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Biomedical subjects

L A Fransson

Publications and source records attributed to L A Fransson.

At least 73 records · Page 4Linked to original sources
PubMed1982

Demonstration of a direct anti-factor Xa activity in certain heparin-related glycosaminoglycans.

Heparan sulphate/heparin subfractions with high plasma anti-Xa activity have an unusual uronate composition, i.e. high proportions of both glucuronate and sulphated iduronate. These preparations inhibit the amidase activity of factor Xa in an uncompetitive mode and the prothrombin-activation catalyzed by Xa, both in the absence of antithrombin III. Subfractions of low affinity for antithrombin III are equally potent against Xa. The anti-X activity is destroyed by a 3-h periodate oxidation.

Animals↗
PubMed1982

Self-association of scleral proteodermatan sulfate. Evidence for interaction via the dermatan sulfate side chains.

Previous studies on scleral proteoglycans (proteodermatan sulfate) using light scattering and ultracentrifugation techniques have shown that the molecules form aggregates in 0.15 M NaCl (Cöster, L., Fransson, L.-A., Sheehan, J. K., Nieduszynski, I. A., and Phelps, C. F. (1981) Biochem. J., 197, 483-490). Aggregation was not promoted by hyaluronate but addition of free scleral dermatan sulfate chains enhanced multimerization. To investigate the possibility that scleral proteoglycans interact via their dermatan sulfate side chains, we have adopted an affinity chromatography procedure where binding of proteoglycans to various dermatan sulfate-agaroses may be studied. The evidence for an interaction between the side chains of the macromolecules and the immobilized dermatan sulfates are as follows: (a) the dermatan sulfate chains released from the proteoglycan by proteolysis display affinity for dermatan sulfate-agarose, (b) a significant proportion of the [3H]acetylated proteoglycans that were bound to the dermatan sulfate gel can be displaced by eluting with a solution of dermatan sulfate chains, (c) selective periodate oxidation of L-iduronate in the dermatan sulfate chains of the proteoglycans abolishes the affinity, (d) the core protein prepared by chondroitinase ABC digestion of the proteoglycan does not bind to dermatan sulfate-agarose, and (e) binding is retained after reduction-alkylation of the protein core. Furthermore, free [3H]dermatan sulfate chains co-elute with the proteoglycan upon gel filtration in 0.2 M NaCl.

Animals↗
PubMed1982

Co-polymeric glycosaminoglycans in transformed cells. Transformation-dependent changes in the co-polymeric structure of heparan sulphate.

1. Heparan sulphates from normal 3T3 fibroblasts are association-prone as indicated by their affinity for agarose gels substituted with cognate heparan sulphate species. Heparan sulphates from SV40-transformed or polyoma-virus-transformed cells have no affinity for the same gels. 2. Heparan sulphates from the medium, the pericellular and intracellular pools of normal, SV40-transformed and polyoma-transformed 3T3 cells were separated into four subfractions (HS1-HS4) by ion-exchange chromatography. In general, HS1-HS3 were found in cell-derived heparan sulphates, whereas HS3-HS4 were present in the medium. The heparan sulphates from transformed cells were more heterogeneous and of lower charge density than those from the normal counterpart. 3. Degradations via periodate oxidation/alkaline elimination yielded the oligomers glucosamine-(hexuronate-glucosamine)(n)-R with n=1-5 and a large proportion of N-sulphate groups. There was a large contribution of fragments n=4-5 from heparan sulphates of normal cells. These fragments were less common in low-sulphated heparan sulphates of transformed cells. In the case of medium-drived heparan sulphates all species had a low content of fragments n=4-5. 4. The size distribution of (glucuronate-N-acetylglucosamine)(n) regions was assessed after deaminative cleavage. It was broad and ranged from n=1-10 for all heparan sulphate species. In the case of medium-derived heparan sulphates there were distinct differences between normal and transformed cells. In the latter chains the N-acetyl-rich segments were both shorter and longer than in the normal case. The shape of the disaccharide peak was consistent with a lower content of O-sulphate in the heparan sulphates from transformed cells. 5. It was concluded that heparan sulphates from medium or transformed cells exhibit the greatest structural deviation from the normal case. The finding of lower proportions of extended, iduronate/glucuronate-bearing, N-sulphate-rich segments in heparan sulphates of transformed cells was particularly interesting in view of the fact that these elements have been associated with ability to self-interact.

