Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase.

The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho[(35)S]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-](n)(), resulted in 2-O-sulfation of iduronic acid. When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure [(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-](n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid. Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid. In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).

Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST) gene. Structure, expression, and function in the formation of the tracheal system.

Heparan sulfate, one of the most abundant components of the cell surface and the extracellular matrix, is involved in a variety of biological processes such as growth factor signaling, cell adhesion, and enzymatic catalysis. The heparan sulfate chains have markedly heterogeneous structures in which distinct sequences of sulfate groups determine specific binding properties. Sulfation at each different position of heparan sulfate is catalyzed by distinct enzymes, sulfotransferases. In this study, we identified and characterized Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST). The deduced primary structure of dHS6ST exhibited several common features found in those of mammalian HS6STs. We confirmed that, when the protein encoded by the cDNA was expressed in COS-7 cells, it showed HS6ST activity. Whole mount in situ hybridization revealed highly specific expression of dHS6ST mRNA in embryonic tracheal cells. The spatial and temporal pattern of dHS6ST expression in these cells clearly resembles that of the Drosophila fibroblast growth factor (FGF) receptor, breathless (btl). RNA interference experiments demonstrated that reduced dHS6ST activity caused embryonic lethality and disruption of the primary branching of the tracheal system. These phenotypes were reminiscent of the defects observed in mutants of FGF signaling components. We also show that FGF-dependent mitogen-activated protein kinase activation is significantly reduced in dHS6ST double-stranded RNA-injected embryos. These findings indicate that dHS6ST is required for tracheal development in Drosophila and suggest the evolutionally conserved roles of 6-O-sulfated heparan sulfate in FGF signaling.

Expression of heparan sulphate L-iduronyl 2-O-sulphotransferase in human kidney 293 cells results in increased D-glucuronyl 2-O-sulphation.

Functionally important interactions between heparan sulphate and a variety of proteins depend on the precise location of O-sulphate groups. Such residues occur at C-2 of L-iduronic (IdoA) and D-glucuronic acid (GlcA) units, and at C-3 and C-6 of D-glucosamine (GlcN) units. Stable transfection of human embryonic kidney 293 cells with a cDNA encoding mouse mastocytoma IdoA 2-O-sulphotransferase resulted in an approx. 6-fold increase in O-sulphotransferase activity, compared with control cells, as determined using O-desulphated heparin as an acceptor. Structural analysis of endogenous heparan sulphate in the transfected cells, following metabolic labelling with either [(3)H]GlcN or [(35)S]sulphate, showed appreciable formation of -GlcA(2-OSO(3))-GlcNSO(3)- disaccharide units (6% of total disaccharide units; 17% of total O-sulphated disaccharide units) that were essentially absent from heparan sulphate from control cells. The increase in GlcA 2-O-sulphation was accompanied by a decrease in the amount of IdoA formed, whereas overall 2-O-sulphation or 6-O-sulphation remained largely unaffected. These findings indicate that 2-O-sulphation of IdoA and GlcA residues is catalysed by the same enzyme in heparan sulphate biosynthesis.

Molecular cloning and expression of chondroitin 4-sulfotransferase.

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine residue of chondroitin. The enzyme has been previously purified to apparent homogeneity from the serum-free culture medium of rat chondrosarcoma cells (Yamauchi, A., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J. H., and Habuchi, O. (1999) J. Biol. Chem. 274, 2456-2463). The purified enzyme also catalyzed the sulfation of partially desulfated dermatan sulfate. We have now cloned the cDNA of the mouse C4ST on the basis of the amino acid sequences of peptides obtained from the purified enzyme by protease digestion. This cDNA contains a single open reading frame that predicts a protein composed of 352 amino acid residues. The protein predicts a Type II transmembrane topology. The predicted sequence of the protein contains all of the known amino acid sequence and four potential sites for N-glycosylation, which corresponds to the observation that the purified C4ST is an N-linked glycoprotein. The amino acid sequence of mouse C4ST showed significant sequence homology to HNK-1 sulfotransferase. Comparison of the sequence of mouse C4ST with human HNK-1 sulfotransferase revealed approximately 29% identity and approximately 48% similarity at the amino acid level. When the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that catalyzes the transfer of sulfate to position 4 of GalNAc residue of both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis showed that, among various mouse adult tissues, 5.7-kilobase message of C4ST was mainly expressed in the brain and kidney.

