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L A Fransson

Publications and source records attributed to L A Fransson.

At least 55 records · Page 3Linked to original sources
PubMed1990

A fibroblast heparan sulphate proteoglycan with a 70 kDa core protein is linked to membrane phosphatidylinositol.

Here we present evidence that a fibroblast heparan sulphate proteoglycan of approx. 300 kDa and with a core protein of apparent molecular mass 70 kDa is covalently linked to the plasma membrane via a linkage structure involving phosphatidylinositol. Phosphatidylinositol-specific phospholipase C releases such a heparan sulphate proteoglycan only from cells labelled with [35S]sulphate in the absence of serum. Cell cultures labelled with [3H]myo-inositol in the absence or presence of serum produce a radiolabelled heparan sulphate proteoglycan which was purified by gel-permeation chromatography and ion-exchange chromatography on MonoQ. Digestion with heparan sulphate lyase and analysis by gel-permeation chromatography and sodium dodecylsulphate-polyacrylamide gel-electrophoresis revealed that the 3H-label is associated with a core protein of apparent mass 70 kDa.

Cells, Cultured↗
PubMed1990

TGF-beta enhances the production of hyaluronan in human lung but not in skin fibroblasts.

Transforming growth factor-beta (TGF-beta) enhances the production of extracellular matrix components, such as type I and type III collagen, fibronectin, proteoglycans, in various cell types. The effect on hyaluronan synthesis in relation to proteoglycan synthesis has not been investigated. Human lung or skin fibroblast cultures were treated with TGF-beta in serum-free medium for various periods of time. 35SO4 or [3H]glucosamine was then added to the cultures in the absence of TGF-beta for up to 48 h. Hyaluronan and proteoglycans were isolated by ion-exchange chromatography and quantitated. TGF-beta induced a three- to fourfold increase in hyaluronan production by lung cells but had no effect on skin fibroblasts. In contrast, proteoglycan synthesis was enhanced in both cell types, although skin fibroblasts responded at lower concentrations of TGF-beta. Increased accumulation of hyaluronan was noted only in the cell medium, whereas proteoglycan accumulation was observed both in the medium and in the cell layer. The ED50 for TGF-beta on hyaluronan accumulation in lung cells was the same as that for proteoglycan accumulation, i.e., 40 pM. In skin fibroblasts the ED50 was considerably lower (4 pM). The induction time needed to attain full effect of TGF-beta was 6 h for both hyaluronan and proteoglycan synthesis. These results indicate that TGF-beta has tissue-specific effects on matrix production which may be of importance for control of cell proliferation in various disease states.

Cells, Cultured↗
PubMed1989

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

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.

Aggrecans↗
PubMed1986

Structure of proteoheparan sulfates from fibroblasts. Confluent and proliferating fibroblasts produce at least three types of proteoheparan sulfates with functionally different core proteins.

[3H]Leucine- and [35S]sulfate-labeled proteoheparan sulfates were isolated from postconfluent or proliferating cultures of human skin fibroblasts. Cell layers were solubilized by Triton X-100, and transferrin-binding macromolecules were isolated by affinity chromatography. Proteoglycans with no affinity for transferrin were purified by using ion-exchange and gel permeation chromatography. Postconfluent cells synthesize a proteoheparan sulfate of Mr 350,000 (as determined by gel permeation chromatography) which has affinity for transferrin as well as for octyl-Sepharose. Its core protein (Mr 180,000) consists of two disulfide-bonded polypeptides of Mr 90,000. This species was not detected in cultures of proliferating cells. Proliferating and confluent cells also synthesize other forms of proteoheparan sulfates (Mr 200,000-400,000) which have no affinity for transferrin. However, most of them have affinity for octyl-Sepharose. The core protein of proteoheparan sulfates made by proliferating cells has Mr 50,000. A smaller form (Mr 250,000) of this proteoglycan was solubilized by Triton X-100, whereas a larger form (Mr 400,000) remained associated with the pericellular matrix. A third type of proteoheparan sulfate (Mr 200,000) without affinity for transferrin nor octyl-Sepharose was associated with postconfluent cell layers but not with proliferating ones. Its core protein has Mr 35,000. Heparan sulfate oligosaccharides (Mr 6,000 or higher) were found in proliferating cells but not in postconfluent ones.