Animals↗
PubMed1981

Self-association of heparan sulfate. Demonstration of binding by affinity chromatography of free chains on heparan sulfate-substituted agarose gels.

We have developed an affinity chromatography procedure which measures binding of heparan sulfate species to agarose gels substituted with different heparan sulfates. Three major subfractions of bovine lung heparan sulfate (HS2, HS3, and HS4) which differ in sulfate content and hexuronate composition have been used. Association-prone variants of these species (HS2-A, HS3-A, and HS4-A) were prepared by gel chromatography. Free heparan sulfate chains were applied to columns of various heparan sulfate-agaroses which were eluted with a linear guanidine gradient and binding was assessed by measuring the hexuronate content of the effluent. Associating heparan sulfate of a particular subfraction was chiefly bound to gels that were substituted with chains of the same kind, i.e. HS2-A to HS2-A-agarose, HS3-A and HS4-A to HS3-A-agarose, and HS4-A to HS4-A-agarose. N-Desulfation and N-acetylation of HS2-A markedly reduced binding to HS2-A-agarose and periodate oxidation of glucuronate in HS3-A completely abolished binding to HS3-A-agarose. Partially oxidized HS2-A was separated into bound and unbound material by affinity chromatography on HS2-A-agarose. Gel chromatography of these fractions indicated that unbound chains were significantly smaller than bound ones. It is concluded that association between heparan sulfate chains may be quite specific and that the strength of binding is dependent on co-operative interactions between a number of contact zones. The latter may correspond to the N-sulfated and iduronate- and glucuronate-containing segments.

Animals↗
PubMed1981

Isopycnic-centrifugation studies in caesium chloride and in caesium sulphate on dermatan sulphate proteoglycans from bovine sclera.

1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs(2)SO(4) both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density rho=1.75g/ml whereas fraction PG-II banded sharply at rho=1.64g/ml. However, in Cs(2)SO(4), fraction PG-II banded at rho=1.51g/ml and fraction PG-I at rho=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs(2)SO(4) fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs(2)SO(4) gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88-42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs(2)SO(4) were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1x10(6)) than were those obtained previously for their ;monomeric' states (fraction PG-I 160000-410000, and fraction PG-II 70000-130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs(2)SO(4) (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs(2)SO(4). Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.

Animals↗
PubMed1981

Bovine aortic chondroitin sulphate- and dermatan sulphate-containing proteoglycans. Isolation, fractionation and chemical characterization.

1. Guanidinium chloride (4M) in the presence of proteinase inhibitors extracted 90% of bovine aorta galactosaminoglycans as proteoglycans that were subsequently purified by ion-exchange and gel chromatography. 2. Fractionation of the calcium salts of the purified proteoglycans with increasing concentration of ethanol yielded fractions PG-25 (28%), PG-35 (45%) and PG-50 (37%). 3. Fraction PG-50 contained proteochondroitin 6-sulphate, whereas fractions PG-25 and PG-35 were proteodermatan sulphates of greatly different carbohydrate composition; the molar proportions of L-iduronate-N-acetylgalactosamine 4-sulphate, D-glucuronate-N-acetyl-galactosamine 4-sulphate and D-glucuronate-N-acetylgalactosamine 6-sulphate were 75: 18 :7 in fraction PG-25 and 14 :46 :40 in fraction PG-35. 4. The presence of alternating or mixed sequences with L-iduronate- and D-glucuronate-containing repeating disaccharides was indicated by the formation of tetrasaccharides after chondroitinase AC digestion (single L-iduronate residues) and by the release of fragments containing four or five consecutive D-glucuronate-N-acetylgalactosamine repeats after periodate oxidation and alkaline elimination. 5. The amino acid compositions of fractions PG-25 and PG-35 were similar and markedly different from that of fraction PG-50, which also contained more side chains.