The occurrence of three isoforms of heparan sulfate 6-O-sulfotransferase having different specificities for hexuronic acid adjacent to the targeted N-sulfoglucosamine.

We previously cloned heparan sulfate 6-O-sulfotransferase (HS6ST) (Habuchi, H., Kobayashi, M., and Kimata, K. (1998) J. Biol. Chem. 273, 9208-9213). In this study, we report the cloning and characterization of three mouse isoforms of HS6ST, a mouse homologue to the original human HS6ST (HS6ST-1) and two novel HS6STs (HS6ST-2 and HS6ST-3). The cDNAs have been obtained from mouse brain cDNA library by cross-hybridization with human HS6ST cDNA. The three cDNAs contained single open reading frames that predicted type II transmembrane proteins composed of 401, 506, and 470 amino acid residues, respectively. Amino acid sequence of HS6ST-1 was 51 and 57% identical to those of HS6ST-2 and HS6ST-3, respectively. HS6ST-2 and HS6ST-3 had the 50% identity. Overexpression of each isoform in COS-7 cells resulted in about 10-fold increase of HS6ST activity. The three isoforms purified with anti-FLAG antibody affinity column transferred sulfate to heparan sulfate and heparin but not to other glycosaminoglycans. Each isoform showed different specificity toward the isomeric hexuronic acid adjacent to the targeted N-sulfoglucosamine; HS6ST-1 appeared to prefer the iduronosyl N-sulfoglucosamine while HS6ST-2 had a different preference, depending upon the substrate concentrations, and HS6ST-3 acted on either substrate. Northern analysis showed that the expression of each message in various tissues was characteristic to the respective isoform. HS6ST-1 was expressed strongly in liver, and HS6ST-2 was expressed mainly in brain and spleen. In contrast, HS6ST-3 was expressed rather ubiquitously. These results suggest that the expression of these isoforms may be regulated in tissue-specific manners and that each isoform may be involved in the synthesis of heparan sulfates with tissue-specific structures and functions.

Solution structure of human acidic fibroblast growth factor and interaction with heparin-derived hexasaccharide.

Fibroblast growth factors (FGFs) bind to extracellular matrices, especially heparin-like carbohydrates of heparan-sulfate proteoglycans which stabilize FGFs to protect against inactivation by heat, acid, proteolysis and oxidation. Moreover, binding of FGFs to cell surface proteoglycans promotes to form oligomers, which is essential for receptor oligomerization and activation. In the present study, we determined the solution structure of acidic FGF using a series of triple resonance multi-dimensional NMR experiments and simulated annealing calculations. Furthermore, we prepared the sample complexed with a heparin-derived hexasaccharide which is a minimum unit for aFGF binding. From the chemical shift differences between free aFGF and aFGF-heparin complex, we concluded that the major heparin binding site was located on the regions 110-131 and 17-21. The binding sites are quite similar to those observed for bFGF-heparin hexasaccharide complex, showing that both FGFs recognize heparin-oligosaccharides in a similar manner.

Molecular characterization and expression of heparan-sulfate 6-sulfotransferase. Complete cDNA cloning in human and partial cloning in Chinese hamster ovary cells.