Cell Division↗
PubMed1986

The functions of the heparan sulphate proteoglycans.

Heparan sulphate (HS)-containing proteoglycans (HS-PGs) are present at the surface of nearly all adherent mammalian cells. The principal mode of attachment is by way of the protein core which is inserted into the plasma membrane. Other forms of HS-PG may be components of pericellular matrices, notably basement membranes. The core proteins of HS-PGs can be small (35K) as in hepatocytes, intermediate (50K) as in many mesenchymal cells, or very large (400K) as in basement membranes. A special case is the HS-PG synthesized by postconfluent fibroblasts. This proteoglycan has a core protein that closely resembles the transferrin receptor glycoprotein. It is possible that this HS-PG is a pro-form of the receptor. Low molecular weight, carbohydrate-rich HS-PG forms are probably derived from larger forms by partial degradation. The HS side-chains can contain 24 different disaccharides in an unknown number of arrangements. The biosynthetic machinery can impose considerable restrictions; for example, the extent of N-sulphation rarely exceeds 40-50%, whereas O-sulphation may range from 20% to 75% of potential sites. Nevertheless, the informational capacity of HS is formidable. By way of the HS chains, HS-PG at the surface of endothelial cells can interact specifically or selectively with a number of plasma proteins. HS-PG at the surface of matrix-producing cells is similarly in a position to interact with matrix proteins, notably collagen, fibronectin and laminin. As the cytoplasmic portion of the HS-PG core protein can bind actin, this proteoglycan can provide a connection between extracellular matrices and the cytoskeleton. A number of studies support a role for HS-PGs in the control of cell growth, and this could be one of their major functions. Whether the HS side-chains or the core protein or both are carrying out such a function remains to be determined.

Animals↗
PubMed1985

Structure of the heparan sulfate-protein linkage region. Demonstration of the sequence galactosyl-galactosyl-xylose-2-phosphate.

We have treated bovine lung heparan sulfate with alkaline [3H]borohydride to end label the chains with [3H]xylitol. After subsequent periodate oxidation-alkaline elimination products were separated by gel permeation and ion exchange chromatography. The linkage region fragment expected to have 2 galactoses and 1 [3H]xylitol residue appeared in the tetra-/trisaccharide region after gel filtration and was bound to the anion exchange resin. A similar negatively charged fragment, expected to have 2 galactoses, 1 xylose and 1 serine, was isolated after periodate oxidation-alkaline elimination of unlabeled heparan sulfate. The negative charge was due to the presence of alkaline phosphatase-labile phosphate ester. The molar ratio of galactose:phosphate:xylose was 2.17:1.19:1.00. The phosphate ester was associated with the xylose/[3H] xylitol moiety as indicated by the formation of phosphoxylose/-xylitol by beta-galactosidase digestion of the phosphorylated trisaccharide. Furthermore, orcinol reactivity disappeared after periodate oxidation of the dephosphorylated trisaccharide. The phosphate ester must be located to C-2 of xylose/xylitol as the 1-3H radioactivity could be released by periodate oxidation when it was preceded by alkaline phosphatase treatment. It is estimated that almost every chain of heparan sulfate carries 2-phosphoxylose. It would be of interest to know if glycosaminoglycan chains that are artificially initiated onto exogeneous beta-D-xylosides also acquire the 2-phosphoxylose moiety.

Animals↗
PubMed1985

Domain structure of proteoheparan sulphate from confluent cultures of human embryonic skin fibroblasts.

