A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis.

Mutants of Chinese hamster ovary cells have been found that no longer produce heparan sulfate. Characterization of one of the mutants, pgsD-677, showed that it lacks both N-acetylglucosaminyl- and glucuronosyltransferase, enzymes required for the polymerization of heparan sulfate chains. pgsD-677 also accumulates 3- to 4-fold more chondroitin sulfate than the wild type. Cell hybrids derived from pgsD-677 and wild type regained both transferase activities and the capacity to synthesize heparan sulfate. Two segregants from one of the hybrids reexpressed the dual enzyme deficiency, the lack of heparan sulfate synthesis, and the enhanced accumulation of chondroitin sulfate, suggesting that all of the traits were genetically linked. These findings indicate that the pgsD locus may represent a gene involved in the coordinate control of glycosaminoglycan formation.     

Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation

Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome.     

The 3-O-sulfation of heparan sulfate modulates protein binding and lyase degradation

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

Heparan sulfates (HSs) are highly sulfated polysaccharides that reside on the surface and in the extracellular matrix of virtually all cells of multicellular organisms (1, 2). A large number of proteins, including blood coagulation factors, growth factors and morphogens, chemokines and cytokines, proteins involved in complement activation, and cell adhesion and signaling proteins can bind to HS, resulting in conformational changes, stabilization of receptor−ligand complexes, protein oligomerization, sequestration, and protection against degradation (3, 4). These molecular recognition events regulate many physiological processes, including embryogenesis, angiogenesis, blood coagulation, and inflammation. These interactions are also important for many disease processes such a cancer, viral and bacterial infections, neurological disorders, and a number of genetic diseases (1–4).The biosynthesis of HS starts with the assembly of a protein-bound polymer composed of alternating N-acetyl glucosamine (GlcNAc) and glucuronic acid (GlcA) residues. Discrete regions of this polymer are modified by N-deacetylase/N-sulfotransferases to replace N-acetyl by N-sulfate moieties. Subsequently, the regions of N-sulfation are further modified by a C-5 epimerase that converts GlcA into iduronic acid (IdoA), followed by O-sulfation by iduronosyl 2-O-sulfotransferase (2-OST), glucosaminyl 6-O-sulfotransferases (6-OST), and 3-O-sulfotransferases (3-OST) (5).HS modifications are often incomplete, resulting in at least 20 different HS disaccharide moieties, which can be combined in different manners, creating considerable structural diversity (4, 6). The way these disaccharides are arranged is not random but dictated by the substrate specificities of the HS biosynthetic enzymes. These enzymes are present in multiple isoforms, each having unique substrate specificity. It has been hypothesized that, by regulating the expression of the isoforms of HS biosynthetic enzymes, cells can create unique HS epitopes (4, 7). The so-called “HS sulfate code hypothesis” is based on the notion that such epitopes can recruit specific HS-binding proteins, thereby mediating multiple biological and disease processes.Vertebrates express seven 3-OST isozymes in a cell- and tissue-selective manner. It is the largest family of HS-modifying enzymes, indicating that, in concert with other sulfotransferases, they have an ability to create unique epitopes for recruitment of specific proteins (8). A prototypic example of a protein requiring binding to 3-O-sulfated HS is antithrombin-III (AT-III) to confer anticoagulant activity. The pentasaccharide GlcNAc6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S has been identified as high-affinity ligand for AT-III (9). Removal of the sulfate at C-3 of N-sulfoglucosamine (GlcNS3S) results in a 10⁵-fold reduction in binding affinity. The context in which the 3-O-sulfate (3-OS) is presented is also important, and removal of any other sulfate leads to a substantial reduction in binding affinity. Glycoprotein D (gD) of HSV-1 is another example of an early discovered protein that binds to HS epitopes having a 3-O-sulfated GlcNS residue (10). The interaction of gD with HS in concert with other viral envelope proteins is critical for triggering fusion with the host cell surface membrane. Although HS oligosaccharides have been used to probe ligand requirements of gD, the optimal carbohydrate sequence remains to be determined. A number of other proteins are known to require a 3-O-sulfated HS epitope for binding and biological activity, and examples