On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.     

Structural Properties of the Heparan Sulfate Proteoglycans of Brain

The heparan sulfate proteoglycans present in a deoxycholate extract of rat brain were purified by ion exchange chromatography, affinity chromatography on lipoprotein lipase agarose, and gel filtration. Heparitinase treatment of the heparan sulfate proteoglycan fraction (containing 86% heparan sulfate and 10% chondroitin sulfate) that was eluted from the lipoprotein lipase affinity column with 1 M NaCl led to the appearance of a major protein core with a molecular size of 55,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the effects of heparinase and heparitinase treatment revealed that the heparan sulfate proteoglycans of brain contain a significant proportion of relatively short N-sulfoglucosaminyl 6-O-sulfate [or N-sulfoglucosaminyl](alpha 1-4)iduronosyl 2-O-sulfate(alpha 1-4) repeating units and that the portions of the heparan sulfate chains in the vicinity of the carbohydrate-protein linkage region are characterized by the presence of D-glucuronic acid rather than L-iduronic acid. After chondroitinase treatment of a proteoglycan fraction that contained 62% chondroitin sulfate and 21% heparan sulfate (eluted from lipoprotein lipase with 0.4 M NaCl), the charge and density of a portion of the heparan sulfate-containing proteoglycans decreased significantly. These results indicate that a population of "hybrid" brain proteoglycans exists that contain both chondroitin sulfate and heparan sulfate chains covalently linked to a common protein core.     

Reduced Sulfation of Chondroitin Sulfate but Not Heparan Sulfate in Kidneys of Diabetic db/db Mice

Heparan sulfate proteoglycans are hypothesized to contribute to the filtration barrier in kidney glomeruli and the glycocalyx of endothelial cells. To investigate potential changes in proteoglycans in diabetic kidney, we isolated glycosaminoglycans from kidney cortex from healthy db/+ and diabetic db/db mice. Disaccharide analysis of chondroitin sulfate revealed a significant decrease in the 4-O-sulfated disaccharides (D0a4) from 65% to 40%, whereas 6-O-sulfated disaccharides (D0a6) were reduced from 11% to 6%, with a corresponding increase in unsulfated disaccharides. In contrast, no structural differences were observed in heparan sulfate. Furthermore, no difference was found in the molar amount of glycosaminoglycans, or in the ratio of hyaluronan/heparan sulfate/chondroitin sulfate. Immunohistochemical staining for the heparan sulfate proteoglycan perlecan was similar in both types of material but reduced staining of 4-O-sulfated chondroitin and dermatan was observed in kidney sections from diabetic mice. In support of this, using qRT-PCR, a 53.5% decrease in the expression level of Chst-11 (chondroitin 4-O sulfotransferase) was demonstrated in diabetic kidney. These results suggest that changes in the sulfation of chondroitin need to be addressed in future studies on proteoglycans and kidney function in diabetes.     

Regulation of Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycan Biosynthesis by 4-Fluoro-glucosamine in Murine Airway Smooth Muscle Cells

