Regulation of heparan sulfate 6-O-sulfation by beta-secretase activity

The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable beta-secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of beta-secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent beta-secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GlcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by beta-secretase via an indirect mechanism.     

Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate

Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a β-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles.     

乙酰肝素酶的生物学特性的研究进展

乙酰肝素酶是切割哺乳动物细胞中硫酸肝素蛋白多糖侧链——硫酸乙酰肝素的内源性糖苷酶,是抗肿瘤转移的理想靶点。本就乙酰肝素酶的分子结构特点、亚细胞定位、活性调控机制、与肿瘤转移的关系、底物特异性和抑制剂开发等方面的研究进展进行了综述。     

Inhibition of intracellular degradation of proteoglycans by leupeptin in rat ovarian granulosa cells

Previous work (Yanagishita, M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 10270-10283) has indicated that heparan sulfate (HS) proteoglycans in rat ovarian granulosa cells are degraded by two kinetically distinct pathways. Pathway 1 degrades proteoglycans rapidly with a t 1/2 approximately 25 min without generating appreciable degradative intermediates. Pathway 2 degrades proteoglycans more slowly with a t 1/2 approximately 4 h, generating distinct degradative intermediates: single HS chains of Mr = approximately 10,000 and approximately 5,000. Effects of leupeptin, an inhibitor of thiol proteases, on the intracellular degradation of proteoglycans in the rat ovarian granulosa cell culture were examined using various chase protocols after labeling cells with [35S]sulfate. The presence of leupeptin at 100 micrograms/ml in the culture medium inhibited the intracellular degradation of proteoglycans by approximately 80% during a 7-h chase period after a 20-h labeling. Leupeptin affected neither the cellular content nor the in vitro activities of beta-hexosaminidase and arylsulfatase. Structural analyses of heparan sulfate species in leupeptin-treated cells demonstrated that the drug inhibited the degradation of HS proteoglycans at two distinct points. First, degradation of the core protein was partially inhibited and delayed before the start of glycosaminoglycan degradation. This resulted in the accumulation of degradative intermediates with partially degraded core proteins bearing intact glycosaminoglycan chains. This establishes the initial sequence for HS proteoglycan degradation, with proteolysis preceding endoglycosidase digestion, and suggests that these two degradation steps may occur in physically separate compartments. Second, the final depolymerization of HS fragments through pathway 2 was totally inhibited, resulting in the continuous accumulation of Mr = 5,000 HS chains. This is not due to the direct inhibition of the lysosomal exoglycosidase and sulfatase enzymes responsible for the complete depolymerization of HS chains, since pathway 1, while slowed, continued to completely depolymerize the HS chains in the presence of leupeptin. The results suggest that the intracellular compartment which completely degrades heparan sulfate chains is separate from those containing partially, endoglycosidically processed heparan sulfate chains and that leupeptin interfered with the translocation of glycosaminoglycans to the final degradation site.     

An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure

Heparan sulfate (HS), a glycosaminoglycan present on the surface of cells, has been postulated to have important roles in driving both normal and pathological physiologies. The chemical structure and sulfation pattern (domain structure) of HS is believed to determine its biological function, to vary across tissue types, and to be modified in the context of disease. Characterization of HS requires isolation and purification of cell surface HS as a complex mixture. This process may introduce additional chemical modification of the native residues. In this study, we describe an approach towards thorough characterization of bovine kidney heparan sulfate (BKHS) that utilizes a variety of orthogonal analytical techniques (e.g. NMR, IP-RPHPLC, LC-MS). These techniques are applied to characterize this mixture at various levels including composition, fragment level, and overall chain properties. The combination of these techniques in many instances provides orthogonal views into the fine structure of HS, and in other instances provides overlapping / confirmatory information from different perspectives. Specifically, this approach enables quantitative determination of natural and modified saccharide residues in the HS chains, and identifies unusual structures. Analysis of partially digested HS chains allows for a better understanding of the domain structures within this mixture, and yields specific insights into the non-reducing end and reducing end structures of the chains. This approach outlines a useful framework that can be applied to elucidate HS structure and thereby provides means to advance understanding of its biological role and potential involvement in disease progression. In addition, the techniques described here can be applied to characterization of heparin from different sources.     

