Biomedical subjects

L A Fransson

Publications and source records attributed to L A Fransson.

At least 37 records · Page 2Linked to original sources

PubMed1994

Patterns of uronosyl epimerization and 4-/6-O-sulphation in chondroitin/dermatan sulphate from decorin and biglycan of various bovine tissues.

Dermatan sulphate is a co-polymer of two types of disaccharide repeats: D-glucuronate-N-acetylgalactosamine and L-iduronate-N-acetylgalactosamine. The former can be O-sulphated at C-4 or C-6 of the galactosamine, whereas the latter contains almost exclusively 4-O-sulphated galactosamine. A minor proportion of the L-iduronate may be O-sulphated at C-2. Chondroitin sulphate has no L-iduronate-containing repeats. We have used our recently developed methods for sequence analysis of galactosaminoglycans to investigate the structure of dermatan/chondroitin sulphates of the proteoglycans decorin and biglycan derived from various bovine tissues, like dermis, sclera, tendon, aorta, cartilage and bone. The glycan chains, radioiodinated at the reducing end, were partially cleaved with specific enzymes (chondroitin lyases), and subjected to high-resolution polyacrylamide gel electrophoresis, blotting and autoradiography to identify fragments extending from the labelled reducing end to the point of cleavage. We used chondroitin B lyase to identify the location of L-iduronate, chondroitin AC-I lyase to locate D-glucuronate and chondroitin C lyase to cleave where D-glucuronate residues were succeeded by 6-O-sulphated N-acetylgalactosamine. We could demonstrate tissue-specific, periodic and wave-like patterns of distribution for the two epimeric uronic acids, as well as specific patterns of sulphation in dermatan sulphates derived from either decorin or biglycan. For example, some dermatan sulphates contained D-glucuronate-rich domains that were always 6-sulphated (scleral decorin), others were always 4-sulphated (decorin from bovine dermis, cartilage and bone; biglycan from aorta) or 6-sulphated near the linkage region, but 4-sulphated in more distal domains (decorin from porcine dermis and bovine tendon). Decorin from bone and articular cartilage, as well as biglycan from articular and nasal cartilage, carried largely chondroitin sulphate chains, but also some dermatan sulphate, whereas galactosaminoglycan chains derived from aggrecan of nasal cartilage were free of L-iduronate. Decorin and biglycan from the same tissue (articular cartilage or sclera) had similar glycan chains. The two side chains in a biglycan molecule are probably also similar to one another. The portion of the glycan chains nearest to the core protein was substituted with charged groups to a variable degree, which may correlate with the structural features of the main chain.

Animals↗

PubMed1993

Characterization of two different glycosylated domains from the insoluble mucin complex of rat small intestine.

The highly glycosylated domains of rat small intestinal mucins were isolated after reduction and trypsin digestion and separated into two populations (A and B) by gel chromatography. The molecular mass values were 650 and 335 kDa, respectively, and the relative yields suggest that the two glycopeptides occur in equimolar proportions. Electron microscopy revealed linear structures with weight average lengths of 230 nm (A) and 110 nm (B) corresponding to a mass/unit length of about 3 kDa/nm. The protein cores (17-19%) contain large amounts of threonine (over 40%), serine (17-24%), and proline (18-19%). Carbohydrate and sulfate account for approximately 80 and 0.5%, respectively, and gas chromatography-mass spectrometry showed that the patterns of neutral and sialic acid-containing glycans are very similar in the two glycopeptides. Both contain a significant amount (7-10 mol %) of single GalNAc residues, the average oligosaccharide is about 4 sugar residues long, and the largest species observed are heptasaccharides. The major neutral and sialic acid-containing oligosaccharides are Fuc1-2Gal1-3GalNAcol and GlcNAc1-6(NeuGc2-Gal1-3)GalNAcol, respectively. Sialic acid is present as both N-acetyl- and N-glycoloyl-neuraminic acid. Repeated extractions of the tissue with guanidinium chloride left approximately 80% of the mucus glycoproteins as an insoluble glycoprotein complex whereas exposure to dithiothreitol or high speed homogenization accomplished complete solubilization. The "subunits" obtained after reduction with dithiothreitol are larger than glycopeptides A and B, and fragments corresponding in size to the latter are obtained after cleavage with trypsin. Most of the mucins from rat small intestine thus occurs as an insoluble glycoprotein complex composed of subunits joined with disulfide bonds. The subunits contain two highly glycosylated regions with different lengths substituted with very similar oligosaccharides.