Heparan-sulfate 6-sulfotransferase (HS6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-sulfoglucosamine residue of heparan sulfate. The enzyme was purified to apparent homogeneity from the serum-free culture medium of Chinese hamster ovary (CHO) cells (Habuchi, H., Habuchi, O., and Kimata, K. (1995) J. Biol Chem. 270, 4172-4179). From the amino acid sequence data of the purified enzyme, degenerate oligonucleotides were designed and used as primers for the reverse transcriptase-polymerase chain reaction using poly(A)+ RNA from CHO cells as a template. The amplified cDNA fragment was then used as a probe to screen a cDNA library of CHO cells. The cDNA clone thus obtained encoded a partial peptide sequence composed of 236 amino acid residues that included the sequences of six peptides obtained after endoproteinase digestion of the purified enzyme. This cDNA clone was applied to the screening of a human fetal brain cDNA library by cross-hybridization. The isolated cDNA clones contained a whole open reading frame that predicts a type II transmembrane protein composed of 401 amino acid residues. No significant amino acid sequence identity to any other proteins, including heparan-sulfate 2-sulfotransferases, was observed. When the cDNA for the entire coding sequence of the protein was inserted into a eukaryotic expression vector and transfected into COS-7 cells, the HS6ST activity increased 7-fold over the control. The FLAG fusion protein purified by anti-FLAG affinity chromatography showed the HS6ST activity alone. Northern blot analysis revealed the occurrence of a single transcript of 3.9 kilobases in both human fetal brain and CHO cells. The results, together with the ones from our recent cDNA analysis of heparan-sulfate 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985), suggest that at least two different gene products are responsible for 6- and 2-O-sulfations of heparan sulfate.

Heparan/chondroitin/dermatan sulfate primer 2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside preferentially inhibits growth of transformed cells.

Xylose forms the direct carbohydrate-protein link in extra- or pericellular proteoglycans (PGs) that are substituted with either chondroitin sulfate (CS)/dermatan sulfate (DS) and/or heparan sulfate (HS). Cell surface PGs carrying HS are important regulators of cell growth. Xylose coupled to an aromatic compound can enter cells and initiate either CS/DS synthesis or both HS and CS/DS synthesis, depending on the nature of the aromatic adduct. Here, we show that 2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside, which can prime both types of glycan chains, inhibits growth of a set of normal and transformed cells. Transformed cells are preferentially inhibited, and at a concentration of 0.15-0.20 mM xyloside, transformed cells are totally growth arrested, whereas normal cells are only < or = 50% inhibited. No inhibition of growth is observed with the stereoisomeric 2-(6-hydroxynaphthyl)-O-beta-L-xylopyranoside, which does not prime glycosaminoglycan synthesis at all; with the nonhydroxylated 2-naphthyl-O-beta-D-xylopyranoside, which only primes CS/DS synthesis under these conditions; or with p-nitrophenyl-O-beta-D-xylopyranoside, which is known to prime only CS/DS synthesis. We conclude that growth inhibition is due to priming of HS and/or CS/DS synthesis, which may either lead to the formation of specific antiproliferative glycans or glycan fragments or to interference with endogenous PG synthesis and turnover.

Widespread expression of chondroitin sulfate-type serglycins with CD44 binding ability in hematopoietic cells.

Serglycin is a family of small proteoglycans with Ser-Gly dipeptide repeats and is modified with various types of glycosaminoglycan side chains. We previously demonstrated that chondroitin sulfate-modified serglycin is a novel ligand for CD44 involved in the adherence and activation of lymphoid cells. In this study, we investigated the production and distribution of CD44 binding serglycins in various hematopoietic cells and characterized their carbohydrate side chains. Immunoprecipitation analysis using CD44-IgG and polyclonal antibody against the serglycin core peptide demonstrated that various serglycin species capable of binding CD44 are produced by a variety of hematopoietic cells including lymphoid cells, myeloid cells, and a few tumor cell lines. Glycosaminoglycans on these serglycins, which are essential for CD44 binding, are composed of chondroitin 4-sulfate or a mixture of chondroitin 4-sulfate and chondroitin 6-sulfate, but no heparin or heparan sulfate side chain was detected. The serglycins are also secreted by normal splenocytes, lymph node lymphocytes, and bone marrow cells, whereas they are secreted in very small amounts by normal thymocytes. Secretion of serglycins is greatly enhanced by mitogenic stimulation with concanavalin A or lipopolysaccharide. Our results showed that serglycin, unlike hyaluronate, is produced and secreted in a functional (CD44 binding) form by many members of the hematopoietic system including various lymphocyte subsets. Our data suggest that serglycin may serve as a major ligand for CD44 in various events in the lymphohematopoietic system.

