Molecular organization and antiproliferative domains of arterial tissue heparan sulfate.

Heparan sulfate isolated from mammalian arterial tissue inhibits the growth of homologous arterial smooth muscle cells when added to subconfluent cell cultures at a concentration of 50 to 100 micrograms/ml culture medium. Disintegration of the heparan sulfate molecule by hydrazinolysis that deacetylates N-acetylglucosaminyl residues and by subsequent treatment with nitrous acid at pH 3.9 results in the formation of a mixture of oligosaccharides which was further resolved into sulfate-enriched oligosaccharides with antiproliferative activity in an in vitro bioassay system. A decasaccharide and dodeca/tetradecasaccharide fraction had a significantly higher antiproliferative effect on arterial smooth muscle cells than the native heparan sulfate molecule. The antiproliferative oligosaccharides have a sulfate content of 0.9 to 1.2 sulfate groups/disaccharide unit and consist of 60 to 70% monosulfated, disulfated, and trisulfated disaccharide units. Up to 32% of the sulfate groups were in 2-position of the uronic acid. In contrast, nitrous acid degradation of heparan sulfate at pH 1.5, which cleaves glycosidic linkages of N-sulfoglucosaminyl residues, results in the formation of sulfate-poor or sulfate-free oligosaccharides without antiproliferative potency. The results indicate that (a) heparan sulfate has a heterogeneous molecular organization where sulfate-rich domains are separated by sulfate-poor sequences and that (b) the antiproliferative activity of heparan sulfate resides in domains enriched with 2-O-sulfated uronic acid residues.     

Human follicular fluid heparan sulfate contains abundant 3-O-sulfated chains with anticoagulant activity

Anticoagulant heparan sulfate proteoglycans bind and activate antithrombin by virtue of a specific 3-O-sulfated pentasaccharide. They not only occur in the vascular wall but also in extravascular tissues, such as the ovary, where their functions remain unknown. The rupture of the ovarian follicle at ovulation is one of the most striking examples of tissue remodeling in adult mammals. It involves tightly controlled inflammation, proteolysis, and fibrin deposition. We hypothesized that ovarian heparan sulfates may modulate these processes through interactions with effector proteins. Our previous work has shown that anticoagulant heparan sulfates are synthesized by rodent ovarian granulosa cells, and we now have set out to characterize heparan sulfates from human follicular fluid. Here we report the first anticoagulant heparan sulfate purified from a natural human extravascular source. Heparan sulfate chains were fractionated according to their affinity for antithrombin, and their structure was analyzed by 1H NMR and MS/MS. We find that human follicular fluid is a rich source of anticoagulant heparan sulfate, comprising 50.4% of total heparan sulfate. These antithrombin-binding chains contain more than 6% 3-O-sulfated glucosamine residues, convey an anticoagulant activity of 2.5 IU/ml to human follicular fluid, and have an anti-Factor Xa specific activity of 167 IU/mg. The heparan sulfate chains that do not bind antithrombin surprisingly exhibit an extremely high content in 3-O-sulfated glucosamine residues, which suggest that they may exhibit biological activities through interactions with other proteins.     

Role of 3-O-sulfated heparan sulfate in virus-induced polykaryocyte formation

One way herpes simplex virus type-1 (HSV-1) spreads in vivo is by polykaryocytes formation. Here we demonstrate that polykaryocyte production during HSV-1 spread in cultured human corneal fibroblasts (CF) required heparan sulfate (HS) and more specifically 3-O sulfated HS (3-OS HS). The polykaryocyte formation heavily depended on the expression of HS on target (CF) cells but not on glycoprotein expressing effector cells. Furthermore, we provide the first visual evidence of 3-OS HS and HSV-1 gD colocalization during the membrane fusion process. Taken together our results provide novel insight into the significance of HS in polykaryocyte formation.     

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry.

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.     

Differential expression of heparan sulfate domains in rat spleen.