The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 μm) reduced the quantity (by 64.1–76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K_av shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7–73.0%, and the size showed a K_av shift from 0.30 to 0.50. Treatment of ASM cells with 45 μm and 179 μm 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-β-d-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K_av of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.The synthesis and physical properties (size and charge) of proteoglycans are altered under some pathological conditions such as cancer (1), spinal cord injury (2), atherosclerosis (3), and asthma (4). The importance of these pathological changes in proteoglycans has driven the need to study and design novel chemical tools which can control proteoglycan biosynthesis. Thus, we have studied the effect of a fluorinated analogue of glucosamine on proteoglycan synthesis in murine airway smooth muscle cells.Mono-, di-, and oligosaccharides that contain fluorine have been developed to study the enzymes involved in carbohydrate metabolism, and some of these have been shown to be inhibitors. The atomic size of fluorine is only slightly smaller (van der Waals'' radius (r′) of 135 pm) than that of oxygen (140 pm), and the C-F bond has a higher energy (485 kJ/mol) compared with that of C-O (370 kJ/mol) (5). The substitution of fluorine for oxygen at the 4-position of N-acetylglucosamine (4-F-GlcNAc)² confers a greater electronegativity on the bond and makes it less likely to be removed from the GlcN carbon ring. It is the properties of fluorine that contribute to the unique characteristics of 4-F-GlcNAc.4-F-GlcNAc used for cell culture experiments has O-acetyl groups at several of its ring positions, which in effect increases its cell permeability compared with that of unmodified forms (6). After hydrolysis to remove the O-acetyl residues, 4-F-GlcNAc, like GlcNAc, must be converted to UDP-4-F-GlcNAc, which in turn can be a substrate (or inhibitor) of enzyme reactions that use UDP-GlcNAc. GlcN is typically used as a control compound for 4-F-GlcNAc in vitro because of its superior cell permeability characteristics when compared with acetylated GlcN derivatives. Although acetylated GlcN derivatives enter the cell via passive diffusion, GlcN can enter by both passive diffusion and through the glucose transporter 4 (7).4-F-GlcNAc and 4-F-N-acetylgalactosamine (4-F-GalNAc) have been specifically studied as potential inhibitors of cell growth for the treatment of leukemia. The IC₅₀ values for 4-F-GlcNAc and 4F-GalNAc inhibition of leukemic cell proliferation are 34 and 35 μm, respectively (8). Moreover, by blocking polylactosamine synthesis necessary for elaboration of selectin ligands, 4-F-GlcNAc exhibits anti-inflammatory effects by reducing leukocyte homing to areas of contact allergic hypersensitivity in mice in vivo (9). Beyond effects on cell membrane glycoproteins, it has been proposed that the 4-fluorinated analogue of glucosamine truncates the GlcNAc-hexuronic acid chains on heparan sulfate (HS) by preventing the formation of the normal 1,4-glycosidic linkage between glucuronate (GlcUA) and on the nonreducing end of the growing chain (10). Thus, 4-F-GlcNAc has been suggested as a therapy for reducing amyloid deposition, which can feature HS accumulation (10, 11). Treatment of cultured hepatocytes in vitro with 4-F-GlcNAc and 4F-GalNAc (10–1000 μm) for 24 h reduced [³H]glucosamine and [³⁵S]sulfate incorporation into cellular glycosaminoglycans (11). However, total protein synthesis was also reduced at 1000 μm (11).Although the effects of 4-F-GlcNAc on HS production have been described (10), its effects on other extracellular matrix glycosaminoglycans, chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA), have not been reported.Airway smooth muscle (ASM) cells produce HS- and CS/DS-containing proteoglycans, including perlecan, versican, and decorin (12). Using these cells, we observed that 4F-GlcNAc inhibits CS/DS synthesis nearly as effectively as it inhibits HS synthesis. Although the 4-F on a nonreducing terminal F-GlcNAc-HS chain would block further HS synthesis by preventing the formation of the GlcUAβ1,4 bond required for elongation, the glycosidic bond in CS/DS is β1,3 between hexuronic acid and GalNAc. Thus, UDP-4-F-GlcNAc could not interfere with CS/DS synthesis via the same mechanism because it cannot be 4-epimerized to UDP-4F-GalNAc. Thus, we hypothesized that UDP-4-F-GlcNAc is a potent inhibitor of the 4-epimerase required to convert UDP-GlcNAc to UDP-GalNAc, thereby depleting the cell of UDP-GalNAc, a necessary substrate for CS/DS synthesis. To explore this putative mechanism, we analyzed the inhibitory effects of 4-F-GlcNAc on intrinsic and xyloside-stimulated CS synthesis in ASM cells (13).     

硫酸软骨素对慢性酒精中毒大鼠脑损伤的保护作用

目的:探讨硫酸软骨素对慢性酒精中毒脑损伤的作用及可能机制.方法:雄性 Wistar 大鼠60只随机分为6组,酒精模型组以剂量为8ml·kg-1·d-150%的酒精每天灌胃一次,纳洛酮药物组给予乙醇半小时后腹腔注射纳洛酮0.08mg·k-1·d-1,硫酸软骨素低、中、高剂量干预组在酒精模型组的基础上分别给予硫酸软骨素50、100、150mg·kg-1·d-1,空白对照组给予等体积的蒸馏水,持续2周;第三周把50%的酒精的剂量递增为12mg·kg-1·d-1,持续灌胃6周.在第八周末实验结束后取血,分离血清,留取脑组织.HE染色观察各组大鼠神经细胞的变化.生化测定各组大鼠血清及脑组织匀浆中谷胱甘肽过氧化物酶(GSH-PX)和超氧化物歧化酶(SOD)的活性以及脂质过氧物终未产物丙二醛(MDA);并测定脑皮质中β-内啡肽含量(β-EP).结果:模型组大鼠大脑皮质和海马区神经元数量明显减少,神经细胞排列紊乱.硫酸软骨素中剂量组大鼠大脑皮质和海马区神经细胞排列层次较清晰.与酒精组相比较,硫酸软骨素中剂量组大鼠血清和脑组织匀浆中MDA含量明显降低(P<0.01),脑皮质中β-内啡肽含量明显降低(P<0.01);GSH-PX含量及SOD活性显著升高(P<0.01).结论:硫酸软骨素对大鼠慢性酒精中毒脑损伤具有保护作用.     

