Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5.

In the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level. The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 [Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Rodén (1980) Biochemistry 19, 495-500]. Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ([4GlcAbeta1-4GlcNSO(3)alpha1-](n)), or of O-desulphated heparin (predominantly [4IdoAalpha1-4GlcNSO(3)alpha1-](n)) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx. 1 h of incubation. Similar incubations performed in the presence of (3)H(2)O resulted in progressive labelling at C-5 of the target hexuronic acid units of either substrate polysaccharide. Contrary to chemical D-gluco/L-ido equilibrium, established within 1 h of incubation, the accumulation of (3)H label continued for at least 6 h. This isotope effect suggests that the second stage of the reaction, i.e. the re-addition of a proton to the carbanion intermediate, is the rate-limiting step of the overall process. Analysis of the 5-(3)H-labelled polysaccharide products showed that the (3)H was approximately equally distributed between glucuronic acid and IdoA units, irrespective of incubation time (from 15 min to 72 h) and of the relative proportions of the two epimers in the substrate. This finding points to a catalytic mechanism in which the abstraction and re-addition of C-5 protons are effected by two polyprotic bases, presumably lysine residues. Previous experiments relating to the biosynthesis of dermatan sulphate were similarly interpreted in terms of a two-base epimerization mechanism but differed from the present findings by implicating one monoprotic and one polyprotic base function [Hannesson, Hagner-McWhirter, Tiedemann, Lindahl and Malmström (1996) Biochem. J. 313, 589-596].

Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates.

The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.SO(3)complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its K(m)value, which was significantly lower than that of its partially N-acetylated counterpart. The V(max)values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [(3)H]heparin used in our previous studies. Surprisingly, the apparent K(m)values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent K(m)values with enzyme concentration precluded a detailed comparison of their kinetic properties.

Type I collagen synthesis in cultured human fibroblasts: regulation by cell spreading, platelet-derived growth factor and interactions with collagen fibers.

Type I collagen protein and pro-alpha 1(I) collagen mRNA levels were investigated in human dermal fibroblasts cultured on substrates which induced distinct morphologies. Induction of type I collagen protein synthesis required cell spreading in monolayer cultures; mere attachment to dishes coated with 2-hydroxyethyl methacrylate (poly(HEMA)) did not suffice. Spread cells or round cells cultured on poly(HEMA) differed in collagen type I production, but pro-alpha 1(I) collagen mRNA levels were similar. Recombinant human platelet-derived growth factor (PDGF)-BB could replace cell spreading as a stimulus for collagen synthesis in cells cultured on poly(HEMA). At later time points, pro-alpha 1(I) collagen mRNA levels were down-regulated, although relatively less than type I collagen synthesis. Type I collagen synthesis by fibroblasts cultured in three-dimensional collagen gels was strongly down-regulated at both the protein and RNA levels. In addition to its capacity to stimulate collagen synthesis, PDGF-BB induced elongation and the formation of long processes by fibroblasts cultured in collagen gels. The stimulatory effect by cell spreading and PDGF-BB on collagen synthesis was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. However, inhibition of PI3K only inhibited induction of collagen synthesis by actively spreading cells or by PDGF-BB and did not induce a down-regulation of collagen synthesis in cells which had already spread. These data demonstrate that type I collagen protein synthesis is partly independent of pro-alpha 1(I) collagen mRNA levels but highly regulated by cell shape, although this could be decoupled by PDGF-BB. Both cell shape- and PDGF-BB-induced stimulation of collagen type I synthesis depends on a signalling pathway involving PI3K. Furthermore, levels of pro-alpha 1(I) collagen mRNA in fibroblasts are partly cell shape independent but are down-regulated by fibroblast interactions with native collagen fibers.

Biosynthesis of heparin/heparan sulfate. cDNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung.