The microarchitecture of the spleen is composed of a meshwork of reticulum cells and their matrix. Heparan sulfates (HS) are important components of this meshwork and are involved in processes such as cell adhesion, cell migration, and cytokine/growth factor binding. The expression of HS epitopes was analyzed using anti-HS antibodies. Four different staining patterns were observed, as exemplified by antibodies RB4EA12, HS4E4, AO4B08, and HS4C3. These antibodies recognize different chemical modifications in HS. In adult spleen, RB4EA12 stained only the reticular meshwork and blood vessels in the red pulp and marginal zone. HS4E4 stained blood vessel-associated basal lamina. AO4B08 and HS4C3 stained the reticular meshwork and blood vessels throughout the spleen, but only AO4B08 strongly stained smooth muscle cells and ring fibers. Interleukin-2 localized in the red pulp and marginal zone and was bound to HS. AO4B08, HS4C3, and RB4EA12 but not HS4E4 co-localized with interleukin-2. In 10-day-old spleen, HS4E4 recognized reticular fibers, which were not stained in the adult stage. Immunoelectron microscopy revealed that HS was restricted to basal laminae and reticular fibers. Taken together, data show that HS epitopes are differentially expressed in the spleen and that this may create specific extracellular environments for immunological processes.     

Location of N-unsubstituted glucosamine residues in heparan sulfate

Functional properties of heparan sulfate (HS) are generally ascribed to the sulfation pattern of the polysaccharide. However, recently reported functional implications of rare N-unsubstituted glucosamine (GlcNH(2)) residues in native HS prompted our structural characterization of sequences around such residues. HS preparations were cleaved with nitrous acid at either N-sulfated or N-unsubstituted glucosamine units followed by reduction with NaB(3)H(4). The labeled products were characterized following complementary deamination steps. The proportion of GlcNH(2) units varied from 0.7-4% of total glucosamine in different HS preparations. The GlcNH(2) units occurred largely clustered at the polysaccharide-protein linkage region in intestinal HS, also more peripherally in aortic HS. They were preferentially located within N-acetylated domains, or in transition sequences between N-acetylated and N-sulfated domains, only 20-30% of the adjacent upstream and downstream disaccharide units being N-sulfated. The nearest downstream (toward the polysaccharide-protein linkage) hexuronic acid was invariably GlcUA, whereas the upstream neighbor could be either GlcUA or IdoUA. The highly sulfated but N-unsubstituted disaccharide unit, -IdoUA2S-GlcNH(2)6S-, was detected in human renal and porcine intestinal HS, but not in HS from human aorta. These results are interpreted in terms of a biosynthetic mechanism, whereby GlcNH(2) residues are formed through regulated, incomplete action of an N-deacetylase/N-sulfotransferase enzyme.     

Highly sulfated nonreducing end-derived heparan sulfate domains bind fibroblast growth factor-2 with high affinity and are enriched in biologically active fractions

Human fibroblast growth factor-2 (FGF2) regulates cellular processes including proliferation, adhesion, motility, and angiogenesis. FGF2 exerts its biological function by binding and dimerizing its receptor (FGFR), which activates signal transduction cascades. Effective binding of FGF2 to its receptor requires the presence of heparan sulfate (HS), a linear polysaccharide with N-sulfated domains (NS) localized at the cell surface and extracellular matrix. HS acts as a platform facilitating the formation of a functional FGF-FGFR-HS ternary complex. Crystal structures of the signaling ternary complex revealed two conflicting architectures. In the asymmetrical model, two FGFs and two FGFRs bind a single HS chain. In contrast, the symmetrical model postulates that one FGF and one FGFR bind to the free end of the HS chain and dimerization require these ends to join, bringing the two half-complexes together. In this study, we screened a hexasaccharide HS library for compositions that are able to bind FGF2. The library was composed primarily of NS domains internal to the HS chain with minor presence of non-reducing end (NRE) NS. The binders were categorized into low versus high affinity binders. The low affinity fraction contained primarily hexasaccharides with low degree of sulfation that were internal to the HS chains. In contrast, the high affinity bound fraction was enriched in NRE oligosaccharides that were considerably more sulfated and had the ability to promote FGFR-mediated cell proliferation. The results suggest a role of the NRE of HS in FGF2 signaling and favor the formation of the symmetrical architecture on short NS domains.     