Biophysical investigation of recombinant K5 lyase: structural implications of substrate binding and processing

K5 lyase of coliphage K5A degrades the K5 polysaccharide of encapsulated E. coli strains expressing the K5 antigen thereby contributing to virus binding and infection. We have investigated the affinities of the recombinant enzyme for different GAG ligands by isothermal fluorescence titrations and correlated them with substrate processing and protein structural changes. Chondroitin sulfate (CS) and heparan sulfate (HS) bound to K5 lyase with a Kd of 0.5 microM whereas heparin exhibited a Kd=1.1 microM. The natural substrate K5 polysaccharide displayed a similar apparent affinity as CS and HS but was the only ligand of the enzyme which induced a large structural rearrangement of the protein as detected by far-UV CD spectroscopy. Since significant enzymatic degradation was only found for the K5 polysaccharide peaking at 44 degrees C, but binding was also detected for heparin, we propose that the K5 lyase is able to discriminate between specific (acetylated/non-sulfated) and unspecific (acetylated/sulfated) ligands by its heparin binding motif in the C-terminus. This is proposed to be the origin for the enzyme's residual HS degrading activity.     

Biophysical investigation of human heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A: a mutual effect of enzyme oligomerisation and glycosaminoglycan ligand binding

3-O-sulfation of heparan sulfate (HS) is the rarest modification within heparan sulfate biosynthesis resulting in unique biological activities. Heparan sulfate d-glucosaminyl 3-O-sulfotransferase-3A (3-OST-3A) (EC 2.8.2.23) generates a binding site for the envelope glycoprotein D (gD) of herpes simplex virus 1. We have expressed the sulfotransferase domain of the human heparan sulfate 3-OST-3A isoform in Escherichia coli and subsequently purified the active enzyme which was found to be present as an oligomer under nonreducing conditions. The activity of the enzyme was tested by a novel gD-dependent gel mobility assay. A biophysical characterisation of 3-OST-3A was performed to study ligand binding and ligand-induced structural changes. Interestingly, the natural substrate HS did not cause a secondary structural change in the enzyme, whereas heparin and chondroitin sulfate did, both of which also exhibited similar high affinity binding to 3-OST-3A compared to HS as detected by isothermal fluorescence titrations. In cross-link assays, only HS was found to induce high molecular aggregates of 3-OST-3A whereas other GAG ligands did not or even inhibited enzyme oligomerisation like the K5 polysaccharide, which was nevertheless found to bind to the enzyme. We therefore conclude that since 3-OST-3A is able to bind also non-substrate GAG ligands with high affinity, discrimination among ligands is triggered by protein oligomerisation.     

Identification and characterization of heparin/heparan sulfate binding domains of the endoglycosidase heparanase

The endo-beta-glucuronidase, heparanase, is an enzyme that cleaves heparan sulfate at specific intra-chain sites, yielding heparan sulfate fragments with appreciable size and biological activities. Heparanase activity has been traditionally correlated with cell invasion associated with cancer metastasis, angiogenesis, and inflammation. In addition, heparanase up-regulation has been documented in a variety of primary human tumors, correlating with increased vascular density and poor postoperative survival, suggesting that heparanase may be considered as a target for anticancer drugs. In an attempt to identify the protein motif that would serve as a target for the development of heparanase inhibitors, we looked for protein domains that mediate the interaction of heparanase with its heparan sulfate substrate. We have identified three potential heparin binding domains and provided evidence that one of these is mapped at the N terminus of the 50-kDa active heparanase subunit. A peptide corresponding to this region (Lys(158)-Asp(171)) physically associates with heparin and heparan sulfate. Moreover, the peptide inhibited heparanase enzymatic activity in a dose-responsive manner, presumably through competition with the heparan sulfate substrate. Furthermore, antibodies directed to this region inhibited heparanase activity, and a deletion construct lacking this domain exhibited no enzymatic activity. NMR titration experiments confirmed residues Lys(158)-Asn(162) as amino acids that firmly bound heparin. Deletion of a second heparin binding domain sequence (Gln(270)-Lys(280)) yielded an inactive enzyme that failed to interact with cell surface heparan sulfate and hence accumulated in the culture medium of transfected HEK 293 cells to exceptionally high levels. The two heparin/heparan sulfate recognition domains are potentially attractive targets for the development of heparanase inhibitors.     

