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

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

Structural requirements for heparan sulphate self-association

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

Interactions between glycosaminoglycans and blood coagulation factors: 1. Affinity chromatography of glycosaminoglycans on thrombin-substituted agarose

Various glycosaminoglycans have been subjected to affinity chromatography on immobilized bovine thrombin. Chondroitin sulphate, dermatan sulphate and heparan sulphate variants with a sulphate-to-hexosamine molar ratio of ～ 1 exhibited weak affinities. Heparan sulphate/heparin fractions of higher sulphate content could be separated into material with high and low affinity for thrombin. Removal of N-sulphate followed by N-acetylation did not affect binding, whereas oxidation and cleavage of non-sulphated hexuronate abolished the interaction. Heparan-related molecules of high thrombin-affinity comprised sequences where large blocks of sulphated iduronate-containing repeats were joined via a few repeats carrying non-sulphated iduronate or glucuronate to form continuous segments that were larger than decasaccharide.     

Molecular organization of heparan sulphate from human skin fibroblasts.

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.     

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.     

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

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

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

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

Glycosaminoglycan — lipoprotein interactions: 1. Effect of uronic acid composition and charge density of the glycan

Interactions between glycosaminoglycans and lipoproteins have been studied by affinity chromatography of various modified glycans on agarose substituted with low density lipoprotein (LDL). Elution was performed with increasing concentrations of NaCl. The electrostatic attraction between ligand and polyanion generally increased with increasing sulphate content. However, at equal charge density l-iduronic acid-containing glycans displayed higher affinity than D-glucuronic acid-containing ones. Within a population of heparin-related glycosaminoglycans, material containing 1.23 sulphate groups per hexosamine had higher affinity for LDL than did commercial heparin (2.40 sulphate/hexosamine). Decasaccharides or higher oligosaccharides from heparin-related glycans retained affinity only when they contained sulphate groups, while all fragments smaller than decasaccharide did not bind to LDL. Oligosaccharides that contained both sulphated and non-sulphated l-iduronic acid exhibited higher affinity than did fragments (of corresponding size) that contained only sulphated l-iduronic acid. Heparin-related glycans with the highest LDL-affinity contained 55% d-glucuronic acid. 11% non-sulphated l-iduronic acid and 34% l-iduronic acid-O-sulphate of total uronic acid.     

Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites

Cell surface heparan sulfate proteoglycans facilitate uptake of growth-promoting polyamines (Belting, M., Persson, S., and Fransson, L.-A. (1999) Biochem. J. 338, 317-323; Belting, M., Borsig, L., Fuster, M. M., Brown, J. R., Persson, L., Fransson, L.-A., and Esko, J. D. (2001) Proc. Natl. Acad. Sci. U. S. A., in press). Here, we have analyzed the effect of polyamine deprivation on the structure and polyamine affinity of the heparan sulfate chains in various glypican-1 glycoforms synthesized by a transformed cell line (ECV 304). Heparan sulfate chains of glypican-1 were either cleaved with heparanase at sites embracing the highly modified regions or with nitrite at N-unsubstituted glucosamine residues. The products were separated and further degraded by heparin lyase to identify sulfated iduronic acid. Polyamine affinity was assessed by chromatography on agarose substituted with the polyamine spermine. In heparan sulfate made by cells with undisturbed endogenous polyamine synthesis, free amino groups were restricted to the unmodified, unsulfated segments, especially near the core protein. Spermine high affinity binding sites were located to the modified and highly sulfated segments that were released by heparanase. In cells with up-regulated polyamine uptake, heparan sulfate contained an increased number of clustered N-unsubstituted glucosamines and sulfated iduronic acid residues. This resulted in a greater number of NO/nitrite-sensitive cleavage sites near the potential spermine-binding sites. Endogenous degradation by heparanase and NO-derived nitrite in polyamine-deprived cells generated a separate pool of heparan sulfate oligosaccharides with an exceptionally high affinity for spermine. Spermine uptake in polyamine-deprived cells was reduced when NO/nitrite-generated degradation of heparan sulfate was inhibited. The results suggest a functional interplay between glypican recycling, NO/nitrite-generated heparan sulfate degradation, and polyamine uptake.     