include neuropilin-1 (Nrp-1) (11), cyclophilin B (12), stabilin (13), receptor for advanced glycosylation end product (RAGE) (8), and fibroblast growth factor-7 (FGF-7) (14). The context in which the 3-OS group needs to be presented for optimal binding is largely unknown. It is the expectation that many other proteins need a 3-O-sulfoglucosamine moiety for binding and biological activity. In this respect, this modification has been implicated in many physiological and disease processes, including cell differentiation, axon guidance and growth of neurons, inflammation, vascular diseases, and tumor progression, yet HS-binding proteins that are involved in these diseases are often not known (8).There are indications that 3-O-sulfation can interfere in the degradation of HS by heparin lyases (15, 16). These enzymes are critical for the analysis of heparin/HS (Hep/HS) by controlled depolymerization into smaller fragments, which can then be more readily analyzed by various methods including mass spectrometry (MS) (17). Furthermore, a mixture of lyases I, II, and III can digest heparin into disaccharides facilitating compositional analysis (18). Lyases are also employed for the production of low molecular weight heparins (LMWHs) with higher anticoagulant activities and improved pharmacokinetic profiles (19). Treatment of heparin with a mixture of lyases I, II, and III results in the formation of “resistant” trisaccharides and tetrasaccharides that usually contain a 3-OS moiety (15). These studies have been performed with heparin, which is structurally less diverse compared to HS, and, as a result, there is limited knowledge of how a 3-O-sulfation impacts the degradation of HS by lyases (19).The rudimentary understanding of ligand requirements of HS-binding proteins requiring a GlcNS3S moiety is, in part, due to the fact that this modification is relatively rare and difficult to detect and analyze. This is compounded by a lack of robust technologies that can establish the importance of a 3-OS moiety for binding and biological activity. It is the expectation that a sufficiently large collection of synthetic HS oligosaccharides with and without a 3-OS will provide a powerful discovery tool to establish ligand requirements of 3-OS−binding proteins. Such a collection of compounds will also be valuable to define substrate specificities of heparin lyases and other HS-processing enzymes.Although synthetic approaches to prepare HS oligosaccharides have progressed (7, 20), the preparation of a sufficiently large collection of compounds is still challenging, and careful consideration should be given to compounds selection. A literature survey indicates that GlcNS3S moieties can be flanked by different types of uronic acids, and typical sequences include GlcA-GlcNS3S-GlcA (13, 21), IdoA-GlcNS3S-GlcA (22), IdoA2S-GlcNS3S-GlcA (23–25), GlcA-GlcNS3S-IdoA (13, 22, 23), IdoA-GlcNS3S-IdoA (22), IdoA2S-GlcNS3S-IdoA (25), GlcA-GlcNS3S-IdoA2S (13, 23, 26–28), and IdoA2S-GlcNS3S-IdoA2S (24, 29, 30) (Fig. 1A). In these sequences, the GlcNS3S moieties can be further modified by a sulfate ester at C-6. Such a modification is preferentially installed by specific isozymes, and, for example, it is known that 3-OST-1 prefers substrate having 6-OS on GlcNS, whereas 3-OST-3 has a higher activity for compounds with a hydroxyl at this position (24). To corroborate the importance of 3-OS for binding, it is also important to prepare structural counterparts without such a functionality.Open in a separate windowFig. 1.(A) Identified substructures having 3-OS−bearing glucosamine. (B) General structure of target HS hexasaccharides, featuring structurally diverse sequence (shaded pyranose rings), site of sulfation (text in red), uronic acid composition (wavy bond at C-5 carboxylic acid), constant reducing end GlcN and nonreducing end disaccharide, and an anomeric linker for fabrication of HS arrays. (C) Hexasaccharides numbering and backbone composition, variable core trisaccharide in red color; NS, N-sulfate; 2S: 2-OS; 3S, 3-OS; 6S, 6-OS. (D) Disaccharide building blocks comprising acceptors 10 to 12 and donors 13 to 19 for modular assembly of hexasaccharides.Based on these considerations, 27 synthetic HS oligosaccharides were designed based on nine different core trisaccharides encompassing all relevant uronic acid modifications (Fig. 1 B and C). The central GlcNAc moiety of the nine templates is modified by either a 3-OS, 6-OS, or 3,6-OS and extended at the reducing and nonreducing end by GlcNS6S and GlcA-GlcNS6S, respectively to give sufficiently large set of compounds for binding and enzymology studies. It is expected that, after hit identification, the constant regions can be further optimized in a systematic manner to identify the optimal ligand.     