The heparin/heparan sulfate sequence that interacts with cyclophilin B contains a 3-O-sulfated N-unsubstituted glucosamine residue

Many of the biological functions of heparan sulfate (HS) proteoglycans can be attributed to specialized structures within HS moieties, which are thought to modulate binding and function of various effector proteins. Cyclophilin B (CyPB), which was initially identified as a cyclosporin A-binding protein, triggers migration and integrin-mediated adhesion of peripheral blood T lymphocytes by a mechanism dependent on interaction with cell surface HS. Here we determined the structural features of HS that are responsible for the specific binding of CyPB. In addition to the involvement of 2-O,6-O, and N-sulfate groups, we also demonstrated that binding of CyPB was dependent on the presence of N-unsubstituted glucosamine residues (GlcNH2), which have been reported to be precursors for sulfation by 3-O-sulfotransferases-3 (3-OST-3). Interestingly, 3-OST-3B isoform was found to be the main 3-OST isoenzyme expressed in peripheral blood T lymphocytes and Jurkat T cells. Moreover, down-regulation of the expression of 3-OST-3 by RNA interference potently reduced CyPB binding and consequent activation of p44/42 mitogen-activated protein kinases. Altogether, our results strongly support the hypothesis that 3-O-sulfation of GlcNH2 residues could be a key modification that provides specialized HS structures for CyPB binding to responsive cells. Given that 3-O-sulfation of GlcNH2-containing HS by 3-OST-3 also provides binding sites for glycoprotein gD of herpes simplex virus type I, these findings suggest an intriguing structural linkage between the HS sequences involved in CyPB binding and viral infection.     

Detection of 2-O-sulfated iduronate and N-acetylglucosamine units in heparan sulfate by an antibody selected against acharan sulfate (IdoA2S-GlcNAc)n

The snail glycosaminoglycan acharan sulfate (AS) is structurally related to heparan sulfates (HS) and has a repeating disaccharide structure of alpha-d-N-acetylglucosaminyl-2-O-sulfo-alpha-l-iduronic acid (GlcNAc-IdoA2S) residues. Using the phage display technology, a unique antibody (MW3G3) was selected against AS with a V(H)3, DP 47, and a CDR3 amino acid sequence of QKKRPRF. Antibody MW3G3 did not react with desulfated, N-deacetylated or N-sulfated AS, indicating that reactivity depends on N-acetyl and 2-O-sulfate groups. Antibody MW3G3 also had a high preference for (modified) heparin oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. In tissues, antibody MW3G3 identified a HS oligosaccharide epitope containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues as enzymatic N-deacetylation of HS in situ prevented staining, and 2-O-sulfotransferase-deficient Chinese hamster ovary cells were not reactive. An immunohistochemical survey using various rat organs revealed a distinct distribution of the MW3G3 epitope, which was primarily present in the basal laminae of most (but not all) blood vessels and of some epithelia, including human skin. No staining was observed in the glycosaminoglycan-rich tumor matrix of metastatic melanoma. In conclusion, we have selected an antibody that identifies HS oligosaccharides containing N-acetylated glucosamine and 2-O-sulfated iduronic acid residues. This antibody may be instrumental in identifying structural alterations in HS in health and disease.     

Highly sensitive sequencing of the sulfated domains of heparan sulfate.

The heparan sulfates (HS) are hypervariable linear polysaccharides that act as membrane co-receptors for growth factors, chemokines, and extracellular matrix proteins. In most instances, the molecular basis of protein recognition by HS is poorly understood. We have sequenced 75% of the sulfated domains (S-domains) of fibroblast HS, including all of the major ones. This analysis revealed tight coupling of N- and 2-O-sulfation and a low frequency but precise positioning of 6-O-sulfates, which are required functional groups for HS-mediated activation of the fibroblast growth factors. S-domain sequencing was conducted using a novel and highly sensitive method based on a new way of reading the sequence from high performance liquid chromatography separation profiles of metabolically labeled HS-saccharides following specific chemical and enzymatic scission. The implications of the patterns seen in the sulfated domains for better understanding of the synthesis and function of HS are discussed.     