Characterization of the structure of antithrombin-binding heparan sulfate generated by heparan sulfate 3-O-sulfotransferase 5

The 3-O-sulfation of glucosamine is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). Both heparan sulfate 3-O-sulfotransferase -1 (3-OST-1) and 3-O-sulfotransferase-5 (3-OST-5) transfer sulfate to the 3-OH group of glucosamine to generate antithrombin-binding heparan sulfate (HS(act)). Here, we reported the isolation and characterization of the antithrombin-binding HS oligosaccharides generated by 3-OST-5 (3-OST-5 oligo(act)). (3)H-labeled HS of Chinese hamster ovary cells was exhaustively modified by 3-OST-1 to remove the 3-OST-1 modification sites followed by antithrombin-affinity fractionation. The non-antithrombin-binding fraction of 3-OST-1 pretreated HS was further modified by 3-OST-5 to generate additional antithrombin-binding HS, which was designated as 3-OST-5 HS(act). Structural analysis of 3-OST-5 HS(act) revealed that the antithrombin-binding site of 3-OST-5 HS(act) is located within a domain clustered with N-sulfated glucosamine units. We also isolated 3-OST-5 antithrombin-binding oligosaccharides (3-OST-5 oligo(act)) from high pH nitrous acid degraded 3-OST-5 HS(act). A disaccharide analysis revealed that 3-OST-5 oligo(act) were composed of multiple 3-O-sulfated glucosamine units. Our results provide additional insights on the relationship between the anticoagulant activity and structure of HS.     

A new model for the domain structure of heparan sulfate based on the novel specificity of K5 lyase

Elucidation of the molecular structure of heparan sulfate (HS) is the key to understanding its functional versatility as a co-receptor for growth factors and morphogens. We have identified and exploited the novel substrate specificity of the coliphage K5 lyase in studies of the domain organization of HS. We show that K5 lyase cleaves HS principally within non-sulfated sequences of four or more N-acetylated disaccharides. Uniquely, sections comprising alternating N-acetylated and N-sulfated units are resistant to the enzyme, as are the highly sulfated S domains. Spacing of the K5 lyase cleavage sites ( approximately 7-8 kDa) is similar to that of the S domains. On the basis of these findings, we propose a refined model of the structure of HS in which N-acetylated sequences of four to five disaccharide units (GlcNAc-GlcUA)(4-5) are positioned centrally between the S domains. The latter are embedded within N-acetylated and N-sulfated sequences, forming extended regions of hypervariable sulfation distributed at regular intervals along the polymer chain. K5 lyase provides a means of excision of these composite sulfated regions for structural and functional analyses.     

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase

Cleavage of amyloid precursor protein (APP) by the Alzheimer's beta-secretase (BACE1) is a key step in generating amyloid beta-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with beta-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by alpha-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.     

Cellular adhesion responses to the heparin-binding (HepII) domain of fibronectin require heparan sulfate with specific properties

Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III(12-14) region of fibronectin (HepII domain) through its HS chains. The fine structure of HS is critical to growth factor responses, and whether this extends to matrix ligands is unknown but is suggested from in vitro experiments. Cell attachment to HepII showed that heparin oligosaccharides of >or=14 sugar residues were required for optimal inhibition. The presence of N-sulfated glucosamine in the HS was essential, whereas 2-O-sulfation of uronic acid or 6-O-sulfation of glucosamine had marginal effects. In the more complex response of focal adhesion formation through syndecan-4, N-sulfates were again required and also glucosamine 6-O-sulfate. The significance of polymer N-sulfation and sulfated domains in HS was confirmed by studies with mutant Chinese hamster ovary cells where heparan sulfation was compromised. Finally, focal adhesion formation was absent in fibroblasts synthesizing short HS chains resulting from a gene trap mutation in one of the two major glucosaminoglycan polymerases (EXT1). Several separate, specific properties of cell surface HS are therefore required in cell adhesion responses to the fibronectin HepII domain.     

Involvement of both heparanase and plasminogen activator in lymphoma cell-mediated degradation of heparan sulfate in the subendothelial extracellular matrix

