Sulfated glycosaminoglycans in protein aggregation diseases

Protein aggregation diseases are characterized by intracellular or extracellular deposition of misfolded and aggregated proteins. These aggregated deposits contain multiple proteinaceous and non-protein components that are thought to play critical roles in the etiology and pathogenesis of protein aggregation diseases in vivo. One of these components, the sulfated glycosaminoglycans (GAGs), includes heparan sulfate, chondroitin sulfate, and keratan sulfate. The sulfated GAGs are negatively charged heteropolysaccharides expressed in all mammalian tissues. Enzymatically generated structural patterns and the degree of sulfation in GAGs determine GAGs’ specific interactions with their protein ligands. Here, we review the potential roles of the sulfated GAGs in the pathogenesis and progression of protein aggregation diseases from a perspective of their sulfation modification. We also discuss the possibility of sulfated GAGs as therapeutic targets for protein aggregation diseases.     

Characterization of proteoglycans synthesized by cultured corneal fibroblasts in response to transforming growth factor beta and fetal calf serum

A culture system was developed to analyze the relationship between proteoglycans and growth factors during corneal injury. Specifically, the effects of transforming growth factor beta-1 (TGF-beta1) and fetal calf serum on proteoglycan synthesis in corneal fibroblasts were examined. Glycosaminoglycan synthesis and sulfation were determined using selective polysaccharidases. Proteoglycan core proteins were analyzed using gel electrophoresis and Western blotting. Cells cultured in 10% dialyzed fetal calf serum exhibited decreased synthesis of more highly sulfated chondroitin sulfate and heparan sulfate compared with cells cultured in 1% dialyzed fetal calf serum. The amount and sulfation of the glycosaminoglycans was not significantly influenced by TGF-beta1. The major proteoglycan species secreted into the media were decorin and perlecan. Decorin was glycanated with chondroitin sulfate. Perlecan was linked to either chondroitin sulfate, heparan sulfate, or both chondroitin sulfate and heparan sulfate. Decorin synthesis was reduced by either TGF-beta1 or serum. At early time points, both TGF-beta1 and serum induced substantial increases in perlecan bearing chondroitin sulfate and/or heparan sulfate chains. In contrast, after extended periods in culture, the amount of perlecan bearing heparan sulfate chains was unaffected by TGF-beta1 and decreased by serum. The levels of perlecan bearing chondroitin sulfate chains were elevated with TGF-beta1 treatment and were decreased with serum. Because both decorin and perlecan bind growth factors and are proposed to modulate their activity, changes in the expression of either of these proteoglycans could substantially affect the cellular response to injury.     

Chemically Oversulfated Glycosaminoglycans Are Potent Modulators of Contact System Activation and Different Cell Signaling Pathways

Contaminated heparin was associated with adverse reactions by activating the contact system. Chemically oversulfated/modified glycosaminoglycans (GAGs) consisting of heparan sulfate, dermatan sulfate, and chondroitin sulfate have been identified as heparin contaminants. Current studies demonstrated that each component of oversulfated GAGs was comparable with oversulfated chondroitin sulfate in activating the contact system. By testing a series of unrelated negatively charged compounds, we found that the contact system recognized negative charges rather than specific chemical structures. We further tested how oversulfated GAGs and contaminated heparins affect different cell signaling pathways. Our data showed that chemically oversulfated GAGs and contaminated heparin had higher activity than the parent compounds and authentic heparin, indicative of sulfation-dominant and GAG sequence-dependent activities in BaF cell-based models of fibroblast growth factor/fibroblast growth factor receptor, glial cell line-derived neurotrophic factor/c-Ret, and hepatocyte growth factor/c-Met signaling. In summary, these data indicate that contaminated heparins intended for blood anticoagulation not only activated the contact system but also modified different GAG-dependent cell signaling pathways.     

Sulf loss influences N-, 2-O-, and 6-O-sulfation of multiple heparan sulfate proteoglycans and modulates fibroblast growth factor signaling

Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes.     

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.     