Amino Acids↗
PubMed1981

Self-association of dermatan sulphate proteoglycans from bovine sclera.

1. Two proteodermatan sulphate fractions (I and II) from bovine sclera were studied by gel chromatography, light-scattering and ultracentrifugation under various conditions. 2. Gel chromatography of proteoglycans in the absence or presence of hyaluronate was performed under associative conditions. No effect on the elution profile was noted. 3. Ultracentrifugation experiments (sedimentation-velocity and sedimentation-equilibrium) with proteoglycan I and II in 6 M-guanidine hydrochloride gave molecular weights (Mw) of 160000-220000 and 70000-100000 respectively. As the protein contents were 45% and 60% respectively, it may be calculated that proteoglycan I contained four to five side chains, whereas proteoglycan II contained one or two. Sedimentation-equilibrium runs performed in 0.15 M-NaCl gave an apparent molecular weight (Mw) of 500000-800000 for proteoglycan I and 90000-110000 for proteoglycan II. 4. In light-scattering experiments both proteoglycans I and II yielded high particle weights in 0.15 M-NaCl (3.1 X 10(6) and 3.4 X 10(6) daltons respectively). In the presence of 6 M-guanidine hydrochloride the molecular weights decreased to 410000 and 130000 respectively. The particle weights in 0.15 M-NaCl were not altered by the addition of hyaluronate or hyaluronate oligosaccharides. 5. The dermatan sulphate side chains of scleral proteoglycans (L-iduronate/D-glucuronate ratio 7:13) gave a particle weight of 100000 daltons in 0.15 M-NaCl. In 1.00 M-KCl/0.02M-EDTA the molecular weight was 24000. Addition of free scleral dermatan sulphate chains to a solution of proteoglycan II promoted further multimerization of the macromolecule.

Animals↗
PubMed1981

Isolation and characterization of dermatan sulphate proteoglycans from bovine sclera.

1. Proteoglycans were extracted from sclera with 4 M-guanidine hydrochloride in the presence of proteinase inhibitors and purified by ion-exchange chromatography and density-gradient centrifugation. 2. The entire proteoglycan pool was characterized by compositional analyses and by specific chemical (periodate oxidation) and enzymic (chondroitinases) degradations. The glycan moieties of the molecules were exclusively galactosaminoglycans (dermatan sulphate-chondroitin sulphate co-polymers). In addition, the preparations contained small amounts of oligosaccharides. 3. The scleral proteodermatan sulphates were fractionated into one larger (I) and one smaller (II) component by gel chromatography. Proteoglycan I was eluted in a more excluded position on gel chromatography in 0.5 M-sodium acetate than in 4.0 M-guanidine hydrochloride. Reduced and alkylated proteoglycan I was eluted in the same position (in 0.5 M-sodium acetate) as was the starting material (in 4.0 M-guanidine hydrochloride). The elution position of proteoglycan II was the same in both solvents. Proteoglycans I and II had s0 20,w values of 2.8 x 10(-13) and 2.2 x 10(-13) s respectively in 6.0 M-guanidine hydrochloride. 4. The two proteoglycans differed with respect to the nature of the protein core and the co-polymeric structure of their side chains. Also proteoglycan I contained more side chains than did proteoglycan II. The dermatan sulphate side chains of proteoglycan I were D-glucuronic acid-rich (80%), whereas those of proteoglycan II contained equal amounts of D-glucuronic acid and L-iduronic acid. Furthermore, the co-polymeric features of the side chains of proteoglycans I and II were different. The protein core of proteoglycan I was of larger size than that of proteoglycan II. The latter had an apparent molecular weight of 46 000 (estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis), whereas the former was greater than 100 000. In addition, the amino-acid composition of the two core preparations was different. 5. As proteoglycan I altered its elution position on gel chromatography in 4 M-guanidine hydrochloride compared with 0.5 M-sodium acetate it is proposed that a change in conformation or a disaggregation took place. If the latter hypothesis is favoured, aggregation may be due to self-association or mediated by an extrinsic molecule, e.g. hyaluronic acid.

