Characterization and binding activity of the chonDroitin / dermatan sulphate chain from Endocan, a soluble endothelial Proteoglycan (original) (raw)
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Glycobiology, 2010
Endocan is a recently identified soluble chondroitin/dermatan sulfate (CS/DS) proteoglycan. Synthesized by endothelial cells, it has been found to be over-expressed in the vasculature surrounding a number of tumors, and by promoting growth factor mitogenic activities, hepatocyte growth factor/scatter factor (HGF/SF) in particular, it supports cellular proliferation. In this work, we characterized the glycosaminoglycan (GAG) chain of Endocan, purified either from the naturally producing human umbilical vein endothelial cells (HUVEC) or from a recombinant over-expression system in human embryonic kidney cells (HEK). Compositional analysis using different chondroitinases as well as nuclear magnetic resonance studies revealed that the GAG chains from both sources share many characteristics, with the exception of size (15 and 40 kDa, respectively, for HUVEC and HEK-293 cells). The DS-specific, IdoA-containing disaccharides contribute 30% of the chain (15% of which are 2-O-sulfated) and a...
The Journal of biological chemistry, 2001
Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458-20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)...
PloS one, 2010
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.
The non glycanated endocan polypeptide slows tumor growth by inducing stromal inflammatory reaction
Oncotarget, 2015
Endocan expression is increasingly studied in various human cancers. Experimental evidence showed that human endocan, through its glycan chain, is implicated in various processes of tumor growth. We functionally characterize mouse endocan which is also a chondroitin sulfate proteoglycan but much less glycanated than human endocan. Distant domains from the O-glycanation site, located within exons 1 and 2 determine the glycanation pattern of endocan. In opposite to the human homologue, overexpression of mouse endocan in HT-29 cells delayed the tumor appearance and reduced the tumor growth rate. This tumor growth inhibition is supported by non glycanated form of mouse endocan. Non glycanated human endocan overexpressed in HT-29, A549 or K1000 cells also exhibited an anti-tumor effect. Moreover, systemic delivery of non glycanated human endocan also results in HT-29 tumor growth delay. In vitro, endocan polypeptide did not affect HT-29 cell proliferation, nor cell viability. In tumor ti...
Journal of Biological Chemistry, 2016
We previously reported that the xyloside 2-(6-hydroxynaphthyl) -D-xylopyranoside (XylNapOH), in contrast to 2-naphthyl -D-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo. Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH-and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH-and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells. The cytotoxic effect appeared to be mediated by induction of apoptosis and was inhibited in a concentration-dependent manner by the XylNap-primed heparan sulfate GAGs. In contrast, neither the chondroitin sulfate/ dermatan sulfate nor the heparan sulfate derived from CCD-1095Sk cells primed on XylNapOH or XylNap had any effect on the growth of HCC70 cells or CCD-105Sk cells. These observations were related to the disaccharide composition of the XylNapOH-and XylNap-primed GAGs, which differed between the two cell lines but was similar when the GAGs were derived from the same cell line. To our knowledge this is the first report on cytotoxic effects mediated by chondroitin sulfate/dermatan sulfate. Proteoglycans (PGs) 2 are macromolecules located in the extracellular matrix, associated to the cell surface, or stored in secretory granules of essentially all mammalian cells where they are involved in a variety of biological processes ranging from cellular homeostasis to development and progression of several pathological conditions such as cancer and inflammation (1). PGs consist of a core protein to which one or more linear polysaccharides, glycosaminoglycans (GAGs), are covalently linked. Chondroitin sulfate/dermatan sulfate (CS/DS) and heparin/ heparan sulfate (HS) are two classes of GAGs, which are O-linked by xylose to a serine residue of the PG core protein (2, 3). They are composed of alternating N-acetyl-D-galactosamine-uronic acid (GalNAc-UA; where UA is either D-glucuronic acid (GlcUA) or L-iduronic acid (IdoUA)) or N-acetyl-Dglucosamine (GlcNAc)-UA units, respectively. The complete CS/DS and HS chains typically result from extensive processing by class-specific epimerases and sulfotransferases. GlcUA of CS/DS and HS can be epimerized to IdoUA, but complete epimerization is rarely observed; instead, copolymers of GlcUA and IdoUA disaccharides are common. CS/DS can be O-sulfated at position 2 of GlcUA or IdoUA, and at position 4 and 6 of GalNAc. HS can be N-deacetylated/N-sulfated, O-sulfated at position 2 of GlcUA and IdoUA, at position 6 of GlcNAc and, more rarely, at position 3 of GlcNAc. Heparin is generally the more modified version of HS. GAGs have an immense structural diversity and have been reported to interact with a broad spectrum of biomolecules such as growth factors, selectins, and receptors involved in signaling pathways (1, 4-6). In addition, tumor cells are often associated with abnormalities in GAG expression such as overor undersulfation or altered chain size (7-9). Because these interactions are often highly dependent on GAG sequence, GAGs are potentially important as diagnostic and therapeutic tools. -D-Xylopyranosides, commonly referred to as xylosides, comprise a group of compounds consisting of a xylose residue in -linkage to an aglycon (10). They can act as substrates for GAG synthesis and can, when exogenously supplied to cells, result in secreted xyloside-primed GAGs as well as in alterations of the endogenous PG expression. Depending on the aglycon structure and the cell type, they may induce synthesis of GAGs with different HS/CS/DS composition. Besides being excellent tools to study the effect of GAGs and PGs on cells, xylosides have been described to affect, for example, coagulation (11), skin regeneration (12), and cell morphology (13, 14) as well as endothelial tube formation (15).