A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells.

Chlamydial attachment to columnar conjunctival or urogenital epithelial cells is an initial and critical step in the pathogenesis of chlamydial mucosal infections. The chlamydial major outer membrane protein (MOMP) has been implicated as a putative chlamydial cytoadhesin; however, direct evidence supporting this hypothesis has not been reported. The function of MOMP as a cytoadhesin was directly investigated by expressing the protein as a fusion with the Escherichia coli maltose binding protein (MBP-MOMP) and studying its interaction with human epithelial cells. The recombinant MBP-MOMP bound specifically to HeLa cells at 4 degrees C but was not internalized after shifting the temperature to 37 degrees C. The MBP-MOMP competitively inhibited the infectivity of viable chlamydiae for epithelial cells, indicating that the MOMP and intact chlamydiae bind the same host receptor. Heparan sulfate markedly reduced binding of the MBP-MOMP to cells, whereas chondroitin sulfate had no effect on binding. Enzymatic treatment of cells with heparitinase but not chondroitinase inhibited the binding of MBP-MOMP. These same treatments were also shown to reduce the infectivity of chlamydiae for epithelial cells. Mutant cell lines defective in heparan sulfate synthesis but not chondroitin sulfate synthesis showed a marked reduction in the binding of MBP-MOMP and were also less susceptible to infection by chlamydiae. Collectively, these findings provide strong evidence that the MOMP functions as a chlamydial cytoadhesin and that heparan sulfate proteoglycans are the host-cell receptors to which the MOMP binds.     

Binding of rat thyroglobulin to heparan sulfate proteoglycans.

We previously showed that rat thyroglobulin (Tg) is a heparin-binding protein and that heparin inhibits Tg binding to megalin (gp330), an endocytic Tg receptor found on the apical surface of thyrocytes. Cooperation between cell surface receptors and heparin-like molecules, namely heparan sulfate proteoglycans (HSPGs), can facilitate cell surface binding of some heparin-binding proteins. Based on our previous findings indicating that heparin and megalin-binding sites of rat Tg are functionally related, here we investigated whether rat Tg binds to HSPGs, which are expressed by thyroid cells. We showed in solid phase assays that unlabeled rat Tg binds to a heparan sulfate (HS) preparation in a dose-dependent, saturable manner, with moderately high affinity (Kd approximately 19 nM, Ki approximately 25 nM). Binding was inhibited by heparin and by HS itself. We then studied the role of HSPGs in Tg binding to FRTL-5 cells, a differentiated Fisher rat thyroid cell line. As previously reported, after incubation of FRTL-5 cells with unlabeled rat Tg at 4 degrees C, heparin released virtually all the cell-bound Tg. Co-incubation of Tg with HS or with a preparation of HSPGs resulted in a reduction of binding by 35%-40%. When FRTL-5 cells were preincubated with heparitinase or heparinase I, which released 20%-30% of cell surface HSPGs, Tg binding was reduced to a similar extent. An antibody against a Tg heparin-binding site functionally related to a major megalin-binding site virtually abolished Tg binding to HS and to FRTL-5 cells, supporting the hypothesis that combined interactions of Tg with HSPGs and with megalin are involved in Tg binding to rat thyroid cells.     