Amino Acids↗

PubMed1993

L-iduronate-rich glycosaminoglycans inhibit growth of normal fibroblasts independently of serum or added growth factors.

The effects of various glycosaminoglycans (GAGs) on the growth rate of normal fibroblasts and a fibrosarcoma cell line (HT 1080) were examined. Cells were grown in 96-well microplates in the absence or presence of serum mitogens, epidermal (EGF), platelet-derived (PDGF), acidic fibroblast (aFGF), or basic fibroblast growth factor (bFGF). Cell number was measured by using crystal violet to stain cell nuclei (Westergren-Thorsson, G., Onnervik, P.-O., Fransson, L.-A., and Malmström, A. J. Cell. Phys. 147, 523-530, 1991) and also by using a Coulter counter. In the presence of serum mitogens, L-iduronate (IdoA)-rich GAGs, such as dermatan sulfate, heparin, and highly sulfated heparan sulfate, inhibited proliferation of normal cells (25-35%), whereas HT 1080 cells were unaffected or slightly stimulated. Ham's F-12 supplemented with insulin and transferrin but without growth factors was able to support growth of both cell types. Under these conditions, the IdoA-rich GAGs still suppressed growth of normal cells (40-55%), whereas HT 1080 cells again responded poorly. When growth factors were added proliferation of normal fibroblasts was further stimulated, EGF being the most effective. In the presence of either EGF, PDGF, or bFGF, IdoA-rich GAGs had a sustained inhibitory effect on normal fibroblasts (30-50% at concentrations at or above 10 micrograms/ml). However, in the presence of aFGF, both IdoA-rich and IdoA-poor heparan sulfates enhanced growth (nearly twofold after prolonged exposure) suggesting a stabilization of this growth factor. In general, IdoA-rich GAGs appear to inhibit proliferation of normal cells irrespective of the type of growth factor used. Therefore, GAGs are likely to act directly on cell-derived regulatory components, either before or after internalization. As fibrosarcoma cells were much less sensitive to growth inhibition, they may contain altered receptors for GAGs.

Blood Proteins↗

PubMed1993

The growth promoter spermine interacts specifically with dermatan sulfate regions that are rich in L-iduronic acid and possess antiproliferative activity.

We have investigated interactions between spermine, a member of the growth promoting polyamine family, and various glycosaminoglycans. By using gel chromatography and equilibrium dialysis experiments we found that spermine binds to L-iduronic acid-rich dermatan sulfate (Kd, approximately 3.9 x 10(-4) M) with an affinity similar to that between spermine and DNA. By digesting spermine-dermatan sulfate complexes with chondroitin ABC lyase, the formation of oligosaccharide fragments (tetra-to-decasaccharides) was demonstrated by polyacrylamide gel electrophoresis. Chondroitin sulfate, which is deficient in L-iduronic acid, generates no spermine-protected fragments. Analysis of protected dermatan sulfate oligosaccharides indicates that the majority of the L-iduronic acid residue is non-sulfated and in a periodate-resistant conformation. The oligosaccharides also possess antiproliferative activity.

Cell Division↗

PubMed1993

Studies on binding of glycosaminoglycans to Streptococcus pyogenes by using 125I-heparan sulphate as a probe.