Glucuronyl C5-epimerases catalyze the conversion of D-glucuronic acid (GlcUA) to L-iduronic acid (IdceA) units during the biosynthesis of glycosaminoglycans. An epimerase implicated in the generation of heparin/heparan sulfate was previously purified to homogeneity from bovine liver (Campbell, P., Hannesson, H. H., Sandbäck, D., Rodén, L., Lindahl, U., and Li, J.-p. (1994) J. Biol. Chem. 269, 26953-26958). The present report describes the molecular cloning and functional expression of the lung enzyme. The cloned enzyme contains 444 amino acid residues and has a molecular mass of 49,905 Da. N-terminal sequence analysis of the isolated liver enzyme showed this species to be a truncated form lacking a 73-residue N-terminal domain of the deduced amino acid sequence. The coding cDNA insert was cloned into a baculovirus expression vector and expressed in Sf9 insect cells. Cells infected with recombinant epimerase showed a 20-30-fold increase in enzyme activity, measured as release of 3H2O from a polysaccharide substrate containing C5-3H-labeled hexuronic acid units. Furthermore, incubation of the expressed protein with the appropriate (GlcUA-GlcNSO3)n substrate resulted in conversion of approximately 20% of the GlcUA units into IdceA residues. Northern analysis implicated two epimerase transcripts in both bovine lung and liver tissues, a dominant approximately 9-kilobase (kb) mRNA and a minor approximately 5-kb species. Mouse mastocytoma cells showed only the approximately 5-kb transcript. A comparison of the cloned epimerase with the enzymes catalyzing an analogous reaction in alginate biosynthesis revealed no apparent amino acid sequence similarity.

Scleroderma lung fibroblasts exhibit elevated and dysregulated type I collagen biosynthesis.

OBJECTIVE: To examine whether scleroderma lung fibroblasts show a pattern of aberrant type I collagen (CI) biosynthesis similar to that observed previously in studies of dermal fibroblasts in this disease. METHODS: CI secretion and steady-state pro alpha1(I) collagen messenger RNA (mRNA) levels and COL1A2 gene activation were examined in fibroblasts grown from lung biopsy specimens obtained from 16 scleroderma patients with lung fibrosis and from 10 histologically normal lung specimens (controls). The effect of culture in a 3-dimensional (3-D) CI gel matrix culture on CI mRNA levels was also examined. RESULTS: The mean (+/- SEM) collagen secretion in monolayer culture for scleroderma lung fibroblasts was 90.9 +/- 56 ng/ml/10(6) cells, significantly greater (P < 0.05) than controls (40.2 +/- 17.5). Pro alpha1(I) collagen mRNA levels in monolayer cultures were higher in scleroderma (mean +/- SEM collagen:GAPDH ratio 3.7 +/- 0.9) compared with control (1.9 +/- 0.8) lung fibroblasts. Transient expression assays confirmed that genes coding for CI are transcriptionally activated in scleroderma lung fibroblasts compared with control strains. Although all lung fibroblasts induced equivalent contraction of 3-D CI gel matrices, scleroderma strains failed to show a reduction in steady-state pro alpha1(I) collagen mRNA levels in gel culture. CONCLUSION: We have demonstrated elevated CI biosynthesis and impaired mRNA down-regulation for CI by scleroderma lung fibroblasts. These properties are likely to be highly relevant to the pathogenesis of scleroderma-associated lung fibrosis.

Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism.

The capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ([GlcA(beta 1-->3)GalNAc(beta 1-->4)]n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units. A Km of 67 x 10(-6) M hexuronic acid (equivalent to disaccharide units) was determined, which is similar to that (80 x 10(-6) M) obtained for dermatan (desulphated dermatan sulphate). Vmax was about 4 times higher with dermatan than with the K4 substrate. A defructosylated K4 polysaccharide isolated after incubation of bacteria with D-[5-3H]glucose released 3H2O on reaction with the epimerase, and thus could be used to assay the enzyme. Incubation of a K4 substrate with solubilized microsomal epimerase for 6 h in the presence of 3H2O resulted in the formation of about 5% IdoA and approximately equal amounts of 3H in GlcA and IdoA. A corresponding incubation of dermatan yielded approx. 22% GlcA, which contained virtually all the 3H label. These results are tentatively explained in terms of a two-base reaction mechanism, involving a monoprotic L-ido-specific base and a polyprotic D-gluco-specific base. Most of the IdoA residues generated by the enzyme occurred singly, although some formation of two or three consecutive IdoA-containing disaccharide units was observed.

