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.     

The influence of acetaldehyde and glycosaminoglycans upon factor Xa- and factor X-deficient plasma

The comparative effects of glycosaminoglycans and acetaldehyde (AcH)--glycosaminoglycan (GAG) mixtures upon Factor Xa- (FXa) and Factor X-deficient plasma (FXDP) have been studied by activated partial thromboplastin time (APTT) studies. Heparin at 0.025, 0.030, 0.04, and 0.05 U statistically prolonged the APTT when pre-incubated with FXa at 37 degrees C for 3 min prior to addition to FXDP and subsequent addition of Ca2+. Upon addition of 0.25, 0.375, and 0.5 microg heparin-6000 (H6k) to FXa, significant increases in APTT were observed. Similarly, profound increases in APTT were observed when 0.5, 0.75, and 1.0 microg heparin-3000 (H3k) was added to FXa. The chondroitin sulfates (CSA, CSB, CSC) had far less impact upon APTT with the FXa-FXDP system. In examining the effects of AcH-GAG mixtures upon the clotting factor, it was observed that 44.3 and 443 mM AcH synergistically prolonged the APTT in a statistically significant manner regardless of the order of premixing the three components. Hence, AcH may play a role in prolonging APTT in alcoholics. It synergistically prolonged APTT in concert with GAGs and FXa at the AcH levels used in this study. The effect of the GAGs upon FXDP is far less than its effect upon FXa.     

Polyelectrolyte complexes of glycol chitosan with some mucopolysaccharides: Dielectric properties and electrical conductivity

The dielectric properties, DC electrical conductivity, and I-V characteristics of thin films of polyelectrolyte complexes formed by glycol chitosan (GlChi) with mucopolysaccharides chondroitin sulfate A (CSA), chondroitin sulfate C (CSC), and hyaluronic acid (HA) have been studied at different temperatures. The dielectric constant and the loss factor were measured at frequencies ranging from 1 to 100 kHz at all temperatures. The behavior of GlChi–CSA and GlChi–CSC complexes resembles that of the GlChi–dextran sulfate complex reported earlier. The GlChi–HA complex behaves differently and is similar to the GlChi–alginic acid complex reported [Srinivasan, R. & Kamalam, R. (1982) Biopolymers 21 , 251–263, 265–275].     

Characterization of the recombinant rat 175-kDa hyaluronan receptor for endocytosis (HARE)

Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained approximately 25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass approximately 133 kDa) to the 175-kDa HARE at 4 degrees C was 4.1 nm with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 degrees C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 degrees C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.     

Spectroscopic studies of interactions of chondroitin sulfates with cisplatin

Complexation of cisplatin (CDDP) and chondroitin sulfate A (CSA) or C (CSC) has been reported to reduce the nephrotoxicity of CDDP. However the mechanism of interaction between CDDP and CSA or CSC was not known. In this study, spectroscopic analyses including NMR were carried out to examine the complexation interactions of CSA and CSC with CDDP. The time-dependent changes in the UV spectra indicate that CSA and CSC effectively complexes with CDDP in aqueous solution and that the reaction occurs subsequent to the hydrolysis of CDDP. The time-dependent change results measured by capillary electrophoresis showed that complexation of chondroitin sulfate (CS) followed first-order reaction kinetics and that the rate of CDDP hydrolysis in the complexation for both CSA and CSC was the same. These results suggested that the mechanism of complexation was a two-step process with monoaqua formation proved to be the first step, which was also the reaction rate controlling step. Moreover, NMR data suggested that the carboxylic and sulfate groups of CS played an important role in its interaction with CDDP.     

Differentiation of murine B cells induced by chondroitin sulfate B

A two-step culture system was used to investigate the role of chondroitin sulfate (CS) B, which is mitogenic to B cells, in differentiation of B cells. Mouse spleen B cells were incubated for 3 days with CSB in the presence of interleukin (IL)-4 and IL-5. After washing, the cells were replated at 10(5) viable cells/well and recultured without CSB in the presence of IL-4 and IL-5. CSB dose-dependently increased IgM production, the greatest enhancement being 450%. Dextran sulfate had a similar effect, whereas other glycosaminoglycans, CSA, CSC, heparin and hyaluronic acid, were marginally effective. Treatment of B cells with CSB resulted in increases in the number of IgM-secreting cells and numbers of CD138-positive cells and CD45R/B220-negative cells. CSB-induced IgM production was inhibited by the protein kinase C (PKC) inhibitor GF109203X but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. These results demonstrated that CSB promoted differentiation of B cells in the presence of IL-4 and IL-5 and suggested that PKC but not PI3K is crucial for CSB-induced IgM production.     