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).     

Surface-exposed amino acid residues of HPV16 L1 protein mediating interaction with cell surface heparan sulfate

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.     

Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.     

Dissociation of heparan sulfate and receptor binding domains of hepatocyte growth factor reveals that heparan sulfate-c-met interaction facilitates signaling.

Hepatocyte growth factor (HGF) is a secreted, heparan sulfate (HS) glycosaminoglycan-binding protein that stimulates mitogenesis, motogenesis, and morphogenesis in a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial cells, and hematopoietic cells. NK1 is an alternative HGF isoform that consists of the N-terminal (N) and first kringle (K1) domains of full-length HGF and stimulates all major HGF biological activities. Within NK1, the N domain retains the HS binding properties of full-length HGF and mediates HS-stimulated ligand oligomerization but lacks significant mitogenic or motogenic activity. In contrast, K1 does not bind HS, but it stimulates receptor and mitogen-activated protein kinase activation, mitogenesis, and motogenesis, demonstrating that structurally distinct and dissociable domains of HGF are the primary mediators of HS binding and receptor activation. Despite the absence of HS-K1 binding, K1 mitogenic activity in HS-negative cells is strictly dependent on added soluble heparin, whereas K1-stimulated motility is not. We also found that, like the receptors for fibroblast growth factors, the HGF receptor c-Met binds tightly to HS. These data suggest that HS can facilitate HGF signaling through interaction with c-Met that is independent of HGF-HS interaction and that the recruitment of specific intracellular effectors that mediate distinct HGF responses such as mitogenesis and motility is regulated by HS-c-Met interaction at the cell surface.     

Reaction of unsaturated uronic acid residues with mercuric salts. Cleavage of the hyaluronic acid disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose.

Degradation of connective-tissue polysaccharides with bacterial or fungal eliminases and subsequent characterization of the reaction products are now part of standard methodology for the analysis of these compounds. However, the scope of preparative and analytical work based on the use of eliminases has been limited by the lack of procedures for specific removal of the unsaturated uronic acid residues generated in the eliminase reactions. In the present investigation, we have shown that these residues are cleaved by mercuric salts under mild conditions that are not likely to affect other structures in an oligo- or poly-saccharide molecule. Thus the disaccharide generated from hyaluronic acid by digestion with chondroitinase AC or ABC was cleaved into a keto acid and free N-acetylglucosamine within 10 min at room temperature upon exposure to 14 mM-mercuric acetate at pH 5. The reaction of the disaccharide with mercuric salts was used for ready determination of the distribution of radioactivity between the glucuronic acid and N-acetylglucosamine moieties in radioactive hyaluronic acid that had been synthesized by IMR-90 fibroblasts from 3H-labelled monosaccharides. When the precursor was [3H]galactose, over 95% of the incorporated radioactivity was found in the glucuronic acid moiety. In contrast, cells grown in the presence of [3H]glucosamine synthesized a polysaccharide in which almost all of the label was located in the N-acetylglucosamine units. It is apparent from these experiments that the reaction of unsaturated uronic acid residues with mercuric salts provides a new tool with potential for many applications in the study of the structure and metabolism of connective-tissue polysaccharides.     

Glycosaminoglycan biosynthesis in arterial wall. Sulfation of heparan sulfate in cell membrane of aortic media-intima

Incubation of microsomal fractions with labelled 3'-phosphoadenylyl sulfate results in incorporation of [35S]sulfate into endogenous glycosaminoglycans. Specific radioactivity observed incorporated into heparan sulfate chains is 10-fold greater than that incorporated into chondro?tin sulfate chains. This is in agreement with the results obtained for glycosylation of glycosaminoglycans in arterial wall membrane fractions. Sulfation of heparan sulfate was studied since it contains N- and O-sulfate groups in contrast with the other sulfated glycosaminoglycans which contain only O-sulfate groups. Sulfation of heparan sulfate occurs rapidly, since sulfate incorporation is detected after exposure for only 0.5 min. Heparan sulfate was identified on the basis of its resistance to hyaluronidase and chondro?tin ABC lyase, its susceptibility to heparitinase, its sensitivity to nitrous acid and the presence of glucosamine as the only hexosamine. The chemical composition of the purified heparan sulfate fractions provides evidence for the high degree of sulfation of its chains. Studies into the distribution of sulfate residues on heparan sulfate at different times of sulfation indicate that N-sulfate groups are not randomly introduced into the polymer. The relationship between the processes of N- and O-sulfation was studied. The present results demonstrate that preferential N-sulfation is obtained for incorporation of labelled precursor over a short period, the O-sulfation occurring on previously N-sulfated heparan sulfate.     