The copolymeric structure of pig skin dermatan sulphate. Characterization of d-glucuronic acid-containing oligosaccharides isolated after controlled degradation of oxydermatan sulphate

Selective periodate oxidation of unsubstituted l-iduronic acid residues in copolymeric dermatan sulphate chains was followed by reduction-hydrolysis or alkaline elimination. By this procedure the glucuronic acid-containing periods were isolated in oligosaccharide form; general formula: [Formula: see text] Further degradation of these oligosaccharides with chondroitinase-AC yielded three types of products: (a) sulphated trisaccharide containing an unsaturated uronosyl moiety in the non-reducing terminal and a C(4) fragment in the reducing terminal, DeltaUA-GalNAc-(-SO(4))-R; (b) monosulphated, unsaturated disaccharide, DeltaUA-GalNAc-SO(4) when n is greater than or equal to 2; and (c) N-acetylgalactosamine with or without sulphate. Oligosaccharides containing a single glucuronic acid residue (n=1) comprised more than half of the glucuronic acid-containing oligosaccharides. The terminal N-acetylgalactosamine moiety of the shortest oligosaccharide was largely 4-sulphated, whereas higher oligosaccharides primarily contained 6-sulphated or unsulphated hexosamine moieties in the same position. Moreover, IdUA-SO(4)-containing oligosaccharides were encountered. These oligosaccharides were resistant to the action of chondroitinase-ABC.     

Initiation of altered heparan sulphate on beta-D-xyloside in rat hepatocytes

Inhibition of protein synthesis by cycloheximide 10(-3)M reduced the incorporation of [35S]sulphate into heparan sulphate to about 5% of untreated hepatocytes. Addition of rho-nitrophenyl beta-D-xyloside could partially revert this inhibitory effect. The sulphated material isolated from the cell layer or secretions of hepatocytes grown in presence of cycloheximide and rho-nitrophenyl beta-D-xyloside were shown to be mostly free heparan sulphate chains not bound to core protein. Covalent association of beta-xylosides to the heparan sulphates was demonstrated for heparan sulphate synthetized in the presence of [35S]sulphate, cycloheximide and the fluorogenic 4-methylumbelliferyl beta-D-xyloside. Beta-Xylosides served as an initiator of heparan sulphate chain synthesis in rat hepatocytes only in the absence of protein synthesis. Heparan sulphates primed on artificial beta-xylosides are slightly smaller in molecular size and are more sulphated than chains linked to core protein.     

Synthesis and immunoadjuvant activity of 2-acetamido-1,5-anhydro-2-deoxy-3-O-[(R)-2-propanoyl-l-alanyl-d-isoglutamine]-d-glucitol (“1-deoxymuramoyl dipeptide”) and its 6-(2-behenoyloxyisobutyrate)

Two heparin-related preparations from beef lung and pig mucosa are able to inhibit the enzymic activity of the clotting factor X_a. These preparations were subjected to deaminative cleavage and periodate oxidation-alkaline elimination. The following structural features were observed: (a) N-acetylated and glucuronate-rich regions are short and frequently intercalated between N-sulphated and iduronate-rich segments of deca- to hexadeca-saccharide size; (b) in the latter segments, sulphated iduronate occurs together with non-sulphated iduronate and glucuronate in a random fashion. These characteristics are distinctly different from those of regular heparan sulphate and of archetypal heparin.     

Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor

Cathepsin L, a lysosomal cysteine protease, is the major excreted protein of transformed mouse NIH 3T3 cells. Previous studies have shown that asparagine-linked oligosaccharides associated with the secreted hydrolase contain mannose 6-phosphate (Man 6-P), the recognition marker for transport of newly synthesized acid hydrolases to lysosomes. To investigate the mechanism by which cathepsin L evades targeting to lysosomes, we determined the structure of the enzyme's oligosaccharides and analyzed its interaction with the cation-independent mannose 6-phosphate (Man 6-PCl) receptor. Oligosaccharides associated with procathepsin L isolated from the medium of [3H]mannose-labeled J774 cells were remarkably homogeneous; all of the radiolabeled structures were high mannose-type units that contained two phosphomonoesters and 7 mannose residues. Both the alpha 1,3- and alpha 1,6-branches of the oligosaccharides were phosphorylated. Oligosaccharides released by endoglycosidase H from [3H]mannose-labeled procathepsin L bound to a Man 6-PCl receptor affinity column. Despite the high affinity binding of these oligosaccharides, the intact glycoprotein was not a good ligand for the Man 6-PCl receptor. Procathepsin L was internalized poorly by Man 6-P receptor-mediated endocytosis and the purified acid protease interacted weakly with a Man 6-PCl affinity column. In contrast, pro-beta-glucuronidase (another acid hydrolase produced by J774 cells) was an excellent ligand for the Man 6-PCl receptor as judged by the endocytosis and affinity chromatographic assays. Phosphorylated oligosaccharides associated with the J774-secreted pro-beta-glucuronidase were heterogeneous and contained both mono- and diphosphorylated species. Tryptic glycopeptides generated from [3H]mannose-labeled procathepsin L, unlike the intact protein, were excellent ligands for the Man 6-PCl receptor. The results indicate that oligosaccharides associated with procathepsin L are processed uniformly to diphosphorylated species that bind with high affinity to the Man 6-PCl receptor. Protein determinants inherent within the intact acid hydrolase, however, inhibit the high affinity binding of these oligosaccharides and, as a result, impair the interaction of procathepsin L with the receptor.     

Oligosaccharide mapping of heparan sulphate by polyacrylamide-gradient-gel electrophoresis and electrotransfer to nylon membrane.

A new method that we have called 'oligosaccharide mapping' is described for the analysis of radiolabelled heparan sulphate and other glycosaminoglycans. The method involves specific enzymic or chemical scission of polysaccharide chains followed by high-resolution separation of the degradation products by polyacrylamide-gradient-gel electrophoresis. The separated oligosaccharides are immobilized on charged nylon membranes by electrotransfer and detected by fluorography. A complex pattern of discrete bands is observed covering an oligosaccharide size range from degree of polymerization (d.p.) 2 (disaccharide) to approximately d.p. 40. Separation is due principally to differences in Mr, though the method also seems to detect variations in conformation of oligosaccharide isomers. Resolution of oligosaccharides is superior to that obtained with isocratic polyacrylamide-gel-electrophoresis systems or gel chromatography, and reveals structural details that are not accessible by other methods. For example, in this paper we demonstrate a distinctive repeating doublet pattern of iduronate-rich oligosaccharides in heparitinase digests of mouse fibroblast heparan sulphate. This pattern may be a general feature of mammalian heparan sulphates. Oligosaccharide mapping should be a valuable method for the analysis of fine structure and sequence of heparan sulphate and other complex polysaccharides, and for making rapid assessments of the molecular distinctions between heparan sulphates from different sources.     

Basement-membrane heparan sulphate with high affinity for antithrombin synthesized by normal and transformed mouse mammary epithelial cells.