几种动物肝脏糖胺聚糖的醋酸纤维素薄膜电泳分析

采用酶解和离子交换色谱的方法,从兔、鸡、猪和羊肝组织中提取和纯化得到了糖胺聚糖（GAGs）.通过比较透明质酸（HA）、硫酸软骨素A（CS-A）、硫酸软骨素C（CS-C）、硫酸皮肤素（DS）、肝素（HP）、硫酸乙酰肝素（HS）等标准品在醋酸钡、醋酸锌、吡啶-甲酸等几种不同缓冲体系下的醋酸纤维素薄膜电泳行为,结合灰度积分建立了适合于微量GAGs定性和定量分析的电泳方法.将从不同动物肝脏组织中提取的GAGs运用该方法进行分析,发现 不同动物肝脏组织中,GAG含量和组成均有较大差异：羊肝中GAGs含量最高（0.52 mg/g 组织干粉）,种类也最丰富,含有HA、HS、DS和CS,其中HA所占比例最高;鸡肝中GAGs含量最少（0.18 mg/g组织干粉）,主要含有HA和DS;兔肝GAGs种类与猪肝相似,均含有HA、HS和DS,但HS是猪肝GAGs的主要成分,DS是兔肝GAGs的主要成分.     

Specificity of glycosaminoglycan suppression of endothelin-1 production by human umbilical vein endothelial cells.

Endothelin-1 (ET-1) is the most potent vasoconstrictor peptide found in nature. Its production is stimulated by thrombin. By inhibiting thrombin we have previously shown that heparin, a highly negatively-charged glycosaminoglycan (GAG), suppresses the production of ET-1 by cultured human umbilical vein endothelial cells (HUVEC). The purpose of our study is to determine the effect of other GAGs and related compounds on ET-1 production. The GAGs and related compounds used in the study were: chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, fucoidin, low molecular weight dextran sulfate, high molecular weight dextran sulfate, and hyaluronan. HUVEC were incubated for 48 hr with media containing these GAGs and related compounds and with media without GAG as control. ET-1 levels were measured by radioimmunoassay. GAGs and related molecules with higher sulfate content, heparin, chondroitin sulfate B, low and high molecular weight dextran sulfates significantly suppressed ET-1 production by HUVEC. Fucoidin also suppressed ET-1 production despite its lower sulfate content, probably because of its structural similarity to heparin. These compounds may be useful for future in vivo studies.     

Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, stimulate myoblast proliferation in vitro

Summary Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 μM) of donor concentrations. However, 50 μM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 μM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.     

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.     

The effect of glycosaminoglycans with acetaldehyde on the activation of prothrombin

Heparin17-19k, (25, 50, and 100 ng), heparin6k (50 and 100 ng), heparin3k (50, 100, and 200 microg), chondroitin sulfates B (dermatan sulfate) (0.25, 0.5, and 1.0 microg), C (1 and 10 microg), and A (1 and 10 microg) each prolong the activated partial thromboplastin time (APTT) when preincubated with prothrombin to a greater extent than when preincubated with Factor II-deficient plasma prior to their mixing and subsequent additions of APTT reagent and Ca2+. In all cases statistical significance (p < or = 0.05) was observed except with the 2 lower levels of heparin3k. These results suggest that the glycosaminoglycans (GAGs) may exert a direct effect upon prothrombin (FII) in their anticoagulant activity. Pre mix tures of [(FII/25 ng H17-19k) + 447 mmol acetaldehyde (AcH)/L] as well as [(AcH/H) + FII] and [(FII/AcH) + H] each exert a synergistic anticoagulant effect upon APTT. At low AcH concentrations (44.7 mmol/L), neither a synergistic nor an additive effect is seen. H6k and H3k, on premixing with 447 mmol AcH/L, exhibit an additive effect on APTT prolongation but no synergism. Similarly, premixtures of CSB/447 mmol AcH/L/FII show a greater anticoagulant effect than do [(CSB/AcH) + FII] or [(FII/AcH) + CSB] premixtures. CSC-AcH and CSA-AcH patterns are analogous to those of CSB (DS). These data suggest the possibility that AcH, the primary product of ethanol metabolism, may serve as a crosslinking adduct with proteins, in this case, prothrombin, as well as GAGs. Thus ternary complexes between the zymogen form of coagulation factors, GAGs, and AcH are possible, further influencing coagulopathy.     

Cell surface glycosaminoglycans (GAGs) of cultured chick fibroblasts : Modifications in relation to the stage of embryo development

We have investigated the changes in glycosaminoglycan (GAG) composition between cultured fibroblasts derived from 8- and 16-day chick embryos. GAG composition has been studied after [3H]glucosamine and [35S]sulfate labeling. Both the 8- and 16-day embryo fibroblasts were found to contain hyaluronic acid (HA), dermatan sulfate (DS), heparan sulfate (HS) and chondroitin sulfates (CS), the latter being the major component in 8- and 16-day cells. These four GAGs were quantified after their separation using cellulose acetate electrophoresis. The amounts of HA and CS were respectively shown to increase 2-fold and 4-fold between the 8th and 16th day of development, whereas the amounts of HS and DS resp. diminished 2.5-fold and 1.2-fold. These results show that the relative proportions of the different GAGs alter during embryo development. The fibroblasts from 8-day-old embryos detached more rapidly from the culture dishes than the cells from 16-day-old embryos when treated with trypsin. However, this difference was not directly related to the different GAG content.     