Turnover of heparan sulfate depends on 2-O-sulfation of uronic acids.

To study how the pattern of sulfation along a heparan sulfate chain affects its turnover, we examined heparan sulfate catabolism in wild-type Chinese hamster ovary cells and mutant pgsF-17, defective in 2-O-sulfation of uronic acid residues (Bai, X., and Esko, J. D. (1996) J. Biol. Chem. 271, 17711-17717). Heparan sulfate from the mutant contains normal amounts of 6-O-sulfated glucosamine residues and iduronic acid and somewhat higher levels of N-sulfated glucosamine residues but lacks any 2-O-sulfated iduronic or glucuronic acid residues. Pulse-chase experiments showed that both mutant and wild-type cells transport newly synthesized heparan sulfate proteoglycans to the plasma membrane, where they shed into the medium or move into the cell through endocytosis. Internalization of the cell-associated molecules leads to sequential endoglycosidase (heparanase) fragmentation of the chains and eventual lysosomal degradation. In wild-type cells, the chains begin to degrade within 1 h, leading to the accumulation of intermediate (10-20-kDa) and small (4-7-kDa) oligosaccharides. Mutant cells did not generate these intermediates, although internalization and intracellular trafficking of the heparan sulfate chains appeared normal, and the chains degraded with normal kinetics. This difference was not due to defective heparanase activities in the mutant, since cytoplasmic extracts from mutant cells cleaved wild-type heparan sulfate chains in vitro. Instead, the heparan sulfate chains from the mutant were relatively resistant to degradation by cellular heparanases. These findings suggest that 2-O-sulfated iduronic acid residues in heparan sulfate are important for cleavage by endogenous heparanases but not for the overall catabolism of the chains.

Molecular cloning and expression of Chinese hamster ovary cell heparan-sulfate 2-sulfotransferase.

Heparan-sulfate 2-sulfotransferase (HS2ST), which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to L-iduronic acid at position 2 in heparan sulfate, was purified from cultured Chinese hamster ovary (CHO) cells to apparent homogeneity (Kobayashi, M., Habuchi, H., Habuchi, O., Saito, M., and Kimata, K. (1996) J. Biol. Chem. 271, 7645-7653). The internal amino acid sequences were obtained from the peptides after digestion of the purified protein with a combination of endoproteinases. Mixed oligonucleotides based on the peptide sequences were used as primers to obtain a probe fragment by reverse transcriptase-polymerase chain reaction using CHO cell poly(A)+ RNA as template. The clone obtained from a CHO cDNA library by screening with the probe is 2.2 kilobases in size and contains an open reading frame of 1068 bases encoding a new protein composed of 356 amino acid residues. The protein predicts a type II transmembrane topology similar to other Golgi membrane proteins. Messages of 5.0 and 3.0 kilobases were observed in Northern analysis. Evidence that the cDNA clone corresponds to the purified HS2ST protein is as follows. (a) The predicted amino acid sequence contains all five peptides obtained after endoproteinase digestion of the purified protein; (b) the characteristics of the predicted protein fit those of the purified protein in terms of molecular mass, membrane localization, and N-glycosylation; and (c) when the cDNA containing the entire coding sequence of the enzyme in a eukaryotic expression vector was transfected into COS-7 cells, the HS2ST activity increased 2.6-fold over controls, and the FLAG-HS2ST fusion protein purified by affinity chromatography showed the HS2ST activity alone.