A glycomic approach to proteoglycan with a two-dimensional polysaccharide chain map

Glycosaminoglycan chains were liberated from proteoglycans (bovine lung, tracheal cartilage, and cerebrum) by successive digestion with actinase and with cellulase from Aspergillus niger, which has endo-beta-xylosidase activity. The glycosaminoglycan chains were fluorescence-labeled with 2-aminopyridine after digestion with Streptomyces hyaluronidase. The resulting pyridylamino-glycosaminoglycans, including heparan sulfate, chondroitin sulfate/dermatan sulfate, and heparin, were separated by high-performance liquid chromatography. Each separated fraction was analyzed by two types of high-performance liquid chromatography: gel-filtration chromatography and anion-exchange chromatography. The correlation between molecular weight and degree of sulfation could be shown on the two-dimensional polysaccharide chain map. Use of a commonly available cellulase with endo-beta-xylosidase activity together with the two-dimensional polysaccharide chain map allows easy analysis of various glycosaminoglycan chains and comprehensive comparison among the structures. These techniques will become useful tools in the further development of glycotechnology and glycome analysis.     

Lacrimal gland development and Fgf10-Fgfr2b signaling are controlled by 2-O- and 6-O-sulfated heparan sulfate

Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Differential binding of platelet-derived growth factor isoforms to glycosaminoglycans

The platelet-derived growth factor (PDGF) family comprises disulfide-bonded dimeric isoforms and plays a key role in the proliferation and migration of mesenchymal cells. Traditionally, it consists of homo- and heterodimers of A and B polypeptide chains that occur as long (A_L and B_L) or short (A_S and B_S) isoforms. Short isoforms lack the basic C-terminal extension that mediates binding to heparin. In the present study, we show that certain PDGF isoforms bind in a specific manner to glycosaminoglycans (GAGs). Experiments performed with wild-type and mutant Chinese hamster ovary cells deficient in the synthesis of GAGs revealed that PDGF long isoforms bind to heparan sulfate and chondroitin sulfate, while PDGF short isoforms only bind to heparan sulfate. This was confirmed by digestion of cell surface GAGs with heparitinase and chondroitinase ABC and by incubation with sodium chloride to prevent GAG sulfation. Furthermore, exogenous GAGs inhibited the binding of long isoforms to the cell membrane more efficiently than that of short isoforms. Additionally, we performed surface plasmon resonance experiments to study the inhibition of PDGF isoforms binding to low molecular weight heparin by GAGs. These experiments showed that PDGF-AA_L and PDGF-BB_S isoforms bound to GAGs with the highest affinity. In conclusion, PDGF activity at the cell surface may depend on the expression of various cellular GAG species.     

Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.     

Immunoelectron microscopic analysis of chondroitin sulfates during calcification in the rat growth plate cartilage

The proximal growth plate cartilage of rat tibia was fixed in the presence of ruthenium hexamine trichloride (RHT) in order to preserve proteoglycans in the tissue. Quantitative changes of chondroitin sulfates during endochondral calcification were investigated by immunoelectron microscopy using mouse monoclonal antibodies 1-B-5, 2-B-6, and 3-B-3, which recognize unsulfated, 4-sulfated, and 6-sulfated chondroitin sulfates, respectively. The content of chondroitin-4-sulfate in the cartilage matrix increased from the proliferative zone to the calcifying zone, while that of unsulfated chondroitin sulfate decreased. Chondroitin-6-sulfate remained constant from the proliferative zone to the upper hypertrophic zone, then decreased in the calcifying zone. The immunoreaction to each antibody increased conspicuously in the cartilagenous core of metaphysial bone trabeculae. The changes of sulfation in chondroitin sulfate chains of proteoglycans may play an important role in inducing and/or promoting calcification in growth plate cartilage.     

Functions of Chondroitin Sulfate and Heparan Sulfate in the Developing Brain

Chondroitin sulfate and heparan sulfate proteoglycans are major components of the cell surface and extracellular matrix in the brain. Both chondroitin sulfate and heparan sulfate are unbranched highly sulfated polysaccharides composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine, and glucuronic acid and N-acetylglucosamine, respectively. During their biosynthesis in the Golgi apparatus, these glycosaminoglycans are highly modified by sulfation and C5 epimerization of glucuronic acid, leading to diverse heterogeneity in structure. Their structures are strictly regulated in a cell type-specific manner during development partly by the expression control of various glycosaminoglycan-modifying enzymes. It has been considered that specific combinations of glycosaminoglycan-modifying enzymes generate specific functional microdomains in the glycosaminoglycan chains, which bind selectively with various growth factors, morphogens, axon guidance molecules and extracellular matrix proteins. Recent studies have begun to reveal that the molecular interactions mediated by such glycosaminoglycan microdomains play critical roles in the various signaling pathways essential for the development of the brain.     