Binding of 125I-heparan sulphate to the cell surface of Streptococcus pyogenes is mediated by proteins, that could be released from the streptococcal cell wall by using alkaline buffer. SDS-electrophoresis revealed two bands with molecular weights of 63 and 58 kDa. Binding of the 125I-labelled heparan sulphate probe to streptococci seems to be due to charge interactions, as the same probe was displaced by unlabelled heparan sulphate, other negatively charged molecules such as heparin, dextran sulphate, dermatan sulphate or by high ionic strength. The interaction was also strongly influenced by pH. The binding constant at pH 7.2 was estimated to be 9.8 x 10(6) mol/l, suggesting a moderate affinity. The presence of collagen of different types enhanced binding of 125I-labelled heparan sulphate to streptococci, whereas fibronectin and vitronectin had an inhibitory effect. The cooperation between heparan sulphate and collagen could be important for the adhesion of streptococci to connective tissue.

Bacterial Outer Membrane Proteins↗

PubMed1993

Affinity of the gastric pathogen Helicobacter pylori for the N-sulphated glycosaminoglycan heparan sulphate.

Binding of 125I-heparan sulphate was a common property of Helicobacter pylori strains isolated from patients with gastroduodenal ulcer diseases. Binding was (i) saturable; (ii) reversible by the addition of unlabelled heparan sulphate and heparin; (iii) inhibited by unlabelled heparan sulphate, heparin, and heparin oligosaccharides but not by other glycosaminoglycans of comparable size (chondroitin sulphate and dermatan sulphate) or by highly glycosylated glycoproteins (hog gastric mucin and fetuin); (iv) reduced by heat treatment (80 degrees C, 10 min) and exposure of the bacteria to pronase E, proteinase K, trypsin and chymotrypsin, but unaffected by treatment with pepsin and neuraminidase; and (v) time-, pH-, and ionic strength-dependent. Scatchard plot analysis of the binding data indicated the presence of one class of high-affinity receptor (Kd = 9 x 10(-9) M) for heparan sulphate.

Binding, Competitive↗

PubMed1992

Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts.

(1) We have isolated radiolabelled proteoglycans and glycosaminoglycans produced by human embryonic skin fibroblasts in the presence of (a) cycloheximide to inhibit protein synthesis or (b) brefeldin A to impede transport between the endoplasmic reticulum and the Golgi complex or (c) suramin, heparin or primaquine to interfere with internalization, recycling and degradation. Effects on glycosaminoglycan synthesis were assayed separately by using exogenous p-nitrophenyl beta-D-xylopyranoside (and [3H]galactose) or 125I-labelled p-hydroxyphenyl beta-D-xylopyranoside as initiators. (2) Inhibition of protein synthesis or blocking of transport to the Golgi complex prevented production of most of the proteoglycans with one exception: Cell-associated heparan sulphate-proteoglycan was still produced at 20% of the control level. (3) Treatment with suramin or heparin resulted in decreased deposition of proteoglycan in the pericellular matrix but increased accumulation of cell-associated proteoglycan. Primaquine blocked all proteoglycan synthesis. (4) In the presence of cycloheximide, exogenous beta-D-xyloside initiated galactosaminoglycan production. In contrast, in brefeldin A-treated cells, synthesis was completely abolished. Not even formation of the linkage-region trisaccharide could be detected. (5) These results suggest that exogenous xyloside enters the endoplasmic reticulum and is subsequently transported to the trans-Golgi complex where all further steps involved in glycosaminoglycan assembly takes place. (6) Heparan sulphate proteoglycan produced by brefeldin A-treated cells could be derived from (a) an intracellular pool of preformed core protein located to the trans-Golgi complex, or (b) resident proteoglycan that was either deglycanated/reglycanated or chain-extended. As combined treatment with suramin and brefeldin A markedly reduced cell-associated proteoglycan production, the latter possibility is favoured.

Anti-Bacterial Agents↗

PubMed1992

Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts.