Radiolabelled proteoheparan sulphates were isolated from confluent monolayers of fibroblasts and from their spent media. The cell-surface-associated proteoglycan (Mr 350 000) has a core protein of Mr 180 000 that is cleaved by reduction of disulphide bonds into polypeptides of Mr 90 000, both of which can bind transferrin [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661]. Thrombin digestion of the proteoglycan yielded two major fragments. The larger one contained the heparan sulphate chains and glycoprotein-type oligosaccharides, whereas the smaller one contained interchain disulphide bond(s) and had affinity for transferrin as well as for octyl-Sepharose. The larger thrombic fragment was cleaved by trypsin into fragments containing the heparan sulphate chains and the oligosaccharides respectively. The smaller proteoheparan sulphate derived from the culture medium (Mr 150 000) had a core protein of Mr 30 000, which contained heparan sulphate-attachment and oligosaccharide-attachment regions, but no domains for binding of transferrin or for hydrophobic interactions.

Alkylation↗
PubMed1985

On the self-affinity of heparan sulfates from quiescent or proliferating normal 3T3 cells and from SV40-transformed cells.

35S-Labelled heparan sulfates derived from the culture medium (extracellular), a trypsinate of the cells (pericellular) and the cell residue (intracellular) of quiescent normal, proliferating normal or SV40-transformed 3T3 cells were analyzed for charge heterogeneity, by ion exchange chromatography and for self-affinity, by chromatography on heparan sulfate-agarose gels. Quiescent normal cells retained most of their heparan sulphate intra- or pericellularly. The surface-exposed material was charge heterogeneous and had a strong affinity for heparan sulfate. In cultures of growing cells and transformed cells most of the heparan sulfate was found in the medium. The heparan sulfate retained on the surface or growing cells had a lower self-affinity than did the corresponding material from normal and transformed cells. Although cell surface heparan sulfates from transformed cells showed affinity for a matrix substituted with the total heparan sulfate pool, the affinity for one particular subtype was much less pronounced or non-existent.

Animals↗
PubMed1985

Chemical heterogeneity of heparan sulfate from a human neuroblastoma cell line.

The chemical heterogeneity of radiolabelled neuroblastoma heparan sulfate has been studied by ion exchange chromatography and by affinity chromatography on heparan sulfate-agarose. Although the entire population of chains shows considerable homogeneity in charge density, the deaminative cleavage products ranged in size from disaccharides to eicosasaccharides. Under appropriate conditions neuroblastoma heparan sulfate could be separated into two pools of low or high affinity for lung heparan sulfate-agarose. Analyses of periodate oxidation-alkaline elimination indicated that the high affinity chains contained larger proportions of heparin-like segments, i.e. iduronate-rich and N-sulfated ones.

Cell Line↗
PubMed1984

Binding of transferrin to the core protein of fibroblast proteoheparan sulfate.

Cell-surface-associated proteoheparan sulfate from confluent human skin fibroblasts appears to consist of two disulfide-bonded polypeptides of Mr approximately equal to 90,000. The transferrin receptor, a ubiquitous cell-surface component of proliferating cells, also consists of two subunits of Mr 90,000 linked by S--S bonds. Radiolabeled proteoheparan sulfate mixed with holotransferrin or apotransferrin at pH 4-5 followed by rabbit anti-human transferrin was adsorbed onto protein A-Sepharose to approximately equal to 80-90%. At pH 7.5 apotransferrin bound approximately equal to 40% of the proteoglycan, whereas approximately equal to 80% was bound to holotransferrin. Trypsin digestion of the proteoglycan markedly lowered its ability to bind transferrin. However, binding was essentially unaffected by heparan-sulfate lyase treatment and after reduction and alkylation. Over 90% of the 3H activity of an L-[3H]leucine-labeled proteoglycan was recovered by immunoprecipitation (transferrin.antitransferrin) of a heparan-sulfate lyase digest of the proteoglycan. The immunoprecipitated core protein had an apparent Mr of 150,000 before reduction and Mr of 90,000 after reduction of disulfide bonds. The core protein of the proteoglycan was recognized by the monoclonal antibody B3/25, which is known to be receptor specific. The present findings suggest that the core polypeptides of proteoheparan sulfate and the transferrin receptor may be identical or closely similar.