Acetates↗
PubMed1981

Self-association of bovine lung heparan sulphates: identification and characterization of contact zones.

Self-association between various heparan sulphate species and oligosaccharide fragments thereof have been studied by affinity chromatography. Polysaccharides or oligosaccharides were coupled to agarose and free chains were applied at low concentrations (less than or equal to 2 mg/ml) in 0.15 M NaCl to minimize self-association between free chains. The results show that the interaction may be specific. Heparan sulphate chains chiefly bind to gels substituted with cognate chains, i.e. the same kind or closely similar ones. Oligosaccharides of the general structure glucosamine-(iduronate/glucuronate-glucosamine)n--O--C(=CH2)--CHO were prepared by periodate oxidation/alkaline elimination and also coupled to agarose via the --CHO group. Cognate heparan sulphate chains were bound to this affinity matrix with the same affinity as in the case of heparan-sulphate--agarose. Free oligosaccharides were not bound to oligosaccharide-agarose, nor to the corresponding heparan-sulphate--agarose. Oligosaccharides of the same size and containing only iduronate were ineffective as affinity ligands. It is concluded that the segments comprising both iduronate and glucuronate may serve as contact zones in the heparan sulphate/heparan sulphate self-association and that the strength of binding is dependent on cooperative interactions between a number of such zones. The putative contact zones, as ligands on the matrix, showed an emerging lack of specificity as non-associating or unrelated and associating chains were bound to this gel. This is ascribed to a randomization of the contact zones which, in the polymeric chains, are placed in their proper register by the intervening (glucuronate-N-acetylglucosamine)n segments.

Animals↗
PubMed1980

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.

1. (3)H- and (35)S-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. (3)H- and (35)S-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.

Alkalies↗
PubMed1980

Interaction between heparan sulphate chains. I. A gel chromatographic, light-scattering and structural study of aggregating and non-aggregating chains.

1. Heparan sulphate from bovine lung was fractionated with cetylpyridinium chloride. Solubilisation of complexes was accomplished by increasing concentrations of NaCl in a step-wise manner. Fractions I-IV, which were low-sulphated, contained more D-glucuronic acid than L-iduronic acid, fraction V contained equal proportions while fraction VI was L-iduronic acid-rich. 2. Gel chromatography of heparan sulphates II-IV in 0.5 M sodium acetate yielded extremely asymmetric profiles, while fractions V, VI and heparin did not. 3. Heparan sulphate IV was separated into aggregatable and non-aggregatable species by gel chromatography in 0.5 M sodium acetate. The particle/molecular weights of the two species were determined by light scattering. In 0.15 M NaCl or KCl the aggregatable chains yielded particle weights of 60 000-100 000 while the molecular weight was 20 000 (in 4.0 M guanidine HCl). Non-aggregatable chains afforded 'monomeric' values in 0.15 M NaCl or KCl. 4. Periodate oxidation of D-glucuronic acid residues in N-acetylated block regions followed by scission in alkali was used to fragment aggregating and non-aggregating heparan sulphate IV. The former chains yielded, on average, shorter oligosaccharides than did the latter. Reoxidation of the remaining D-glucuronic acid residues (adjacent to N-sulphated amino sugars) in the oligosaccharides followed by alkaline cleavage resulted in distinctly different fragmentation patterns in the two cases. The iduronate-containing oligosaccharides derived from aggregatable chains were markedly degraded into fragments ranging from glucosamine-L-iduronic acid-glucosamine-(C-3 fragment) to higher saccharides. Only higher saccharides were obtained from fragments of non-aggregatable chains. 5. It is concluded that self-associating heparan sulphates comprise both D-glucuronic acid- and L-iduronic acid-containing repeating units and that these units are arranged in an alternating or mixed fashion. These characteristics are analogous to those observed with self-associating dermatan sulphate species (Fransson, L.-A. and Cöster, L. (1979) Biochim. Biophys. Acta 582, 132-144).

Animals↗
PubMed1980

Structural studies on heparan sulphates. Characterization of oligosaccharides; obtained by periodate oxidation and alkaline elimination.