1. The structure of chondroitin/dermatan and heparan-sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]-glucosamine and 35SO4(2-), and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmström, A. & Fransson, L.-A. (1990) Biochem. J. 265, 289-300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl-Sepharose in Triton X-100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL-6B, Bio-Gel P-6, ion exchange HPLC and gel electrophoresis. 2. Three types of dermatan-sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell-associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatan-sulphate chains were very similar to those of the small proteoglycans. 3. Heparan-sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of D-glucuronate, L-iduronate (with or without sulphate) and N-sulphate groups. They all displayed heparin-lyase-resistant domains with average molecular mass of 10-15 kDa. The heparan-sulphate chains from proteoglycans with 250-kDa and 350-kDa cores were the largest greater than 50 kDa), containing an average of four or five domains, in contrast to heparan-sulphate chains from the small heparan-sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell-associated heparan-sulphate chains were heterogeneous in size (5-45 kDa). 4. These results suggest that the core protein may have important regulatory functions with regard to dermatan-sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.

Cells, Cultured↗

PubMed1992

Transforming growth factor-beta induces selective increase of proteoglycan production and changes in the copolymeric structure of dermatan sulphate in human skin fibroblasts.

Human embryonic skin fibroblasts were pretreated with transforming growth factor-beta (TGF-beta) for 6 h and then labeled with [35S]sulphate and [3H]leucine for 24 h. Radiolabeled proteoglycans from the culture medium and the cell layer were isolated and separated by isopycnic density-gradient centrifugation, followed by gel, ion-exchange and hydrophobic-interaction chromatography. The major proteoglycan species were examined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate before and after enzymatic degradation of the polysaccharide chains. The results showed that TGF-beta increased the production of several different 35S-labelled proteoglycans. A large chondroitin/dermatan sulphate proteoglycan (with core proteins of approximately 400-500 kDa) increased 5-7-fold and a small dermatan sulphate proteoglycan (PG-S1, also termed biglycan, with a core protein of 43 kDa) increased 3-4-fold both in the medium and in the cell layer. Only a small effect was observed on another dermatan sulphate proteoglycan, PG-S2 (also named decorin). These observations are generally in agreement with results of other studies using similar cell types. In addition, we have found that the major heparan sulphate proteoglycan of the cell layer (protein core approximately 350 kDa) was increased by TGF-beta treatment, whereas all the other smaller heparan sulphate proteoglycans with protein cores from 250 kDa to 30 kDa appeared unaffected. To investigate whether TGF-beta also influences the glycosaminoglycan (GAG) chain-synthesizing machinery, we also characterized GAGs derived from proteoglycans synthesized by TGF-beta-treated cells. There was generally no increase in the size of the GAG chains. However, the dermatan sulphate chains on biglycan and decorin from TGF-beta treated cultures contained a larger proportion of D-glucuronosyl residues than those derived from untreated cultures. No effect was noted on the 4- and 6-sulphation of the GAG chains. By the use of p-nitrophenyl beta-D-xyloside (an initiator of GAG synthesis) it could be demonstrated that chain synthesis was also enhanced in TGF-beta-treated cells (approximately twofold). Furthermore, the dermatan sulphate chains synthesized on the xyloside in TGF-beta-treated fibroblasts contained a larger proportion of D-glucuronosyl residues than those of the control. These novel findings indicate that TGF-beta affects proteoglycan synthesis both quantitatively and qualitatively and that it can also change the copolymeric structure of the GAG by affecting the GAG-synthesizing machinery. Altered proteoglycan structure and production may have profound effects on the properties of extracellular matrices, which can affect cell growth and migration as well as organisation of matrix fibres.

Cells, Cultured↗

PubMed1992

Sequence analysis of p-hydroxyphenyl-O-beta-D-xyloside initiated and radio-iodinated dermatan sulfate from skin fibroblasts.

To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated with p-hydroxyphenyl-beta-D-xylopyranoside and [3H]galactose, and free [3H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After 125I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment delta HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments delta HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on the N-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-A, Havsmark B, Silverberg I (1990) Biochem J 269:381-8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.