The growth promoter spermine interacts specifically with dermatan sulfate regions that are rich inl-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 tol-iduronic acid-rich dermatan sulfate (K _d, approximately 3.9×10^–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 inl-iduronic acid, generates no spermine-protected fragments. Analysis of protected dermatan sulfate oligosaccharides indicates that the majority of thel-iduronic acid residue is non-sulfated and in a periodate-resistant conformation. The oligosaccharides also possess antiproliferative activity.     

Distinct substrate specificities of bacterial heparinases against N-unsubstituted glucosamine residues in heparan sulfate

The rare N-unsubstituted glucosamine (GlcNH(3)(+)) residues in heparan sulfate have important biological and pathophysiological roles. In this study, four GlcNH(3)(+)-containing disaccharides were obtained from partially de-N-sulfated forms of heparin and the N-sulfated K5 polysaccharide by digestion with combined heparinases I, II, and III. These were identified as DeltaHexA-GlcNH(3)(+),DeltaHexA-GlcNH(3)(+)(6S),DeltaHexA(2S)-GlcNH(3)(+), and DeltaHexA(2S)-GlcNH(3)(+)(6S). Digestions with individual enzymes revealed that heparinase I did not cleave at GlcNH(3)(+) residues; however, heparinases II and III showed selective and distinct activities. Heparinase II generated DeltaHexA-GlcNH(3)(+)(6S),DeltaHexA(2S)-GlcNH(3)(+), and DeltaHexA(2S)-GlcNH(3)(+)(6S) disaccharides, whereas heparinase III yielded only the DeltaHexA-GlcNH(3)(+) unit. Thus, the action of heparinase II requires O-sulfation, whereas heparinase III acts only on the corresponding non-sulfated unit. These striking distinctions in substrate specificities of heparinases could be used to isolate oligosaccharides with novel sequences of GlcNH(3)(+) residues. Finally, heparinases were used to identify and quantify GlcNH(3)(+)-containing disaccharides in native bovine kidney and porcine intestinal mucosal heparan sulfates. The relatively high content of O-sulfated GlcNH(3)(+)-disaccharides in kidney HS raises questions about how these sequences are generated.     

Preparation of heparin/heparan sulfate oligosaccharides with internal N-unsubstituted glucosamine residues for functional studies

The rare N-unsubstituted glucosamine (GlcNH (3)(+)) residues in heparan sulfate (HS) have important biological and pathophysiological roles. However, it is difficult to prepare naturally-occuring, GlcNH(3)(+)-containing oligosaccharides from HS because of their low abundance, as well as the inherent problems in both excising and identifying them. Therefore, the ability to chemically generate a series of structurally-defined oligosaccharides containing GlcNH(3)(+) residues would greatly contribute to investigating their natural role in HS. In this study, a series of heparin/HS oligosaccharides, from dp6 up to dp16 in length that possess internal GlcNH(3)(+) residues were prepared by a combination of chemical modification and heparinase I digestion. Purification and structural analysis of the major species derived from the octa- to dodeca-saccharide size fractions indicated the introduction of between 1 and 3 internal GlcNH(3)(+) residues per oligosaccharide. In addition, a GlcNH(3)(+) residue was selectively introduced into an internal position in a tetrasaccharide species by direct chemical modification. This selectivity has potential as an alternative procedure for preparing internally-modified oligosaccharides of various lengths. The utility of such oligosaccharides was demonstrated by a comparison of the binding of three different tetrasaccharide species containing 0, 1 and 2 free amino groups to the NK1 truncated variant of hepatocyte growth factor/scatter factor.