Blood Proteins↗
PubMed1983

Proteoheparan sulfate from human skin fibroblasts. Evidence for self-interaction via the heparan sulfate side chains.

We have studied the affinity between fibroblast proteoheparan sulfate (medium- and cell surface-derived species) and heparan sulfate-agaroses by affinity chromatography. The evidence for an interaction between the heparan sulfate side chains of the proteoglycans and the immobilized heparan sulfate are as follows: (a) the individual side chains released from the proteoglycan by papain bind to the affinity matrix, (b) the bound proteoglycans are desorbed by a solution of cognate heparan sulfate chains, and (c) the core protein obtained by heparan sulfate-lyase digestion of the proteoglycan does not bind to the affinity matrix. The proteoglycans interact only with one subtype of heparan sulfate. The binding of free heparan sulfate chains to the affinity matrix is completely abolished by heparan sulfate oligosaccharides provided they are composed of both iduronate- and glucuronate-containing disaccharide sequences.

Animals↗
PubMed1983

The core protein of fibroblast proteoheparan sulphate consists of disulphide-bonded subunits.

Fibroblast proteoheparan sulphate has a disulphide-bonded subunit structure. The core protein appears to consist of two polypeptides each of Mr 80 000-100 000. As shown elsewhere [Carlstedt, Cöster, Malmström & Fransson (1983) J. Biol. Chem. in the press], both polypeptide molecules carry four to six heparan sulphate side chains (approx. Mr 20 000) and an unknown number of oligosaccharide units, giving the whole macromolecule an Mr in the range 300 000-400 000.

Chondroitin Sulfate Proteoglycans↗
PubMed1983

Proteoheparan sulfate from human skin fibroblasts. Isolation and structural characterization.

Fibroblasts in culture were incubated with [35S]sulfate/[3H]glucosamine or [35S]sulfate/[3H]leucine. Proteoglycans were isolated from the medium and a 4 M guanidinium chloride extract of the cell layer or from a trypsin digest of the cells and an extract of the cell residue. Proteoglycans were isolated by density gradient centrifugation, gel permeation, and ion exchange chromatography after digesting contaminating proteogalactosaminoglycans with chondroitinase ABC. Gel chromatography suggests that the cell-derived protoheparan sulfate had an Mr = 350,000 whereas the trypsin-released and the medium-derived counterparts both had an Mr = 140,000. Reduction and alkylation of the cell-derived proteoglycan gave rise to a component with Mr = 140,000, whilst the medium-derived form was not affected. Degradation of cell-associated proteoheparan sulfate by trypsin followed by papain or alkali suggest that the core protein consists of three types of regions, heparan sulfate-containing regions of Mr = 140,000, oligosaccharide-containing regions, and nonglycosylated peptide regions containing most of the [3H]leucine. The heparan sulfates of the cell- and medium-derived proteoglycans were similar in size distribution and charge density and with regard to the proportions and arrangements of various building blocks.

Cells, Cultured↗
PubMed1982

Structural features of the contact zones for heparan sulphate self-association.

The self-association between heparan sulphate chains has been investigated by using heparan sulphate oligosaccharides for the competitive elution of [3H]heparan sulphate from heparan sulphate-agarose. Partial or complete periodate-oxidation followed by alkali-catalysed scission afforded oligomers having the general structure GlcN-(HexA-GlcN)n-R. Oligosaccharides with n greater than 5 were able to desorb bound heparan sulphate, provided that mixed or alternating arrangements of iduronate and glucuronate were present in these fragments. Longer fragments were more effective than shorter ones. The present results corroborate previous proposals that the highly copolymeric regions of heparan sulphate serve as contact zones for the chain-chain association.