Enzyme interactions in heparan sulfate biosynthesis: Uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo

The formation of heparan sulfate occurs within the lumen of the endoplasmic reticulum-Golgi complex-trans-Golgi network by the concerted action of several glycosyltransferases, an epimerase, and multiple sulfotransferases. In this report, we have examined the location and interaction of tagged forms of five of the biosynthetic enzymes: galactosyltransferase I and glucuronosyltransferase I, required for the formation of the linkage region, and GlcNAc N-deacetylase/N-sulfotransferase 1, uronosyl 5-epimerase, and uronosyl 2-O-sulfotransferase, the first three enzymes involved in the modification of the chains. All of the enzymes colocalized with the medial-Golgi marker alpha-mannosidase II. To study whether any of these enzymes interacted with each other, they were relocated to the endoplasmic reticulum (ER) by replacing their cytoplasmic N-terminal tails with an ER retention signal derived from the cytoplasmic domain of human invariant chain (p33). Relocating either galactosyltransferase I or glucuronosyltransferase I had no effect on the other's location or activity. However, relocating the epimerase to the ER caused a parallel redistribution of the 2-O-sulfotransferase. Transfected epimerase was also located in the ER in a cell mutant lacking the 2-O-sulfotransferase, but moved to the Golgi when the cells were transfected with 2-O-sulfotransferase cDNA. Epimerase activity was depressed in the mutant, but increased upon restoration of 2-O-sulfotransferase, suggesting that their physical association was required for both epimerase stability and translocation to the Golgi. These findings provide in vivo evidence for the formation of complexes among enzymes involved in heparan sulfate biosynthesis. The functional significance of these complexes may relate to the rapidity of heparan sulfate formation.     

Tumor attenuation by combined heparan sulfate and polyamine depletion.

Cells depend on polyamines for growth and their depletion represents a strategy for the treatment of cancer. Polyamines assemble de novo through a pathway sensitive to the inhibitor, alpha-difluoromethylornithine (DFMO). However, the presence of cell-surface heparan sulfate proteoglycans may provide a salvage pathway for uptake of circulating polyamines, thereby sparing cells from the cytostatic effect of DFMO. Here we show that genetic or pharmacologic manipulation of proteoglycan synthesis in the presence of DFMO inhibits cell proliferation in vitro and in vivo. In cell culture, mutant cells lacking heparan sulfate were more sensitive to the growth inhibitory effects of DFMO than wild-type cells or mutant cells transfected with the cDNA for the missing biosynthetic enzyme. Moreover, extracellular polyamines did not restore growth of mutant cells, but completely reversed the inhibitory effect of DFMO in wild-type cells. In a mouse model of experimental metastasis, DFMO provided in the water supply also dramatically diminished seeding and growth of tumor foci in the lungs by heparan sulfate-deficient mutant cells compared with the controls. Wild-type cells also formed tumors less efficiently in mice fed both DFMO and a xylose-based inhibitor of heparan sulfate proteoglycan assembly. The effect seemed to be specific for heparan sulfate, because a different xyloside known to affect only chondroitin sulfate did not inhibit tumor growth. Hence, combined inhibition of heparan sulfate assembly and polyamine synthesis may represent an additional strategy for cancer therapy.     