Basement-membrane proteoglycans, biosynthetically labelled with [35S]sulphate, were isolated from normal and transformed mouse mammary epithelial cells. Proteoglycans synthesized by normal cells contained mainly heparan sulphate and, in addition, small amounts of chondroitin sulphate chains, whereas transformed cells synthesized a relatively higher proportion of chondroitin sulphate. Polysaccharide chains from transformed cells were of lower average Mr and of lower anionic charge density compared with chains isolated from the untransformed counterparts, confirming results reported previously [David & Van den Berghe (1983) J. Biol. Chem. 258, 7338-7344]. A large proportion of the chains isolated from normal cells bound with high affinity to immobilized antithrombin, and the presence of 3-O-sulphated glucosamine residues, previously identified as unique markers for the antithrombin-binding region of heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555], could be demonstrated. A significantly lower proportion of the chains derived from transformed cells bound with high affinity to antithrombin, and a corresponding decrease in the amount of incorporated 3-O-sulphate was observed.     

New oligosaccharides from heparin and heparan sulfate and their use as substrates for heparin-degrading enzymes

Oligosaccharides were isolated from heparin and heparan sulfate by a procedure consisting of three major steps: (a) acid hydrolysis; (b) gel chromatography; and (c) cation exchange chromatography on an amino acid analyzer. To date, six new oligosaccharides have been isolated by this procedure and have been sequenced by a combination of NaB3H4-labeling and deaminative cleavage with nitrous acid. The structures of these oligosaccharides were as follows: 1. GlcN-GlcUA-GlcN 2. GlcN-IdUA-GlcN 3. GlcN-GlcUA-GlcN-GlcUA-GlcN 4. GlcN-IdUA-GlcN-GlcUA-GlcN 5. GlcN-GlcUA-GlcN-IdUA-GlcN 6. GlcN-IdUA-GlcN-IdUA-GlcN The linkage positions and anomeric configurations were assumed to be the same as in the polysaccharides from which the oligosaccharides originated. The usefulness of some of these oligosaccharides as enzyme substrates was tested after appropriate modifications and radioactive labeling. Oligosaccharides 2 and 3 were N-[35S]sulfated and were found to serve as substrates for heparan N-sulfate sulfatase (heparin sulfamidase), with a homogenate of cultured skin fibroblasts as enzyme source. Similarly, reduction of oligosaccharide 2 with NaB3H4 yielded a substrate for acetyl-CoA:alpha-D-glucosaminide N-acetyltransferase. Finally, the previously known disaccharide, 4-O-alpha-D-glucosaminyl-L-iduronic acid, which was isolated in the course of this work, was N-acetylated with [3H] acetic anhydride and was shown to be a substrate for N-acetyl-alpha-D-glucosaminidase.     

Heparan sulphate/heparin glycosaminoglycans with strong affinity for the growth-promoter spermine have high antiproliferative activity

Depletion of intracellular polyamine pools inhibits cell proliferation.Polyamine pools are maintained by intracellular synthesis andby uptake from the extracellular environment. It may be expectedthat cationic polyamines are sequestered by the polyanionicglycosaminoglycan substituents of extracellular proteoglycans.Moreover, highly sulphated heparin-related glycans inhibit growthof human embryonic lung fibroblasts. We have therefore investigatedinteractions between polyamines and heparin-related glycosaminoglycans.Affinity chromatography of various polyamines on heparin-agaroseindicated that spermine was the only polyamine that bound efficientlyto this type of glycan. By using equilibrium dialysis we foundthat spermine binds to a highly sulphated heparan sulphate/heparinpreparation with a dissociation constant of 3.7x10^–5M.Enzymatic degradation of heparan sulphate using three differentheparan sulphate/heparin lyases, separately or in combinationand in the absence or presence of spermine, was used to generatespermine-binding and degradation-protected oligosaccharides.As indicated by chromato graphic and electrophoretic analysisa size- and charge-heterogeneous collection was obtained. However,protected oligosaccharides derived from antiproliferative heparansulphates were inactive. Highly sulphated, antiproliferativeheparan sulphates were subfractionated on spermine-agarose yieldinghigh-affinity material with increased antiproliferative activity.A very potent material was obtained from pig skin. Althoughthere was generally a clear correlation between high spermine-affinityand strong growth-inhibition, no correlation with sulphate contentor oligosaccharide mapping patterns could be detected. Beeflung heparan sulphate comprised naturally occurring fragmentsof eicosasaccharide size with substantially increased specificactivity. As these fragments were longer than oligosaccharidesgenerated by enzymatic degradation in the presence of spermine(hexa- to tetradecasaccharide), multiple spermine-binding sitesin tandem may be necessary to induce antiproliferative activity. heparan sulphate spermine interaction antiproliferation     