Carbohydrate Conformation↗

PubMed1992

A new method for sequence analysis of glycosaminoglycans from heavily substituted proteoglycans reveals non-random positioning of 4- and 6-O-sulphated N-acetylgalactosamine in aggrecan-derived chondroitin sulphate.

We have developed a new procedure for the sequence analysis of glycosaminoglycans, which is particularly suitable for the analysis of chains from heavily substituted proteoglycans. The procedure has been applied to various aggrecan-derived chondroitin sulphates. The glycans are released from the core protein by alkaline scission of the xylose-serine bond, subjected to reductive amination using p-aminobenzoic acid and finally radioiodinated at an acidic pH. Sequence analysis is performed by using various enzymic degradations, partial or complete, followed by high-resolution polyacrylamide gel electrophoresis, blotting and autoradiography to identify segments extending from the labelled reducing end to the point of cleavage. By using chondroitin C lyase to identify the location of 6-O-sulphated hexosamines, we find that chondroitin sulphate from tracheal cartilage has its 6-O-sulphated repeats concentrated to the extreme non-reducing terminal portion of the chain. In chondroitin sulphates derived from intervertebral discs (nucleus pulposus), the 6-O-sulphated repeats have a biphasic distribution; they occur mostly near the linkage region (i.e. the reducing end), but also in the non-reducing portion of the chain. Chondroitin sulphate from nasal cartilage, which is mostly 4-O-sulphated, displays considerable heterogeneity in the linkage region. Three or possibly more charge variants are observed.

4-Aminobenzoic Acid↗

PubMed1992

Binding of heparan sulfate to Staphylococcus aureus.

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.

Amino Acid Sequence↗

PubMed1991

Domain structure of endothelial heparan sulphate.

The domain structure of heparan sulphate chains from an endothelial low-density proteoglycan was examined using specific degradations of the chains while attached to the intact proteoglycan. 'Inner' chain fragments, remaining on the protein core, were separated from 'outer' fragments by gel chromatography, and were subsequently released from the protein core by alkaline cleavage. The structure of 'inner' and 'outer' chain fragments was then examined and compared. Using deaminative cleavage we obtained evidence that the first N-sulphated glucosamine residue is variably positioned some 10-17 disaccharides from the xylose-serine linkage of the proteoglycan. Digestion with heparinase yielded 'inner' and 'outer' fragments covering a broad range of different sizes, indicating a scarce and variable distribution of sulphated iduronic acid in the native chains. N-sulphated glucosamine occurred more frequently in the 'outer' fragments. We also studied the affinity of the endothelial heparan sulphate chains towards two presumptive biological ligands, namely antithrombin III and lipoprotein lipase. A major part of the endothelial heparan sulphate chains showed a weak affinity for antithrombin III and the affinity was essentially lost on heparinase digestion. On lipoprotein lipase-agarose the endothelial heparan sulphate chains were eluted at the same salt concentration as heparin, and the binding persisted, although with decreased strength, after digestion with heparinase.

Chromatography, Affinity↗

PubMed1991

Proliferation of cultured fibroblasts is inhibited by L-iduronate-containing glycosaminoglycans.

We have modified a method (Gilles et al: Anal. Biochem., 159:109-113, 1986) for measuring cell number, that is based on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in the absence of serum can be detected. Human lung fibroblasts were growth arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1-100 micrograms/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed growth (20%-50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates, with moderately high sulfate and L-iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate and L-iduronate contents. The antiproliferative effect of exogenous glycans appeared after a lag period of 3-4 days, suggesting that they interfered with factors produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous growth-promoting effects in densely seeded cultures.

Cell Count↗

PubMed1991

Oligosaccharide mapping of proteoglycan-bound and xyloside-initiated dermatan sulfate from fibroblasts.