Animals↗
PubMed1982

Structural requirements for heparan sulphate self-association.

To investigate heparan sulphate self-association, various sub-fractions of beef-lung heparan sulphate have been subjected to affinity chromatography on heparan sulphate-agarose. A particular variant of heparan sulphate was chiefly bound to matrices substituted with the same or cognate heparan sulphates. N-desulphation and N-acetylation abolished the chain-chain interaction. Also, dermatan sulphates and chondroitin sulphates showed affinity for heparan sulphate-agarose. [3H]Heparan sulphates that were bound to a heparan sulphate-agarose were desorbed by elution with the corresponding heparan sulphate chains and also with unrelated heparan sulphates, heparin, and the galactosaminoglycans to various degrees. However, the corresponding heparan sulphate species was the most efficient at low concentrations. Dextran sulphate was unable to desorb bound heparan sulphate. When the corresponding heparan sulphate was N-desulphated/N-acetylated, carboxyl-reduced, or periodate-oxidised (D-glucuronate), the modified polymer was unable to displace [3H]heparan sulphate from heparan sulphate-agarose. The displacing ability of heparin was also destroyed by periodate oxidation. It is concluded that self-interaction between heparan sulphate chains is strongly dependent on the overall molecular conformation. The N-sulphate and carboxylate groups as well as the integrity of the D-glucuronate residue are all essential for maintaining the proper secondary structure.

Animals↗
PubMed1982

Chemical features of aggregatable heparan sulphates from human-lung fibroblasts.

The ability of fibroblast heparan sulphate to aggregate was examined by affinity chromatography on agarose gels substituted with heparan sulphate variants from beef-lung tissue (designated HS2-A, HS3-A, and HS4-A) having different tendencies for association. Fibroblast heparan sulphate bound only to HS4-A agarose. The cell-derived material was subdivided into low-, medium-, and high-affinity species by affinity chromatography on HS4-A-agarose. High-affinity variants contained a larger proportion of chains of high molecular weight. Degradation of heparan sulphate by selective periodate-oxidation of glucuronate in regions rich in 2-acetamido-2-deoxyglucose followed by scission in alkali produced fragments comprising the highly irregular, iduronate-rich and N-sulphate-rich domains. The latter were slightly larger in the high-affinity form. By re-oxidation of the irregular domains, the location of glucuronate residues was identified. The degradation pattern, i.e., the distribution of iduronate-containing repeats, was markedly similar in the high-affinity variant and in the HS4-A chains that were used as affinity ligands. It is suggested that the strength and specificity of heparan sulphate self-association is dependent on complementary and co-operative associations between a number of contact zones.

Chromatography, Affinity↗
PubMed1982

Structural differences between beef-lung heparan sulphates with specific self-associations.

Three, specifically self-associating variants of heparan sulphate (HS2-A, HS3-A, and HS4-A) from beef lung were subjected to (a) deaminative cleavage of bonds between 2-deoxy-2-sulphoaminoglucose and uronic acid and (b) periodate oxidation of glucuronic acid residues in fully N-acetylated block-regions. In addition, the periodate-oxidised and alkali-cleaved chains were re-oxidised with periodate to identify the glucuronic acid residues in the N-sulphate-containing regions. The results showed that HS2-A was distinguished by much longer (GlcA-GlcNAc)n-segments than HS3-A and HS4-A. The latter two species were characterised by the structure of the variously N-acetyl- and N-sulphate-containing regions. In HS3-A, there was a significant contribution from segments composed of both N-acetylated and N-sulphated 2-amino-2-deoxyglucose residues. The N-sulphate-rich regions contained chiefly iduronic acid. In contrast, HS4-A had mixed or alternating arrangements of the two epimeric uronic acids in the N-sulphate-rich regions. These differences may be the basis for specific self-associations between heparan sulphate chains.

Animals↗