The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor

We have analyzed the interaction of phosphorylated oligosaccharides and lysosomal enzymes with immobilized bovine liver cation-dependent mannose-6-P receptor. Oligosaccharides with phosphomonoesters were the only species that interacted with the receptor, and molecules with two phosphomonoesters showed the best binding. Lysosomal enzymes with several oligosaccharides containing only one phosphomonoester had a higher affinity for the receptor than did the isolated oligosaccharides, indicating the possible importance of multivalent interactions between weakly binding ligands and the receptor. The binding of a mixture of phosphorylated lysosomal enzymes to the cation-dependent Man-6-P receptor was markedly influenced by pH. At pH 6.3, almost all of the lysosomal enzymes bound to the receptor; whereas at pH 7.0-7.5, approximately one-third of the material passed through the column, one-third interacted weakly, and one-third bound tightly. The distribution of individual lysosomal enzyme activities was similar to that of the total material. The species of phosphorylated oligosaccharides present on the lysosomal enzymes which interacted poorly with the receptor were similar to those found on the tightly bound material and included species of oligosaccharides with two phosphomonoester groups. Isolated oligosaccharides of this type bound to the receptor over the entire pH range tested. These findings indicate that at neutral pH the phosphorylated oligosaccharides on some lysosomal enzyme molecules are oriented in a manner which makes them inaccessible to the binding site of the cation-dependent Man-6-P receptor. Since the same enzymes bind to the cation-independent Man-6-P receptor at neutral pH, at least a portion of the phosphomannosyl residues must be exposed. We conclude that small variations in the pH of the Golgi compartment where lysosomal enzymes bind to the receptors could potentially modulate the extent of binding to the two receptors.     

Structural features in heparin which modulate specific biological activities mediated by basic fibroblast growth factor

The biological activity of basic fibroblast growth factor (bFGF)is influenced greatly by direct binding to heparin and heparansulphate (HS). Heparin-derived oligosaccharides have been utilizedto determine the structural requirements present in the polymerthat account for bind ing to bFGF. We had previously demonstratedthat fragments >6 mer can inhibit the interaction betweencell surface heparan sulphate proteoglycan (HSPG) and bFGF,and bFGF-induced proliferation of adrenocortical endothelial(ACE) cells. In contrast, oligosaccharides > 10 mer can enhancethe binding of bFGF to its high-affinity receptor or supportbFGF-induced mitogenesis in ACE cells (Ishihara et al., J. Biol.Chem., 268, 4675–4683, 1993). We have extended these studiesto size- and structure-defined oligosaccharides from heparin,2-O-desulphated (2-O-DS-) heparin, 6-O-desulphated (6-O-DS-)heparin, carboxyreduced (CR-) heparin and carboxy-amidomethylsulphonated(AMS-) heparin. Oligosaccharides from these polymers were fractionatedon a bFGF-affinity column and were assessed as inhibitors orenhancers of specific bFGF-derived biological activities. Theresults of these studies indicate that both 2-O-sulphate andthe negative charge of the carboxy group [L-iduronic acid (IdoA)residues] are required for specific interactions of heparin-derivedoligosaccharides with bFGF and for modulation of bFGF mitogenicactivity. In addition, the charge of the carboxy groups in uronicacids can be replaced by other functional groups with a negativecharge, such as the amidomethyl sulphonate moiety describedhere. basic fibroblast growth factor heparan sulphate heparin oligosaccharides