Collagen type I is not under autocrine control by transforming growth factor-beta 1 in normal and scleroderma fibroblasts.

BACKGROUND: The ability of transforming growth factor-beta (TGF-beta) to induce synthesis of extracellular matrix proteins stimulated this study in which we address the hypothesis that TGF-beta can induce, in normal fibroblasts, the sustained, elevated collagen synthesis characteristic of the scleroderma fibroblast. EXPERIMENTAL DESIGN: Fibroblasts were studied for synthesis of and responsiveness to TGF-beta. Secreted TGF-beta levels were determined in a bioassay and at the transcriptional level in a series of scleroderma (SSc) and normal fibroblasts. The ability of cells to interact functionally with a 3-dimensional collagen matrix after TGF-beta treatment was examined. The kinetics of TGF-beta-induced fibrosis in fibroblasts was studied. RESULTS: SSc fibroblasts were not characterized by elevated TGF-beta synthesis. There was no evidence of coordinate regulation of TGF-beta and collagen over passage number. Repeated pulses of 200 pM of TGF-beta did not significantly induce sustained procollagen alpha 1(I) mRNA synthesis in normal fibroblasts, and this treatment did not significantly alter the characteristics of normal fibroblasts in a collagen gel. mRNA for both collagen and TGF-beta type II receptor was induced by TGF-beta in both SSc and control cells. SSc fibroblasts were found to have an impaired ability to activate the small latent complex of TGF-beta. CONCLUSIONS: Our data give no support to the hypothesis that TGF-beta can maintain the SSc phenotype in vitro or that it is able to induce this phenotype. The inducibility of TGF-beta receptor mRNA in SSc fibroblasts after exposure to TGF-beta suggests that the lack of sustained elevation in collagen synthesis is not due to lack of responsiveness by the fibroblasts but is rather a reflection of the transient nature of TGF-beta-induced fibrosis.

Impaired regulation of collagen pro-alpha 1(I) mRNA and change in pattern of collagen-binding integrins on scleroderma fibroblasts.

We explored the hypothesis that dermal fibroblasts isolated from patients suffering from systemic sclerosis are disturbed in their ability to interact functionally with native collagen fibers. Additionally, we investigated the expression of one collagen-binding integrin matrix receptor, alpha 1 beta 1 on those cells. Two populations of primary dermal fibroblasts were established, one from patients with systemic sclerosis and one from normal subjects. When cultured for 24 h in free-floating collagen gels, both types of fibroblasts down-regulated the cellular content of collagen pro-alpha 1(I) messenger ribonucleic acid, the systemic sclerosis fibroblasts less markedly than the normals. In normal, but not in systemic sclerosis fibroblasts, the kinetics of collagen gel contraction were directly proportional to the extent of the down-regulation. Fetal bovine serum stimulated collagen gel contraction in both populations. When grown in collagen gels in the presence of fetal bovine serum, no difference between systemic sclerosis and normal fibroblasts in capacity to down-regulate pro-alpha 1(I) was observed. Collagen-binding beta 1 integrins mediate the functional interactions between fibroblasts and the collagen fibers. To assess the cell surface expression of collagen-binding beta 1 integrins on fibroblasts, we labeled cells with 125I and subjected Triton X-100 extracts from them to immunoprecipitation with anti-beta 1 integrin immunoglobulin G. Among the systemic sclerosis fibroblasts, a larger number of isolates expressed low amount of alpha 1 beta 1 than did the fibroblasts isolated from normal individuals. Our data are compatible with the hypothesis that systemic sclerosis fibroblasts have a disturbed interaction with collagen fibers; this disturbance may in part be the result of an aberrant expression of collagen-binding beta 1 integrins.