Effects of primer-concentration on uronosyl-epimerization and sulfation patterns in p-hydroxyphenyl-O-beta-D-xylopyranoside-primed galactosaminoglycans produced by skin fibroblasts.

By supplying skin fibroblasts with different concentrations of the galactosaminoglycan chain-primer p-hydroxyphenyl-O-beta-D-xylopyranoside we have produced and recovered glycan-chains that were subsequently radio-iodinated in the hydroxyphenyl group and subjected to sequence analysis by using graded enzymic treatment followed by a combination of gel chromatography and electrophoresis. Fragments extending from the tagged reducing end to the cleavage-point were identified and quantified. Degradation by chondroitin B lyase of chains primed at 0.1 or 0.5 mM xyloside gave profiles indicating a periodic and wave-like distribution of iduronate-containing repeats, with high incidence around positions 2, 5 and onwards, whereas in chains produced at 1.0 mM xyloside the incidence of iduronate was similar in positions 1-4 and then declined. Degradation by chondroitin AC lyase indicated a high incidence of glucuronate in or near the linkage-region. There was a relatively uniform degree of sulfation in chains primed at low xyloside concentration, whereas chains primed at 1.0 mM xyloside gave very heterogeneous charge-patterns in all segments of the chain, including the linkage-region, giving the impression that adequate sulfation, probably at C-4 and at the first opportunity, is necessary to obtain an ordered and periodic epimerization pattern.

Heparan sulfate proteoglycan on leukemic cells is primarily involved in integrin triggering and its mediated adhesion to endothelial cells.

Leukocyte migration from circulation into tissue depends on leukocyte integrin-mediated adhesion to endothelium, but integrins cannot function until activated. However, it remains to be understood how tumor cells adhere to endothelium and infiltrate into underlying tissue. We studied mechanisms of extravasation of leukemic cells using adult T cell leukemia (ATL) cells and report the following novel features of cell surface heparan sulfate proteoglycan on ATL cells in ATL cell adhesion to endothelium: ATL cells adhere to endothelial cells through already activated integrins without exogenous stimulation; different from any other hematopoietic cells, ATL cells express a characteristic heparan sulfate capable of immobilizing heparin-binding chemokine macrophage inflammatory protein (MIP)-1 beta, a potent T cell integrin trigger, produced by the cells themselves; competitive interruption of endogenous heparan sulfate proteoglycan synthesis reduces cell surface MIP-1 beta and prevents ATL cells from integrin-mediated adhesion to endothelial cells or intercellular adhesion molecule-1 triggered through G-protein. We propose that leukemic cells adhere to endothelial cells through the adhesion cascade, similar to normal leukocyte, and that the cell surface heparan sulfate, particularly on ATL cells, is pivotally involved in chemokine-dependent autocrine stimulation of integrin triggering by immobilizing the chemokine on them.

Purification and characterization of heparan sulfate 2-sulfotransferase from cultured Chinese hamster ovary cells.

Heparan sulfate 2-sulfotransferase, which catalyzes the transfer of sulfate from adenosine 3'-phosphate 5'-phosphosulfate to position 2 of L-iduronic acid residue in heparan sulfate, was purified 51,700-fold to apparent homogeneity with a 6% yield from cultured Chinese hamster ovary cells. The isolation procedure included a combination of affinity chromatography on heparin-Sepharose CL-6B and 3',5'-ADP-agarose, which was repeated twice for each, and finally gel chromatography on Superose 12 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 47 and 44 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin and mouse Engelbreth-Holm-Swarm tumor heparan sulfate were used as acceptors, the purified enzyme transferred sulfate to position 2 of L-iduronic acid residue but did not transfer sulfate to the amino group of glucosamine residue or to position 6 of N-sulfoglucosamine residue. Heparan sulfates from pig aorta and bovine liver, however, were poor acceptors. The enzyme showed no activities toward chondroitin, chondroitin sulfate, dermatan sulfate, and keratan sulfate. The optimal pH for the enzyme activity was around 5.5. The enzyme activity was minimally affected by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.20 microM.