Characterization of the binding site on heparan sulfate for macrophage inflammatory protein 1alpha

The CC chemokine macrophage inflammatory protein 1alpha (MIP1alpha) is a key regulator of the proliferation and differentiation of hematopoietic progenitor cells. The activity of MIP1alpha appears to be modulated by its binding to heparan sulfate (HS) proteoglycans, ubiquitous components of the mammalian cell surface and extracellular matrix. In this study we show that HS has highest affinity for the dimeric form of MIP1alpha. The predominantly dimeric BB10010 MIP1alpha interacts with an 8.3-kDa sequence in the HS polysaccharide chain, which it protects from degradation by heparinase enzymes. The major structural motif of this HS fragment appears to consist of 2 sulfate-rich S-domains separated by a short central N-acetylated region. The optimum lengths of these S-domains seem to be 12 to 14 saccharides. We propose that this binding fragment may wrap around the MIP1alpha dimer in a horseshoe shape, facilitating the interaction of the S-domains with the heparin-binding domains on each monomer. Molecular modeling suggests that these S-domains are likely to interact with basic residues Arg 17, Arg 45, and Arg 47 and possibly with Lys 44 on MIP1alpha and that the interconnecting N-acetylated region is of sufficient length to allow the 2 S-domains to bind to these sites on opposite faces of the dimer. Elucidation of the structure of the HS-binding site for MIP1alpha may enable us to devise ways of enhancing its myeloprotective or peripheral blood stem cell mobilization properties, which can be used to improve cancer chemotherapy treatments.     

A strategy for rapid sequencing of heparan sulfate and heparin saccharides

Sulfated glycosaminoglycans (GAGs) are linear polysaccharides of repeating disaccharide sequences on which are superimposed highly complex and variable patterns of sulfation, especially in heparan sulfate (HS). HS and the structurally related heparin exert important biological functions, primarily by interacting with proteins and regulating their activities. Evidence is accumulating that these interactions depend on specific saccharide sequences, but the lack of simple, direct techniques for sequencing GAG saccharides has been a major obstacle to progress. We describe how HS and heparin saccharides can be sequenced rapidly by using an integrated strategy with chemical and enzymic steps. Attachment of a reducing-end fluorescent tag establishes a reading frame. Partial selective chemical cleavage at internal N-sulfoglucosamine residues with nitrous acid then creates a set of fragments of defined sizes. Subsequent digestion of these fragments with combinations of exosulfatases and exoglycosidases permits the selective removal of specific sulfates and monosaccharides from their nonreducing ends. PAGE of the products yields a pattern of fluorescent bands from which the saccharide sequence can be read directly. Data are presented on sequencing of heparin tetrasaccharides and hexasaccharides of known structure; these data show the accuracy and versatility of this sequencing strategy. Data also are presented on the application of the strategy to the sequencing of an HS decasaccharide of unknown structure. Application and further development of this sequencing strategy, called integral glycan sequencing, will accelerate progress in defining the structure-activity relationships of these complex GAGs and lead to important insights into their biological functions.     

A single intermolecular contact mediates intramolecular stabilization of both RNA and protein

An arginine-rich peptide from the Jembrana disease virus (JDV) Tat protein is a structural "chameleon" that binds bovine immunodeficiency virus (BIV) or HIV TAR RNAs in two different binding modes, with an affinity for BIV TAR even higher than the cognate BIV peptide. We determined the NMR structure of the JDV Tat-BIV TAR high-affinity complex and found that the C-terminal tyrosine in JDV Tat forms a network of inter- and intramolecular hydrogen bonding and stacking interactions that simultaneously stabilize the beta-hairpin conformation of the peptide and a base triple in the RNA. A neighboring histidine also appears to help stabilize the peptide conformation. Induced fit binding is recurrent in protein-protein and protein-nucleic acid interactions, and the JDV Tat complex demonstrates how high affinity can be achieved not only by optimization of the binding interface but also by inducing new intramolecular contacts that stabilize each binding partner. Comparison to the cognate BIV Tat peptide-TAR complex shows how such a costabilization mechanism can evolve with only small changes to the peptide sequence. In addition, the bound structure of BIV TAR in the chameleon peptide complex is strikingly similar to the bound conformation of HIV TAR, suggesting new strategies for the development of HIV TAR binding molecules.     