Synthetic Heparan Sulfate Oligosaccharides Inhibit Endothelial Cell Functions Essential for Angiogenesis

Background

Heparan sulfate (HS) is an important regulator of the assembly and activity of various angiogenic signalling complexes. However, the significance of precisely defined HS structures in regulating cytokine-dependent angiogenic cellular functions and signalling through receptors regulating angiogenic responses remains unclear. Understanding such structure-activity relationships is important for the rational design of HS fragments that inhibit HS-dependent angiogenic signalling complexes.

Methodology/Principal Findings

We synthesized a series of HS oligosaccharides ranging from 7 to 12 saccharide residues that contained a repeating disaccharide unit consisting of iduronate 2-O-sulfate linked to glucosamine with or without N-sulfate. The ability of oligosaccharides to compete with HS for FGF2 and VEGF₁₆₅ binding significantly increased with oligosaccharide length and sulfation. Correspondingly, the inhibitory potential of oligosaccharides against FGF2- and VEGF₁₆₅-induced endothelial cell responses was greater in longer oligosaccharide species that were comprised of disaccharides bearing both 2-O- and N-sulfation (2SNS). FGF2- and VEGF₁₆₅-induced endothelial cell migration were inhibited by longer 2SNS oligosaccharide species with 2SNS dodecasaccharide activity being comparable to that of receptor tyrosine kinase inhibitors targeting FGFR or VEGFR-2. Moreover, the 2SNS dodecasaccharide ablated FGF2- or VEGF₁₆₅-induced phosphorylation of FAK and assembly of F-actin in peripheral lamellipodia-like structures. In contrast, FGF2-induced endothelial cell proliferation was only moderately inhibited by longer 2SNS oligosaccharides. Inhibition of FGF2- and VEGF₁₆₅-dependent endothelial tube formation strongly correlated with oligosaccharide length and sulfation with 10-mer and 12-mer 2SNS oligosaccharides being the most potent species. FGF2- and VEGF₁₆₅-induced activation of MAPK pathway was inhibited by biologically active oligosaccharides correlating with the specific phosphorylation events in FRS2 and VEGFR-2, respectively.

Conclusion/Significance

These results demonstrate structure-function relationships for synthetic HS saccharides that suppress endothelial cell migration, tube formation and signalling induced by key angiogenic cytokines.     

Chondroitin Sulfate Proteoglycan Tenascin-R Regulates Glutamate Uptake by Adult Brain Astrocytes

In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.     

Isolation and Immunohistochemical Localization of a Chondroitin Sulfate Proteoglycan from Adult Rat Brain

A chondroitin sulfate proteoglycan called PGM1 has been isolated from the particulate fraction of adult rat forebrain. Delipidation of the material, solubilization of proteoglycans in guanidinium chloride, precipitation at low ionic strength, and final extraction at pH 5.0 were used for its isolation. Proteoglycans were subjected to further purification by diethylaminoethyl-cellulose chromatography. Individual components were separated by gel filtration. PGM1 appeared to be a high-molecular-weight chondroitin sulfate proteoglycan, capable of strong interaction with hyaluronic acid. It was finally isolated by gel filtration on Ultrogel AcA 22 in the presence of 4 M guanidinium chloride. Monospecific antibodies obtained in rabbits against the purified molecule did not cross-react with other brain proteoglycans. Immunocytochemical techniques revealed an almost unique association of this compound with axons, particularly those known to contain neurofilaments. However, not all these axons and all parts of these axons contained PGM1. This component was not detectable in liver, intestine, spleen, kidney, lung, heart, skin, hair, lens, and muscle, a finding suggesting a specificity for the nervous tissue. This component is expressed in neural cell cultures. Despite the preservation of the neuronal specificity, it seems to lose its specific axonal localization in vitro.     