Expression and function of surface antigens on scleroderma fibroblasts.

Dermal fibroblasts from patients with systemic sclerosis (SSc) bound a much greater number of T lymphocytes than did normal dermal fibroblasts. Monoclonal antibodies (MAb) against classes I and II antigens of the major histocompatibility complex (MHC) and their receptors, CD8 and CD4, had no effect on T cell interaction with SSc and normal cells, while MAb against lymphocyte function-associated antigen type 3 (LFA-3) and CD2 both strongly inhibited lymphocyte attachment. MAb against intercellular adhesion molecule type 1 (ICAM-1) and LFA-1 also prevented binding of T lymphocytes, but had a more marked effect on adhesion to SSc fibroblasts than to normal fibroblasts; they also completely abolished the increased binding to fibroblasts treated with interleukin-1 alpha, tumor necrosis factor alpha, and interferon-gamma. No difference was found in the proportion of normal and SSc fibroblasts that expressed MHC classes I and II and LFA-3, but more SSc cells expressed ICAM-1, and at a higher level, than did normal fibroblasts. These results show that cultured SSc cells have elevated binding to T lymphocytes, which possibly results from expansion of a subset of fibroblasts that produces high levels of ICAM-1.

Antireticulin antibody in systemic sclerosis.

The prevalence, immunoglobulin class, and IgG subclass of antireticulin antibody in the serum samples of 32 patients with systemic sclerosis were investigated by indirect immunofluorescence on unfixed rodent tissue. Antireticulin antibody was present in 22/32 (69%) of patients and belonged to the IgG class in 19/22 (86%), the IgA class in 13/22 (59%), and the IgM class in 6/22 (27%) of positive sera. IgG1 was the predominant subclass of IgG antireticulin antibody, occurring either alone or in association with IgG3 in 12/19 cases (63%). Thus antireticulin antibody of the IgG and IgA classes is found in most patients with systemic sclerosis. The finding of an autoantibody with reactivity for collagen-like fibres in systemic sclerosis indicates that the antibody has a potential role in the pathogenesis of the disease, and as it belongs to the IgA class this suggests that it arises in response to antigens presented to the immune system at the mucosal level.

Serum type III procollagen peptide concentrations in systemic sclerosis and Raynaud's phenomenon: relationship to disease activity and duration.

Serum levels of the amino-terminal type III procollagen peptide (P-3-NP) have been used as an index of collagen synthesis. Systemic sclerosis (SS) is characterized by uncontrolled production of collagen of several types. This study aimed to explore the profile of P-3-NP in patients with SS and Raynaud's phenomenon, a common forerunner of the disease. Using a radioimmunoassay, the mean level for P-3-NP was found to be raised in SS compared with both the control (p less than 0.001) and Raynaud's groups (p less than 0.001). Analysis of serial samples from the patients with SS suggested that the P-3-NP level reflected changing clinical activity. Three groups emerged: a group with stable disease which showed a less than 20% change in P-3-NP level (mean 5.7%); a group with increasing activity which showed an increase of greater than 20% (mean 35.8%) and a group of decreasing activity which showed a decrease of greater than 20% (mean 33.6%). These data suggest that there is an increase in collagen metabolism in SS and that changes in P-3-NP levels may reflect the clinical course of the disease.

Genetic susceptibility to scleroderma-like syndrome in symptomatic and asymptomatic workers exposed to vinyl chloride.

We have previously reported a genetic susceptibility to a scleroderma-like disorder in a group of workers exposed to polyvinyl chloride and this susceptibility could be mapped to the major histocompatibility complex area of the 6th chromosome. To date no genetic studies have been reported comparing affected with unaffected workers. We report such data and discuss it in the light of our previous findings in vinyl chloride exposed patients and in patients with classical scleroderma. Our results suggest that susceptibility to this disorder is increased in the presence of HLA-DR5 or of a gene in linkage disequilibrium with it and an antigen associated with the haplotype A1 B8, while DR3 favours progression of the disease.