The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate

Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses.     

Differential distribution of heparan sulfate glycoforms and elevated expression of heparan sulfate biosynthetic enzyme genes in the brain of mucopolysaccharidosis IIIB mice

The primary pathology in mucopolysaccharidosis (MPS) IIIB is lysosomal storage of heparan sulfate (HS) glycosaminoglycans, leading to complex neuropathology and dysfunction, for which the detailed mechanisms remain unclear. Using antibodies that recognize specific HS glycoforms, we demonstrate differential cell-specific and domain-specific lysosomal HS-GAG distribution in MPS IIIB mouse brain. We also describe a novel neuron-specific brain HS epitope with broad, non-specific increase in the expression in all neurons in MPS IIIB mouse brain, including cerebellar granule neurons, which do not exhibit lysosomal storage pathology. This suggests that biosynthesis of certain HS glycoforms is enhanced throughout the CNS of MPS IIIB mice. Such a conclusion is further supported by demonstration of increased expression of multiple genes encoding enzymes essential in HS biosynthesis, including HS sulfotransferases and epimerases, as well as FGFs, for which HS serves as a co-receptor, in MPS IIIB brain. These data suggest that lysosomal storage of HS may lead to the increase in HS biosyntheses, which may contribute to the neuropathology of MPS IIIB by exacerbating the lysosomal HS storage.     

In vivo fragmentation of heparan sulfate by heparanase overexpression renders mice resistant to amyloid protein A amyloidosis

Amyloid diseases encompass >20 medical disorders that include amyloid protein A (AA) amyloidosis, Alzheimer's disease, and type 2 diabetes. A common feature of these conditions is the selective organ deposition of disease-specific fibrillar proteins, along with the sulfated glycosaminoglycan, heparan sulfate. We have generated transgenic mice that overexpress human heparanase and have tested their susceptibility to amyloid induction. Drastic shortening of heparan sulfate chains was observed in heparanase-overproducing organs, such as liver and kidney. These sites selectively escaped amyloid deposition on experimental induction of inflammation-associated AA amyloidosis, as verified by lack of material staining with Congo Red, as well as lack of associated polysaccharide, whereas the same tissues from control animals were heavily infiltrated with amyloid. By contrast, the spleens of transgenic mice that failed to significantly overexpress heparanase contained heparan sulfate chains similar in size to those of control spleen and remained susceptible to amyloid deposition. Our findings provide direct in vivo evidence that heparan sulfate is essential for the development of amyloid disease.     

Presence of heparan sulfate in the glomerular basement membrane

The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. The findings were the same both in situ and in vitro. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and testicular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation-procedures specific for heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent major components of these sites since the latter are not affected by digestion with neuraminidase and other glycosaminoglycan-specific enzymes. Identical findings were obtained on basement membranes in other locations (Bowman's capsule, tubule epithelium, and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury.     

Drosophila syndecan: conservation of a cell-surface heparan sulfate proteoglycan.

In mammals, cell-surface heparan sulfate is required for the action of basic fibroblast growth factor, fibronectin, antithrombin III, as well as other effectors. The syndecans, a gene family of four transmembrane proteoglycans that participates in these interactions, are the major source of this heparan sulfate. Based on the conserved transmembrane and cytoplasmic domains of the mammalian syndecans, a single syndecan-like gene was detected and localized in the Drosophila genome. As in mammals, Drosophila syndecan is a heparan sulfate proteoglycan expressed at the cell surface that can be shed from cultured cells. The single Drosophila syndecan is expressed in embryonic tissues that correspond with those tissues in mammals that express distinct members of the syndecan family predominantly. Conservation of this class of molecules suggests that Drosophila, like mammals, uses cell-surface heparan sulfate as a receptor or coreceptor for extracellular effector molecules.     

Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan.

The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. This proteoglycan inhibits the thromboplastin-activated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.