Three heparan sulphate fractions were subjected to degradation by periodate oxidation and alkaline elimination. The starting materials were one low-sulphated fraction rich in glucuronic acid and N-acetylglucosamine (I), one fraction (IV) that was medium-sulphated, contained similar proportions of N-acetyl and N-sulphate as well as of glucuronic and iduronic acid and one fraction (V) that was oversulphated, contained an excess of N-sulphate and was rich in iduronic acid. Selective periodate oxidation of glucuronic acid residues within N-acetylated regions followed by scission in alkali yielded three categories of fragments that were isolated by gel and ion-exchange chromatography. (a) N-Acetylglucosamine-R compounds where R is the remnant of an oxidised and degraded glucuronic acid residue. (b) Medium-sized oligosaccharides, of the general structure glucosamine-(glycuronic acid-glucosamine)n-R where n = 1--4, contained largely glucuronic acid associated with glucosamines that were N-sulphated, N-acetylated or unsubstituted. In saccharides where n = 4 indications of an alternating arrangement of N-sulphate and N-acetyl groups as well as of iduronic and glucuronic acid residues were obtained. The glucuronic acid residues of the oligosaccharide fragments were susceptible to reoxidation with periodate. The smaller saccharides were not depolymerised by HNO2 despite the presence of N-sulphate groups. (c) Longer oligosaccharide fragments (n greater than or equal to 5) that were highly sulphated and contained heparin-like repeating units, i.e. iduronic acid--glucosamine-N-sulphate with ester-sulphate on both sugars. These oligosaccharides were susceptible to deaminative cleavage.

Animals↗
PubMed1979

Structure and metabolism of sulphated glycosaminoglycans in cultures of human fibroblasts. Structural characteristics of co-polymeric galactosaminoglycans in sequential extracts of fibroblasts during pulse-chase experiments.

1. Human embryonic lung and skin fibroblasts were allowed to incorporate 32SO42- or 35SO42- and D-[1-3H]glucosamine. After removal of the medium the monolayer was subjected to sequential extractions by using EDTA, brief trypsin digestion, extraction with dithiothreitol ofllowed by freeze--thawing and extraction with trichloroacetic acid. The heparan sulphate and galactosaminoglycan contents of the various extracts were estimated after deaminative cleavage of the former component. Heparan sulphate was the major component of the trypsin digest, whereas galactosaminoglycans were the dominant component of other fractions. 2. Galactosaminoglycans of the various fractions were subjected to chemical (periodate oxidation/alkaline elimination) and enzymic (chondroitinase-AC and -ABC, as well as testicular hyaluronidase) degradations. Galactosaminoglycans from the insoluble cell fraction and the dithiothreitol extract contained larger amounts of L-iduronic acid than did those of other fractions. 3. Pulse-chase experiments were performed with and without replating of the cells at the start of the chase period. Radioactive glycans were isolated from the various extracts during the chase period. The half-lives of glycans of the insoluble cell fraction and the dithioreitol extract were shorter (5--8h) than were those of the trypsin digest and the EDTA extract (22h and 11h respectively). After replating of the cells in chase medium, radioactive cell-associated glycans were secreted from the cells and could be recovered in the trypsin digest, the EDTA extract and the medium. Furthermore, 35S/3H ratios of glycans from all these fractions decreased during the chase period. The following conclusions were reached. The insoluble cell fraction contains the synthesis pool and some structural material, whereas the soluble cell fraction is the storage and degradation pool. The dithiothreitol extract appears to contain the immediate precursors of secreted material. The trypsin-released glycans comprise structural components as well as material destined for pinocytosis or secretion into the medium. The EDTA extract is considered to consist of glycans en route to the medium. 4. The two presumptive precursor pools were preferentially depleted of L-iduronic acid-rich galactosaminoglycans during the chase. Glycans recovered from the trypsin digest, the EDTA extract and the medium during the chase contained larger amounts of periodate-resistant uronic acid residues (D-glucuronic acid and/or L-iduronic acid O-sulphate) than did their precursors. It is proposed that polymer-level modifications of secreted glycans are partly responsible for the results.

Cells, Cultured↗