The copolymeric structure of dermatan sulfate chains synthesized by skin fibroblasts has been examined. Chains initiated onto exogeneous p-nitrophenyl-beta-D-xylopyranoside or attached to protein in a large proteoglycan, PG-L, and two small proteoglycans, PG-S1 and PG-S2, have been compared by using high resolution electrophoresis and gel chromatography of oligosaccharides generated by specific enzymatic or chemical degradations. The results confirm that chains attached to PG-L are glucuronate-rich, whereas novel findings indicate that chains attached to either of the two PG-S variants yield closely similar oligosaccharide maps, have approximately equal glucuronate and iduronate content and contain over 90% 4-sulfated disaccharide repeating units. Dermatan sulfate chains built onto xyloside at concentrations of 50 microM and below have a copolymeric structure similar to that of chains from the two PG-S variants. These findings indicate that the polymer-modifying machinery can generate chains with extended iduronate-containing repeats also when the xylose primer is not linked to core protein.

Carbohydrate Sequence↗

PubMed1990

A method for the sequence analysis of dermatan sulphate.

We are attempting to develop methods for the sequencing of glycosaminoglycans from their reducing end. Here we describe a procedure for the analysis of dermatan sulphate from pig skin. The glycosaminoglycan is released from its parent proteoglycan by exhaustive proteolysis by using both endo- and exo-peptidases. The amino group of the residual serine residue is conjugated with a p-hydroxyphenyl group, which in turn is iodinated with 125I (the Bolton-Hunter reagent, BHR). The ion-exchange-purified end-labelled dermatan sulphate is then degraded partially or completely by various enzymic or chemical means to yield fragments extending from the labelled serine residue to the point of cleavage. The various products are separated by gradient PAGE, detected by autoradiography and quantified by videodensitometry. Complete digestion with chondroitin ABC lyase affords the labelled fragment delta HexA-GalNAc(-SO4)-GlcA-Gal-Gal-Xyl-Ser(-BHR). The structure was confirmed by sequential degradation from the non-reducing end by chondroitin AC lyase, HgCl2, and beta-galactosidase. Periodate oxidation cleaves most of the Xyl even without treatment with alkaline phosphatase, showing that Xyl is not substituted with phosphate. Results from partial and selective periodate oxidation indicate that most of the non-sulphated IdoA residues are located towards the non-reducing end. Partial or complete digestions with testicular hyaluronidase (in the presence of an excess of beta-glucuronidase) or chondroitin AC lyase identify the positions of GlcA residues. The results confirm that HexA next to Gal is always GlcA. Moreover, GlcA is common in the first three disaccharide repeats. Results with testicular hyaluronidase indicate that the distribution of clustered GlcA-GalNAc repeats is periodic and peaks at positions 1-3, 8-9 and around 25. Although there must be chains that contain IdoA in nearly all of the available positions, regions that have not been fully processed during biosynthesis are markedly non-random.

Animals↗

PubMed1990

Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans.

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining 'matrix', and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).

Chondroitin Sulfate Proteoglycans↗

PubMed1990

Endothelial heparan sulphate: compositional analysis and comparison of chains from different proteoglycan populations.

From cultures of human umbilical vein endothelial cells incubated with 3H-glucosamine or 35S-sulphate, we have purified three heparan sulphate proteoglycans: 1) a low density (1.31 g/ml) proteoglycan from the cell extract, 2) a low density proteoglycan from the medium, and 3) a high density (greater than 1.4 g/ml) proteoglycan from the medium. The disaccharide composition of heparan sulphate chains from the low density proteoglycan of the medium was examined, using specific chemical and enzymic degradations followed by gel chromatography and strong anion exchange HPLC. Chains released from each of the different proteoglycan populations were then compared by gel chromatography and gradient polyacrylamide gel electrophoresis before and after various specific degradations. The results indicate that heparan sulphate from human endothelial cells are large polymers (MW greater than 50,000) of low overall sulphation (32-35% N-sulphated glucosamine and an N/O-linked sulphate ratio of 2.0) with rare and solitary heparin-like disaccharides. Heparan sulphate from the different proteoglycan populations appeared to have similar structure except that chains from the high density fraction were larger polymers.

Carbohydrate Sequence↗