The effect of plasminogen on the ability of highly metastatic ESb mouse lymphoma cells to degrade heparan sulfate (HS) in the subendothelial extracellular matrix (ECM) was studied. A metabolically sulfate-labeled ECM was incubated with the lymphoma cells, and labeled degradation products were analyzed by gel filtration on Sepharose 6B. Heparanase-mediated release of low-Mr (0.5 less than Kav less than 0.85) HS cleavage products was stimulated fourfold in the presence of plasminogen. Incubation of plasminogen alone with the ECM resulted in its conversion into plasmin, which released high-Mr (Kav less than 0.33) labeled proteoglycans from the ECM. Heating the ECM (80 degrees C, 1 hr) abolished its ability to convert plasminogen into plasmin, yet plasminogen stimulated, through its activation by the ESb plasminogen activator, heparanase-mediated release of low-Mr HS fragments. Heparin inhibited both the basal and plasminogen-stimulated degradation of HS side chains but not the total amount of labeled material released from the ECM. In contrast, aprotinin inhibited the plasminogen-stimulated release of high- as well as low-Mr material. In the absence of plasminogen, degradation of heated ECM by ESb cells was completely inhibited by aprotinin, but there was only a partial inhibition of the degradation of native ECM and no effect on the degradation of soluble HS proteoglycan. These results demonstrate that proteolytic activity and heparanase participate synergistically in the sequential degradation of ECM HS and that the ESb proteolytic activity is crucial for this degradation when the ECM-associated protease is inactivated. Plasminogen may serve as a source for the proteolytic activity that produces a more accessible substrate to the heparanase.     

The heparan and heparin metabolism pathway is involved in regulation of fatty acid composition

Six genes involved in the heparan sulfate and heparin metabolism pathway, DSEL (dermatan sulfate epimerase-like), EXTL1 (exostoses (multiple)-like 1), HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), HS6ST3 (heparan sulfate 6-O-sulfotransferase 3), NDST3 (N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3), and SULT1A1 (sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1), were investigated for their associations with muscle lipid composition using cattle as a model organism. Nineteen single nucleotide polymorphisms (SNPs)/multiple nucleotide length polymorphisms (MNLPs) were identified in five of these six genes. Six of these mutations were then genotyped on 246 Wagyu x Limousin F(2) animals, which were measured for 5 carcass, 6 eating quality and 8 fatty acid composition traits. Association analysis revealed that DSEL, EXTL1 and HS6ST1 significantly affected two stearoyl-CoA desaturase activity indices, the amount of conjugated linoleic acid (CLA), and the relative amount of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in skeletal muscle (P<0.05). In particular, HS6ST1 joined our previously reported SCD1 and UQCRC1 genes to form a three gene network for one of the stearoyl-CoA desaturase activity indices. These results provide evidence that genes involved in heparan sulfate and heparin metabolism are also involved in regulation of lipid metabolism in bovine muscle. Whether the SNPs affected heparan sulfate proteoglycan structure is unknown and warrants further investigation.     

Crystal structure and mutational analysis of heparan sulfate 3-O-sulfotransferase isoform 1

Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO(3)) from 3'-phosphoadenosine 5'-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate. This structure reveals residues critical for 3'-phosphoadenosine 5'-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases.     

Heparanase degrades syndecan-1 and perlecan heparan sulfate: functional implications for tumor cell invasion

Heparanase (HPSE-1) is involved in the degradation of both cell-surface and extracellular matrix (ECM) heparan sulfate (HS) in normal and neoplastic tissues. Degradation of heparan sulfate proteoglycans (HSPG) in mammalian cells is dependent upon the enzymatic activity of HPSE-1, an endo-beta-d-glucuronidase, which cleaves HS using a specific endoglycosidic hydrolysis rather than an eliminase type of action. Elevated HPSE-1 levels are associated with metastatic cancers, directly implicating HPSE-1 in tumor progression. The mechanism of HPSE-1 action to promote tumor progression may involve multiple substrates because HS is present on both cell-surface and ECM proteoglycans. However, the specific targets of HPSE-1 action are not known. Of particular interest is the relationship between HPSE-1 and HSPG, known for their involvement in tumor progression. Syndecan-1, an HSPG, is ubiquitously expressed at the cell surface, and its role in cancer progression may depend upon its degradation. Conversely, another HSPG, perlecan, is an important component of basement membranes and ECM, which can promote invasive behavior. Down-regulation of perlecan expression suppresses the invasive behavior of neoplastic cells in vitro and inhibits tumor growth and angiogenesis in vivo. In this work we demonstrate the following. 1) HPSE-1 cleaves HS present on the cell surface of metastatic melanoma cells. 2) HPSE-1 specifically degrades HS chains of purified syndecan-1 or perlecan HS. 3) Syndecan-1 does not directly inhibit HPSE-1 enzymatic activity. 4) The presence of exogenous syndecan-1 inhibits HPSE-1-mediated invasive behavior of melanoma cells by in vitro chemoinvasion assays. 5) Inhibition of HPSE-1-induced invasion requires syndecan-1 HS chains. These results demonstrate that cell-surface syndecan-1 and ECM perlecan are degradative targets of HPSE-1, and syndecan-1 regulates HPSE-1 biological activity. This suggest that expression of syndecan-1 on the melanoma cell surface and its degradation by HPSE-1 are important determinants in the control of tumor cell invasion and metastasis.     