Chondroitin Sulfate and Chondroitin/Keratan Sulfate Proteoglycans of Nervous Tissue: Developmental Changes of Neurocan and Phosphacan

Abstract: We have studied developmental changes in the structure and concentration of the hyaluronic acid-binding proteoglycan, neurocan, and of phosphacan, another major chondroitin sulfate proteoglycan of nervous tissue that represents the extracellular domain of a receptor-type protein tyrosine phosphatase. A new monoclonal antibody (designated 1F6), which recognizes an epitope in the N-terminal portion of neurocan, has been used for the isolation of proteolytic processing fragments that occur together with link protein in a complex with hyaluronic acid. Both link protein and two of the neurocan fragments were identified by amino acid sequencing. The N-terminal fragments of neurocan are also recognized by monoclonal antibodies (5C4, 8A4, and 3B1) to epitopes in the G1 and G2 domains of aggrecan and/or in the hyaluronic acid-binding domain of link protein. The presence in brain of these N-terminal fragments is consistent with the developmentally regulated appearance of the C-terminal half of neurocan, which we described previously. We have also used a slot-blot radioimmunoassay to determine the concentrations of neurocan and phosphacan in developing brain. The levels of both proteoglycans increased rapidly during early brain development, but whereas neurocan reached a peak at approximately postnatal day 4 and then declined to below embryonic levels in adult brain, the concentration of phosphacan remained essentially unchanged after postnatal day 12. Keratan sulfate on phosphacan-KS (a glycoform that contains both chondroitin sulfate and keratan sulfate chains) was not detectable until just before birth, and its peak concentration (at 3 weeks postnatal) was reached ～1 week later than that of the phosphacan core protein. Immunocytochemical studies using monoclonal antibodies to keratan sulfate (3H1 and 5D4) together with specific glycosidases (endo-β-galactosidase, keratanase, and keratanase II) also showed that with the exception of some very localized areas, keratan sulfate is generally not present in the embryonic rat CNS.     

Chemoenzymatic Design of Heparan Sulfate Oligosaccharides

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C₅-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.     

Combinatorial Roles of Heparan Sulfate Proteoglycans and Heparan Sulfates in Caenorhabditis elegans Neural Development

Heparan sulfate proteoglycans (HSPGs) play critical roles in the development and adult physiology of all metazoan organisms. Most of the known molecular interactions of HSPGs are attributed to the structurally highly complex heparan sulfate (HS) glycans. However, whether a specific HSPG (such as syndecan) contains HS modifications that differ from another HSPG (such as glypican) has remained largely unresolved. Here, a neural model in C. elegans is used to demonstrate for the first time the relationship between specific HSPGs and HS modifications in a defined biological process in vivo. HSPGs are critical for the migration of hermaphrodite specific neurons (HSNs) as genetic elimination of multiple HSPGs leads to 80% defect of HSN migration. The effects of genetic elimination of HSPGs are additive, suggesting that multiple HSPGs, present in the migrating neuron and in the matrix, act in parallel to support neuron migration. Genetic analyses suggest that syndecan/sdn-1 and HS 6-O-sulfotransferase, hst-6, function in a linear signaling pathway and glypican/lon-2 and HS 2-O-sulfotransferase, hst-2, function together in a pathway that is parallel to sdn-1 and hst-6. These results suggest core protein specific HS modifications that are critical for HSN migration. In C. elegans, the core protein specificity of distinct HS modifications may be in part regulated at the level of tissue specific expression of genes encoding for HSPGs and HS modifying enzymes. Genetic analysis reveals that there is a delicate balance of HS modifications and eliminating one HS modifying enzyme in a compromised genetic background leads to significant changes in the overall phenotype. These findings are of importance with the view of HS as a critical regulator of cell signaling in normal development and disease.     

Chondroitin Sulfate Promotes Activation of Cathepsin K

Cathepsin K (CatK), a major lysosomal collagenase produced by osteoclasts, plays an important role in bone resorption. Evidence exists that the collagenase activity of CatK is promoted by chondroitin sulfate (CS), a sulfated glycosaminoglycan. This study examines the role of CS in facilitating CatK activation. We have demonstrated that chondroitin 4-sulfate (C4-S) promotes autoprocessing of the pro-domain of CatK at pH ≤ 5, leading to a fully matured enzyme with collagenase and peptidase activities. We present evidence to demonstrate this autoactivation process is a trans-activation event that is efficiently inhibited by both the covalent cysteine protease inhibitor E-64 and the reversible selective CatK inhibitor L-006,235. During bone resorption, CatK and C4-S are co-localized at the ruffled border between osteoclast bone interface, supporting the proposal that CatK activation is accomplished through the combined action of the acidic environment together with the presence of a high concentration of C4-S. Formation of a multimeric complex between C4-S and pro-CatK has been speculated to accelerate CatK autoactivation and promote efficient collagen degradation. Together, these results demonstrate that CS plays an important role in contributing to the enhanced efficiency of CatK collagenase activity in vivo.     

Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development

Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.     

Heparan Sulfate Biosynthesis: Methods for Investigation of the Heparanosome

Heparan sulfate is perhaps the most complex polysaccharide known from animals. The basic repeating disaccharide is extensively modified by sulfation and uronic acid epimerization. Despite this, the fine structure of heparan sulfate is remarkably consistent with a particular cell type. This suggests that the synthesis of heparan sulfate is tightly controlled. Although genomics has identified the enzymes involved in glycosaminoglycan synthesis in a number of vertebrates and invertebrates, the regulation of the process is not understood. Moreover, the localization of the various enzymes in the Golgi apparatus has not been carried out in a detailed way using high-resolution microscopy. We have begun this process, using well-known markers for the various Golgi compartments, coupled with the use of characterized antibodies and cDNA expression. Laser scanning confocal microscopy coupled with line scanning provides high-quality resolution of the distribution of enzymes. The EXT2 protein, which when combined as heterodimers with EXT1 comprises the major polymerase in heparan sulfate synthesis, has been studied in depth. All the data are consistent with a cis-Golgi distribution and provide a starting point to establish whether all the enzymes are clustered in a multimolecular complex or are distributed through the various compartments of the Golgi apparatus.     

Role of 6-O-Sulfated Heparan Sulfate in Chronic Renal Fibrosis

Heparan sulfate (HS) plays a crucial role in the fibrosis associated with chronic allograft dysfunction by binding and presenting cytokines and growth factors to their receptors. These interactions critically depend on the distribution of 6-O-sulfated glucosamine residues, which is generated by glucosaminyl-6-O-sulfotransferases (HS6STs) and selectively removed by cell surface HS-6-O-endosulfatases (SULFs). Using human renal allografts we found increased expression of 6-O-sulfated HS domains in tubular epithelial cells during chronic rejection as compared with the controls. Stimulation of renal epithelial cells with TGF-β induced SULF2 expression. To examine the role of 6-O-sulfated HS in the development of fibrosis, we generated stable HS6ST1 and SULF2 overexpressing renal epithelial cells. Compared with mock transfectants, the HS6ST1 transfectants showed significantly increased binding of FGF2 (p = 0.0086) and pERK activation. HS6ST1 transfectants displayed a relative increase in mono-6-O-sulfated disaccharides accompanied by a decrease in iduronic acid 2-O-sulfated disaccharide structures. In contrast, SULF2 transfectants showed significantly reduced FGF2 binding and phosphorylation of ERK. Structural analysis of HS showed about 40% down-regulation in 6-O-sulfation with a parallel increase in iduronic acid mono-2-O-sulfated disaccharides. To assess the relevance of these data in vivo we established a murine model of fibrosis (unilateral ureteric obstruction (UUO)). HS-specific phage display antibodies (HS3A8 and RB4EA12) showed significant increase in 6-O-sulfation in fibrotic kidney compared with the control. These results suggest an important role of 6-O-sulfation in the pathogenesis of fibrosis associated with chronic rejection.     

Identification of Specific Chondroitin Sulfate Species in Cutaneous Autoimmune Disease

Cutaneous lupus erythematosus and dermatomyositis (DM) are chronic inflammatory diseases of the skin with accumulated dermal mucin. Earlier work has shown chondroitin sulfate (CS) accumulation within the dermis of discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), and DM lesions compared with control skin. Immunohistochemistry for C4S revealed a greater density in DLE and DM lesions, whereas SCLE lesions did not differ from controls. Scleredema and scleromyxedema are attributed to increased hyaluronic acid, and lesional samples from these diseases also demonstrated accumulated dermal C4S. Interferon-γ and interleukin-1α, but not interferon-α, treatment of cultured dermal fibroblasts induced mRNA expression of CHST-11, which attaches sulfates to the 4-position of unsulfated chondroitin. These studies on possible CS core proteins revealed that serglycin, known to have C6S side chains in endothelial cells, had greater density within DM dermal endothelia but not in DLE or SCLE, following the pattern of C6S overexpression reported previously. CD44 variants expand the CS binding repertoire of the glycoprotein; CD44v7 co-localized to the distribution of C4S in DLE lesions, a finding not observed in DM, SCLE lesions, or controls. Because C4S and C6S have immunologic effects, their dysregulation in cutaneous mucinoses may contribute to the pathogenesis of these disorders.     

Immunocytochemical Localization of Chondroitin and Chondroitin 4- and 6-Sulfates in Developing Rat Cerebellum

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of Sindbis Virus to Cell Surface Heparan Sulfate

Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.