Binding of two growth factor families to separate domains of the proteoglycan betaglycan.

Cell surface proteoglycans help present some polypeptide growth factors such as basic fibroblast growth factor (bFGF) to their receptors and may act as reservoirs for others such as transforming growth factor-beta (TGF-beta). Betaglycan, a cell surface heparan sulfate/chondroitin sulfate proteoglycan that binds TGF-beta via its core protein, is shown here to bind bFGF via its heparan sulfate chains. We investigated the potential for regulation of betaglycan by its ligands in osteoblasts, a system in which bFGF and TGF-beta have complementary effects. We report here that the apparent molecular mass of betaglycan from an osteoblast-enriched primary culture of fetal rat calvaria is decreased in response to bFGF, as detected by an increased electrophoretic migration of betaglycan. The betaglycan forms expressed in bFGF-treated osteoblasts have a reduced content of heparan sulfate GAGs, without detectable changes in the content of chondroitin sulfate GAGs or the size of the core protein. bFGF did not affect the overall population of cell-surface-associated proteins identified by sulfate labeling, which contained primarily heparan sulfate, and had only small effects on the major secreted proteoglycans, which were, by contrast, chondroitin sulfate proteoglycans. The effect of bFGF on betaglycan is therefore a selective one. These results suggest that cells can interact with members of the TGF-beta and FGF families through separate domains of the same membrane proteoglycan, and can selectively regulate the bFGF-binding carbohydrate chains of this proteoglycan in response to bFGF.     

Characterization of a neutrophil cell surface glycosaminoglycan that mediates binding of platelet factor 4

Platelet factor 4 (PF-4) is a platelet-derived alpha-chemokine that binds to and activates human neutrophils to undergo specific functions like exocytosis or adhesion. PF-4 binding has been shown to be independent of interleukin-8 receptors and could be inhibited by soluble chondroitin sulfate type glycosaminoglycans or by pretreatment of cells with chondroitinase ABC. Here we present evidence that surface-expressed neutrophil glycosaminoglycans are of chondroitin sulfate type and that this species binds to the tetrameric form of PF-4. The glycosaminoglycans consist of a single type of chain with an average molecular mass of approximately 23 kDa and are composed of approximately 85-90% chondroitin 4-sulfate disaccharide units type CSA (-->4GlcAbeta1-->3GalNAc(4-O-sulfate)beta1-->) and of approximately 10-15% di-O-sulfated disaccharide units. A major part of these di-O-sulfated disaccharide units are CSE units (-->4GlcAbeta1-->3GalNAc(4,6-O-sulfate)beta1-->). Binding studies revealed that the interaction of chondroitin sulfate with PF-4 required at least 20 monosaccharide units for significant binding. The di-O-sulfated disaccharide units in neutrophil glycosaminoglycans clearly promoted the affinity to PF-4, which showed a Kd approximately 0.8 microM, as the affinities of bovine cartilage chondroitin sulfate A, porcine skin dermatan sulfate, or bovine cartilage chondroitin sulfate C, all consisting exclusively of monosulfated disaccharide units, were found to be 3-5-fold lower. Taken together, our data indicate that chondroitin sulfate chains function as physiologically relevant binding sites for PF-4 on neutrophils and that the affinity of these chains for PF-4 is controlled by their degree of sulfation.     

Cell Surface Glycosaminoglycans As Receptors for Adhesion of Candida Spp. To Corneal Cells

The most common causal agents of fungal keratitis are yeasts of the Candida genus. Adhesion constitutes the first stage of pathogenesis. Previous studies have shown that glycosaminoglycans from the corneal cell surface play an essential role in bacterial keratitis, although little is known about their role in fungal infections. The objective of this work is to analyze the role that glycosaminoglycans (GAGs) play in the adhesion of fungi of the Candida genus to corneal epithelial cells. The participation of GAGs in the adhesion of fungi was studied through the specific inhibition of the synthesis of these molecules by enzymatic digestion using specific lyases and the silencing of various genes involved in heparan sulfate sulfation. The results seem to indicate that glycosaminoglycans act to some extent as receptors for this fungus, although there are differences between fungal species. Treatment with inhibitors partially reduced the adherence of fungal species. Digestion of cell surface heparan sulfate further reduced the adherence of Candida albicans and Candida glabrata compared to chondroitin sulfate, indicating that the binding is preferentially mediated by heparan sulfate. Degradation of both heparan sulfate and chondroitin sulfate produced similar effects on the adherence of Candida parapsilosis. However, adhesion of C. albicans hyphae is not dependent on GAGs, suggesting the expression of other adhesins and the recognition of other receptors present in corneal cells. Our results open the door to new strategies for stopping the adhesion of pathogenic fungi, and their subsequent invasion of the cornea; thus, reducing the probability of the keratitis development.     

