M guanidinium chloride. The aggregated proteoglycans are excluded from Sepharose CL4B and 2B, but become included, eluting with a Kav value of 0.53 from Sepharose CL4B, when Triton X-100 is included in the buffer. Conversely, some of the detergent-extractable chondroitin sulfate proteoglycans can be incorporated into liposomes, suggesting the existence of a hydrophobic membrane-intercalated chondroitin sulfate proteoglycan fraction. Purified preparations of hydrophobic chondroitin sulfate proteoglycans contain two major core protein forms of 90 and 52 kD. A monoclonal antibody (F58-7D8) obtained from the fusion of myeloma cells with spleen cells of BALB/c mice that were immunized with hydrophobic proteoglycans recognized the 90- but not the 52-kD core protein. The epitope that is recognized by the antibody is exposed at the surface of cultured human lung fibroblasts and at the surface of several stromal cells in vivo, but also at the surface of Kupffer cells and of epidermal cells. The core proteins of these small membrane-associated chondroitin sulfate proteoglycans are probably distinct from those previously identified in human fibroblasts by biochemical, immunological, and molecular biological approaches.

scholarly journals Biglycan isoforms with differences in polysaccharide substitution and core protein in human lung fibroblasts

2002 ◽  
Vol 269 (15) ◽  
pp. 3688-3696 ◽  
Author(s):  
Ellen Tufvesson ◽  
Johan Malmström ◽  
György Marko-Varga ◽  
Gunilla Westergren-Thorsson
Keyword(s):  
Human Lung ◽  
Core Protein ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts

scholarly journals Identification of the core proteins in proteoglycans synthesized by vascular endothelial cells

1989 ◽  
Vol 261 (1) ◽  
pp. 145-153 ◽  
Author(s):  
A Lindblom ◽  
I Carlstedt ◽  
L Å Fransson
Keyword(s):  
Endothelial Cells ◽  
Molecular Mass ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Buoyant Density ◽  
Cell Extract ◽  
Apparent Molecular Mass ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Proteoglycans, metabolically labelled with [3H]leucine and 35SO4(2-), were isolated from the spent media and from guanidinium chloride extracts of cultured human umbilical-vein endothelial cells by using isopycnic density-gradient centrifugation, gel filtration and ion-exchange h.p.l.c. The major proteoglycan species were subjected to SDS/polyacrylamide-gel electrophoresis before and after enzymic degradation of the polysaccharide chains. The cell extract contained mainly a heparan sulphate proteoglycan that has a buoyant density of 1.31 g/ml and a protein core with apparent molecular mass 300 kDa. The latter was heterogeneous and migrated as one major and one minor band. After reduction, the apparent molecular mass of the major band increased to approx. 350 kDa, indicating the presence of intrachain disulphide bonds. The proteoglycan binds to octyl-Sepharose and its polysaccharide chains are extensively degraded by heparan sulphate lyase. The proteoglycans of the medium contained 90% of all the incorporated 35SO4(2-). Here the predominant heparan sulphate proteoglycan was similar to that of the cell extract, but was more heterogeneous and contained an additional core protein with apparent molecular mass 210 kDa. Furthermore, two different chondroitin sulphate proteoglycans were found: one 200 kDa species with a high buoyant density (approx. 1.45 g/ml) and one 100 kDa species with low buoyant density (approx. 1.3 g/ml). Both these proteoglycans have a core protein of molecular mass approx. 47 kDa.

scholarly journals Isolation and partial characterization of heparan sulphate proteoglycans from human hepatic amyloid

1992 ◽  
Vol 288 (1) ◽  
pp. 225-231 ◽  
Author(s):  
J H Magnus ◽  
T Stenstad ◽  
G Husby ◽  
S O Kolset
Keyword(s):  
Molecular Mass ◽  
Core Protein ◽  
Alkali Treatment ◽  
Heparan Sulphate ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Tissue Sections ◽  
The Core ◽  
Amyloid Deposits ◽  
Sds Page

Proteoglycans were isolated from human amyloidotic liver by extraction with guanidine, followed by trichloroacetic acid precipitation, DEAE-Sephacel ion-exchange chromatography, and Sepharose CL-6B gel chromatography. A significant portion of the material was found to be free chondroitin/dermatan sulphate chains (30%), whereas the predominant part was heparan sulphate proteoglycan (HSPG) (70%). The approx. molecular mass of the HSPG was 200 kDa, as measured by gel electrophoresis and gel chromatography. The molecular mass of the core protein was shown to be 60 kDa by SDS/PAGE following de-aminative cleavage of the heparan sulphate chains. The heparan sulphate chains were liberated from the core protein by alkali treatment and found to have a molecular mass of approx. 35 kDa by Sepharose CL-6B gel chromatography. The core protein was shown, by immunoblotting, to react with a monoclonal antibody against bovine basement membrane HSPG. The presence of HSPG in amyloid deposits was further confirmed by immunohistochemistry on tissue sections from amyloidotic liver using the same antibody.

scholarly journals Molecular cloning of a phosphatidylinositol-anchored membrane heparan sulfate proteoglycan from human lung fibroblasts.

1990 ◽  
Vol 111 (6) ◽  
pp. 3165-3176 ◽  
Author(s):  
G David ◽  
V Lories ◽  
B Decock ◽  
P Marynen ◽  
J J Cassiman ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Human Lung ◽  
Core Protein ◽  
Heparan Sulfate Proteoglycan ◽  
Amino Acid Sequences ◽  
Lung Fibroblasts ◽  
Sulfate Proteoglycan ◽  
Human Lung Fibroblast ◽  
Human Lung Fibroblasts ◽  
Core Proteins

Two mAbs raised against the 64-kD core protein of a membrane heparan sulfate proteoglycan from human lung fibroblasts also recognize a nonhydrophobic proteoglycan which accumulates in the culture medium of the cells. Pulse-chase studies suggest that the hydrophobic cell-associated forms act as precursors for the nonhydrophobic medium-released species. The core proteins of the medium-released proteoglycans are slightly smaller than those of the hydrophobic cell-associated species, but the NH2-terminal amino acid sequences of both forms are identical. The characterization of human lung fibroblast cDNAs that encode the message for these core proteins and the effect of bacterial phosphatidylinositol-specific phospholipase C suggest that the hydrophobic proteoglycan is membrane-anchored through a phospholipid tail. These data identify a novel membrane proteoglycan in human lung fibroblasts and imply that the shedding of this proteoglycan may be related to the presence of the phospholipid anchor.

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