Biosynthesis of 3-O-sulfated heparan sulfate: unique substrate specificity of heparan sulfate 3-O-sulfotransferase isoform 5

Heparan sulfate 3-O-sulfotransferase transfers sulfate to the 3-OH position of a glucosamine to generate 3-O-sulfated heparan sulfate (HS), which is a rare component in HS from natural sources. We previously reported that 3-O- sulfotransferase isoform 5 (3-OST-5) generates both an antithrombin-binding site to exhibit anticoagulant activity and a binding site for herpes simplex virus 1 glycoprotein D to serve as an entry receptor for herpes simplex virus. In this study, we characterize the substrate specificity of 3-OST-5 using the purified enzyme. The enzyme was expressed in insect cells using the baculovirus expression approach and was purified by using heparin-Sepharose and 3',5'-ADP- agarose chromatographies. As expected, the purified enzyme generates both an antithrombin binding site and a glycoprotein D binding site. We isolated IdoUA-AnMan3S and IdoUA-AnMan3S6S from nitrous acid-degraded 3-OST-5-modified HS (pH 1.5), suggesting that 3-OST-5 enzyme sulfates the glucosamine residue that is linked to an iduronic acid residue at the nonreducing end. We also isolated a disaccharide with a structure of DeltaUA2S-GlcNS3S and a tetrasaccharide with a structure of DeltaUA2S-GlcNS-IdoUA2S-GlcNH23S6S from heparin lyases-digested 3-OST-5-modified HS. Our results suggest that 3-OST-5 enzyme sulfates both N-sulfated glucosamine and N-unsubstituted glucosamine residues. Taken together, the results indicate that 3-OST-5 has broader substrate specificity than those of 3-OST-1 and 3-OST-3. The unique substrate specificity of 3-OST-5 serves as an additional tool to study the mechanism for the biosynthesis of biologically active HS.     

Characterization of heparanase from a rat parathyroid cell line

Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-beta-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1-2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.     

Heparan sulfate structure in mice with genetically modified heparan sulfate production

Using a high throughput heparan sulfate (HS) isolation and characterization protocol, we have analyzed HS structure in several tissues from mice/mouse embryos deficient in HS biosynthesis enzymes (N-deacetylase/N-sulfotransferase (NDST)-1, NDST-2, and C5-epimerase, respectively) and in mice lacking syndecan-1. The results have given us new information regarding HS biosynthesis with implications on the role of HS in embryonic development. Our main conclusions are as follows. 1) The HS content, disaccharide composition, and the overall degree of N- and O-sulfation as well as domain organization are characteristic for each individual mouse tissue. 2) Removal of a key biosynthesis enzyme (NDST-1 or C5-epimerase) results in similar structural alterations in all of the tissues analyzed. 3) Essentially no variation in HS tissue structure is detected when individuals of the same genotype are compared. 4) NDST-2, although generally expressed, does not contribute significantly to tissue-specific HS structures. 5) No change in HS structure could be detected in syndecan-1-deficient mice.     

Follicular fluid glycosaminoglycans inhibit degradation of low-density lipoproteins and progesterone production by porcine granulosa cells

Porcine granulosa cells were incubated with commercially available glycosaminoglycans (GAGs) or GAGs purified from porcine follicular fluid to evaluate the effects of GAGs on degradation of low-density lipoproteins (LDL) and progesterone production. Commercially available heparin and chondroitin sulfates (CS) as well as follicular CS and heparan sulfate (HS) inhibited degradation of LDL in dose-dependent manners. Doses of follicular CS and HS required to inhibit 50% of the LDL degradation corresponded to concentrations found in follicular fluid (less than 1 mg/ml). Progesterone production was also inhibited in a dose-dependent fashion by follicular GAGs at concentrations found in follicles. The ability of the follicular GAGs to inhibit degradation of LDL could represent a mechanism by which the utilization of LDL-derived sterol is temporarily restricted following permeabilization of the ovulatory follicle. Follicular GAGs may also modulate utilization of apoprotein E-containing high-density lipoproteins in unruptured follicles.