Matrix disruptions, growth, and degradation of cartilage with impaired sulfation

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.     

Isolation and identification of the major heparan sulfate proteoglycans in the developing bovine rib growth plate

Heparan sulfate proteoglycans are thought to mediate the action of growth factors. The heparan sulfate-containing proteoglycans in extracts of the bovine fetal rib growth plate were detected using the monoclonal antibody 3G10, which recognizes a neoepitope generated by heparitinase digestion (David, G., Bai, X. M., Van der Schueren, B., Cassiman, J. J., and Van den Berghe, H. (1992) J. Cell Biol. 119, 961-975). The heparan sulfate proteoglycans that react with this antibody were identified using antisera to known proteoglycans; purified using CsCl density gradient centrifugation, molecular sieve, and ion exchange chromatography; and then characterized. The major heparan sulfate proteoglycans in the growth plate had core proteins of 200 kDa and larger and were identified as perlecan and aggrecan. These two heparan sulfate proteoglycans could be effectively separated from each other by CsCl density gradient centrifugation alone. Perlecan contained 25% heparan sulfate and 75% chondroitin sulfate. The heparan sulfate chains on growth plate perlecan were considerably smaller than the chondroitin sulfate chains, and the heparan sulfate disaccharide content was different than that found for heparan sulfate from either kidney, tumor tissue, or growth plate aggrecan. Aggrecan contained only 0.1% heparan sulfate, which was localized to the CS-1 domain of aggrecan. These results indicate that perlecan and aggrecan would be the principal candidate proteoglycans involved in the action of heparan sulfate-binding proteins in the developing growth plate.     

Enzymatic Degradation of GlycosaminogIycans

Abstract

Glycosarninoglycans (GAGs) play an intricate role in the extracellular matrix (ECM), not only as soluble components and polyelectrolytes, but also by specific interactions with growth factors and other transient components of the ECM. Modifications of GAG chains, such as isomerization, sulfation, and acetylation, generate the chemical specificity of GAGs. GAGS can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. As such, the enzymes can serve as tools for studying the physiological effect of residue modifications and as models at the molecular level of protein-GAG recognition. This review examines the structure of the substrates, the properties of enzymatic degradation, and the enzyme substrate-interactions at a molecular level. The primary structure of several GAGS is organized macro-scopicallyby segregation into alternating blocks of specific sulfation patterns and microscopicallyby formation of oligosaccharide sequences with specific binding functions. Among GAGs, considerable dermatan sulfate, heparin and heparan sulfate show conformational flexibility in solution. They elicit sequence-specific interactions with enzymes that degrade them, as well as with other proteins, however, the effect of conformational flexibility on protein-GAG interactions is not clear. Recent findings have established empirical rules of substrate specificity and elucidated molecular mechanisms of enzyme-substrate interactions for enzymes that degrade GAGs. Here we propose that local formation of polysaccharide secondary structure is determined by the immediate sequence environment within the GAG polymer, and that this secondary structure, in turn, governs the binding and catalytic interactions between proteins and GAGs.     

Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells

IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.     

Comparative glycomics of connective tissue glycosaminoglycans

Homeostasis of connective joint tissues depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans, and glycosaminoglycans (GAGs). Isomeric chondroitin sulfate (CS) glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work profiles the CS epitopes expressed by different joint tissues as a function of age and osteoarthritis. GAGs were extracted from joint tissues (cartilage, tendon, ligment, muscle, and synovium) and partially depolymerized using chondroitinase enzymes. The oligosaccharide products were differentially stable isotope labeled by reductive amination using 2-anthranilic acid-d(0) or -d(4) and subjected to amide-hydrophilic interaction chromatography (HILIC) online LC-MS/MS. The analysis presented herein enables simultaneous profiling of the expression of nonreducing end, linker region, and Delta-unsaturated interior oligosaccharide domains of the CS chains among the different joint tissues. The results provide important new information on the changes to the expression of CS GAG chains during disease and development.