European Journal of Biochemistry, 1987
A predominant species of heparan sulfate proteoglycan that consisted of at least two subunits linked by disulfide bonding was isolated from cell layers of normal ('cobblestone') bovine vascular endothelial cells in culture. Treatment of the parent molecules with dithiothreitol caused their complete cleavage and permitted the subsequent separation of the larger and smaller subunits on Sepharose CL4B columns. Removal of dithiothreitol by dialysis resulted in the reformation of large disulfide-bonded molecules but such recombination of the subunits was prevented by prior reductive alkylation using iodoacetamide. Buoyant density gradient analysis as well as gel chromatography on Sepharose CL-6B columns, following alkaline borohydride and nitrous acid treatment of individual carbohydrate-rich subunits, showed that the latter consisted of core proteins associated solely with heparan sulfate glycosaminoglycans. The sizes of the latter were estimated by chromatographic techniques to be approximately 50 kDa in the case of the larger and 14 kDa for the smaller subunit. This is the first description of disulfide-bonded proteoheparan sulfates in endothelial cells.
Galactosaminoglycan Function and Oligosaccharide Structure Determination
The Scientific World JOURNAL, 2007
This review will discuss the importance of sequencing long chondroitin sulfate and dermatan sulfate chains specifically derived from decorin. Decorin is a member of the small leucine-rich repeat proteoglycans and ubiquitously expressed primarily in the skin. Sequence information and diverse function of glycosaminoglycans is further influenced by variable expression through the core protein indicating the importance to analyse glycosaminoglycans from specific proteoglycans.
Inhibition of Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis
Journal of Biological Chemistry, 2008
Proteoglycans (PGs) are composed of a protein moiety and a complex glycosaminoglycan (GAG) polysaccharide moiety. GAG chains are responsible for various biological activities. GAG chains are covalently attached to serine residues of the core protein. The first step in PG biosynthesis is xylosylation of certain serine residues of the core protein. A specific linker tetrasaccharide is then assembled and serves as an acceptor for elongation of GAG chains. If the production of endogenous GAG chains is selectively inhibited, one could determine the role of these endogenous molecules in physiological and developmental functions in a spatiotemporal manner. Biosynthesis of PGs is often blocked with the aid of nonspecific agents such as chlorate, a bleaching agent, and brefeldin A, a fungal metabolite, to elucidate the biological roles of GAG chains. Unfortunately, these agents are highly lethal to model organisms. Xylosides are known to prime GAG chains. Therefore, we hypothesized that modified xylose analogs may able to inhibit the biosynthesis of PGs. To test this, we synthesized a library of novel 4-deoxy-4fluoroxylosides with various aglycones using click chemistry and examined each for its ability to inhibit heparan sulfate and chondroitin sulfate using Chinese hamster ovary cells as a model cellular system.
Glycoconjugate Journal, 2006
Neoglycolipid technology is eminently adaptable for microarray design for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Dermatan sulfate (DS) is known to play an important role because of its ability to bind growth factors as well as chemokines and to modulate their biological activities during inflammation and response to injury. We prepared various iduronic acid-rich fragments from DS by complete digestion with chondroitinase ACI, and investigated whether the DS-binding proteins, such as HGF/SF, RANTES, KGF/FGF-7 and HCII, can detect their oligosaccharide ligands in a neoglycolipid microarray. First, a comparison of the intensity of binding signals obtained from chondroitin oligosaccharides with those of heparin oligosaccharides showed that our microarray system is feasible not only to single-out the oligosaccharide ligands, but also to detect the difference between an intrinsic interaction unrelated only to electrostatic interaction and non-specific electrostatic interaction. Second, HGF/SF, KGF/FGF-7 and HCII showed preferential binding to iduronic acid-rich fragments of DS oligosaccharides that are greater than 8-mers in lengths. In contrast, RANTES binding seemed to depend only on the negative charges; their binding intensity towards the DS oligosaccharides was somewhat stronger than the binding of HGF/SF, KGF/FGF-7 and HCII. Third, the use of polyvinylpyrrolidone-40 (PVP-40), ovalbumin (OV) and Tween 20 in place of BSA as a blotting agent was useful in these glycosaminoglycan dependent reactions to minimize background due to non-specific interactions.