Acceptor specificity of the Pasteurella hyaluronan and chondroitin synthases and production of chimeric glycosaminoglycans

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (approximately 10(2-4) sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.     

Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study

Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.     

Anchorage of collagen-tailed acetylcholinesterase to the extracellular matrix is mediated by heparan sulfate proteoglycans

Heparan sulfate and heparin, two sulfated glycosaminoglycans (GAGs), extracted collagen-tailed acetylcholinesterase (AChE) from the extracellular matrix (ECM) of the electric organ of Discopyge tschudii. The effect of heparan sulfate and heparin was abolished by protamine; other GAGs could not extract the esterase. The solubilization of the asymmetric AChE apparently occurs through the formation of a soluble AChE-GAG complex of 30S. Heparitinase treatment but not chondroitinase ABC treatment of the ECM released asymmetric AChE forms. This provides direct evidence for the vivo interaction between asymmetric AChE and heparan sulfate residues of the ECM. Biochemical analysis of the electric organ ECM showed that sulfated GAGs bound to proteoglycans account for 5% of the total basal lamina. Approximately 20% of the total GAGs were susceptible to heparitinase or nitrous acid oxidation which degrades specifically heparan sulfates, and approximately 80% were susceptible to digestion with chondroitinase ABC, which degrades chondroitin-4 and -6 sulfates and dermatan sulfate. Our experiments provide evidence that asymmetric AChE and carbohydrate components of proteoglycans are associated in the ECM; they also indicate that a heparan sulfate proteoglycan is involved in the anchorage of the collagen-tailed AChE to the synaptic basal lamina.     

Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.     

Specific inhibition of binding of antistasin and [A103,106,108] antistasin 93-119 to sulfatide (Gal(3-SO4)beta 1-1Cer) by glycosaminoglycans.

Leech-derived antistasin is a potent anticoagulant and antimetastatic protein that binds sulfatide (Gal(3-SO4)beta 1-1Cer) and sulfated polysaccharides. In this study, the synthetic fragment [A103,106,108] antistasin 93-119, which corresponds to the carboxyl terminus, showed specific and saturable binding to sulfatide. Binding was competitively blocked by glycosaminoglycans (GAGs) in the order: dextran sulfate 5000 congruent to dextran sulfate 500,000 greater than heparin greater than dermatan sulfate much greater than chondroitin sulfates A and C. This rank order of inhibitory potency was identical to that observed with whole antistasin. We suggest that residues 93-119 of antistasin represent a critical domain for binding GAGs and sulfated glycolipids.     

Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.     

Inhibitory effects of glycosaminoglycans on basal and stimulated transforming growth factor-β1 expression in mesangial cells: biochemical and structural considerations

A number of glycosaminoglycan (GAG) species related to heparin, dermatan sulfate (DeS) and chondroitin sulfate were tested for their ability to interfere with the physiological expression and/or pathological overexpression of the TGF-β1 gene. The influence of the molecular weight, molecular weight distribution, degree of sulfation and location of the sulfate groups was examined in an attempt to unveil fine relationships between structure and activity. The nature of the polysaccharide plays a major part, heparins proving able to inhibit both basal and stimulated TGF-β1 gene expression, DeSs being essentially inactive and chondroitin sulfates only inhibiting stimulated TGF-β1 gene expression. Within this frame, the particular physical and chemical properties of some GAGs appear to further modulate TGF-β1 gene response. Judging from our investigation, chondroitin sulfates seem the most promising for potential pharmacological applications in disorders characterized by fibrogenic TGF-β1 overexpression.     

Glycosaminoglycans of Rat Cerebellum: I. Quantitative Analysis of the Main Constituents at Postnatal Day 6

Abstract: Isolated glycosaminoglycans (GAGs) were quantified biochemically in the cerebella of 6-day-old rats. ¹⁴C-Labeled hyaluronic acid (HA) and chondroitin-4-sulfate (C-4-S), added prior to isolation of GAGs from tissue, served as internal standards to allow correction for unknown losses during the purification procedure and exact quantification of GAGs in the intact tissue. Three main constituents—HA, chondroitin sulfate (CS), and heparan sulfate (HS)—were found at concentrations of 1.82, 1.52, and 0.76 μg/mg protein amounting to 44%, 37%, and 19% of the total GAG fraction, respectively. Incorporation of [³H]glucosamine precursor into GAGs was higher for HS (56% of incorporated precursor) and lower for HA (29%) and CS (15%). The specific activities of individual GAGs were 64.7 nCi/μg for HS, 14.2 for HA, and 8.3 for CS.