Characterization of heparan sulfate oligosaccharides that bind to hepatocyte growth factor.

Proteoglycans from rat liver had the ability to bind hepatocyte growth factor (HGF). Digestion of the proteoglycans with heparitinase resulted in the complete loss of the activity, while the digestion with chondroitinase ABC had no effect. Heparan sulfate (HS)-conjugated gel also bound HGF, and the binding was competitively inhibited by heparin and bovine liver HS, but not by Engelbreth-Holm-Swarm sarcoma HS, pig aorta HS, or other glycosaminoglycans, suggesting the specific structural domain in HS for the binding of HGF. Among limited digests with heparitinase I of bovine liver HS, octasaccharide is the minimal size to bind HGF. Comparison of the disaccharide unit compositions revealed a marked difference in IdoA(2SO4)-GlcNSO3(6SO4) unit between the bound and unbound octasaccharides. The contents of this disaccharide unit were calculated to be 2 mol/mol for the bound octasaccharide but 1 mol/mol for the unbound one. Considering both the substrate specificity and properties of heparitinase I, the above results suggest that the bound octasaccharide should contain two units of IdoA(2SO4)-GlcNSO3(6SO4) contiguously or alternately in the vicinity of the reducing end. The bound decasaccharide was more than 20 times as active as the unbound one with regard to the ability to release HGF bound to rat liver HS proteoglycan. The ability was comparable to the one-fourth of that of heparin.

Purification and characterization of heparan sulfate 6-sulfotransferase from the culture medium of Chinese hamster ovary cells.

Heparan sulfate 6-sulfotransferase, which catalyzes the transfer of sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-sulfoglucosamine in heparan sulfate, was purified 10,700-fold to apparent homogeneity with a 40% yield from the serum-free culture medium of Chinese hamster ovary cells. The isolation procedure included affinity chromatography of the first heparin-Sepharose CL-6B column (stepwise elution), 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B column (gradient elution). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 52 and 45 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin was used as acceptor, the purified enzyme transferred sulfate to position 6 of N-sulfoglucosamine residue but did not transfer sulfate to the amino group of glucosamine residue or to position 2 of the iduronic acid residue. Heparan sulfate was also sulfated by the purified enzyme at position 6 of N-sulfoglucosamine residue. Chondroitin and chondroitin sulfate did not serve as acceptors. The optimal pH for enzyme activity was around 6.3. The enzyme activity was inhibited by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.44 microM.

Basic fibroblast growth factor-heparan sulphate complex in the human dialysis-related amyloidosis.

A major constituent of the amyloid fibrils in dialysis-related amyloidosis is beta 2-microglobulin (beta 2-MG). Heparan sulphates (HS) co-localize with the amyloid fibrils and monocytes/macrophages are commonly found around amyloid deposits, but the role of HS in amyloidogenesis is not yet defined. HS have variable saccharide sequences and can interact specifically with basic fibroblast growth factor (bFGF), a potent chemotactic factor for the monocyte/macrophage. The present investigation was undertaken to look for a functional link between co-localized HS and the pathogenesis of dialysis-related amyloidosis. Using amyloid-enriched ligament, immunohistochemical localization was tested for beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS. For the detection of bFGF-binding portions of HS, the ligament sections were incubated with exogenous bFGF and then with anti-bFGF antibody. The specificity of the interaction between bFGF and HS was established by confirming a concomitant loss of immunoreactivity during selective removal of HS with heparitinase. beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS were detected between bundles of collagen. Endogenous bFGF and bFGF-binding portions of HS were not detected in more advanced amyloid lesions, whereas beta 2-MG and other portions of HS were detected. We propose that beta 2-MG, endogenous bFGF, and bFGF-binding portions of HS form a complex and localize in the early amyloid lesions of dialysis-related amyloidosis.