Analysis of oversulfation in biglycan chondroitin/dermatan sulfate oligosaccharides by chip-based nanoelectrospray ionization multistage mass spectrometry

Biglycan (BGN) is a small proteoglycan that consists of a protein core containing leucine-rich repeat regions and two glycosaminoglycan (GAG) chains of either chondroitin sulfate (CS) or dermatan sulfate (DS) type. The development of novel, highly efficient analytical methods for structural identification of BGN-derived CS/DS motifs, possibly implicated in biological events, is currently the focus of research. In this work, an improved analytical method based on fully automated chip-nanoelectrospray ionization (nanoESI) in conjunction with high-capacity ion trap (HCT) multistage mass spectrometry (MS) by collision-induced dissociation (CID) was for the first time applied to BGN CS/DS oligosaccharide analysis. The CS/DS chains were released from transfected 293 BGN by β-elimination. The chain was digested with AC I lyase, and the resulting mixture was purified and subsequently separated by size exclusion chromatography (SEC). Di- and tetrasaccharide fractions were pooled and characterized in detail using the developed chip-nanoESI protocol. The chip-nanoESI MS profile in the negative ion mode revealed the presence of under-, regularly, and oversulfated species in both di- and tetrasaccharide fractions. CID MS(2)-MS(3) yielded sequence patterns consistent with unusual oversulfated 4,5-Δ-GlcA(2S)-GalNAc(4S) and 4,5-Δ-GlcA(2S)-GalNAc(6S)-IdoA(2S)-GalNAc(6S) motifs.     

The +838 C/G MT2A polymorphism, metals, and the inflammatory/immune response in carotid artery stenosis in elderly people

Carotid artery stenosis (CS) is a well-established risk factor for stroke. Increased proinflammatory chemokines, enhanced metallothionein (MT), and altered metal homeostasis may play roles in atherosclerosis progression and plaque destabilization. MT may sequester zinc during chronic inflammation, provoke zinc deficiency, and modulate NK cell cytotoxicity. A recent investigation of older patients with diabetes and atherosclerosis showed an association between the -209 A/G MT2A polymorphism, CS, and zinc status. In this study, we evaluated the relationship between two MT2A polymorphisms (-209 and + 838 locus), metal status, and inflammatory/immune response in older patients with CS only (the CS1 group) or with CS and previous cerebrovascular episodes (transient ischemic attack or stroke) (the CS2 group). A total of 506 individuals (188 CS1, 100 CS2, and 218 healthy controls) were studied. Atherosclerotic patients (CS1 and CS2) showed increased levels of MT, MCP-1, and RANTES, reduced NK cell cytotoxicity, and altered trace element concentrations (zinc, copper, magnesium, iron). The +838 C/G MT2A polymorphism was differently distributed in CS1 and CS2 patients, who displayed the GG genotype (C-) with significantly higher frequency than elderly controls. C- carriers showed increased MCP-1 and decreased NK cell cytotoxicity, CD56+ cells, and intracellular zinc availability along with decreased zinc, copper, and magnesium content in erythrocytes and increased iron in plasma. C- carriers also showed a major incidence of soft carotid plaques. In conclusion, the +838 C/G MT2A polymorphism seems to influence inflammatory markers, zinc availability, NK cell cytotoxicity, and trace element status, all of which may promote CS development.     

Molecular dynamics simulation of dipalmitoylphosphatidylcholine membrane with cholesterol sulfate

Using the molecular dynamics simulation technique, we studied the changes occurring in a dipalmitoylphosphatidylcholine (DPPC):cholesterol (CH) membrane at 50 mol% sterol when cholesterol is replaced with cholesterol sulfate (CS). Our simulations were performed at constant pressure and temperature on a nanosecond time scale. We found that 1) the area per DPPC:CS heterodimer is greater than the area of the DPPC:CH heterodimer; 2) CS increases ordering of DPPC acyl chains, but to a lesser extent than CH; 3) the number of hydrogen bonds between DPPC and water is decreased in a CS-containing membrane, but CS forms more water hydrogen bonds than CH; and 4) the membrane dipole potential reverses its sign for a DPPC-CS membrane compared to a DPPC-CH bilayer. We also studied the changes occurring in lipid headgroup conformations and determined the location of CS molecules in the membrane. Our results are in good agreement with the data available from experiments.     

Diabetes results in structural alteration of chondroitin sulfate/dermatan sulfate in the rat kidney: effects on the binding to extracellular matrix components

Chondroitin sulfate (CS)/dermatan sulfate (DS) is a group of sulfated polymers, which play an essential role in various biological phenomena. In the kidney, they are present in small but significant amounts. Studies on their structure-function relationship in the kidney and their changes during diabetic conditions have not been rigorously looked into, which is the focus of this paper. The CS/DS content decreased significantly (14%) during diabetic conditions. This was accompanied by a decrease in the CS/heparan sulfate ratio. Disaccharide composition analysis revealed fine structural changes especially with respect to the E unit [glucuronic acid β1-3 N-acetyl d-galactosamine (4,6-O-sulfate)] and the degree of sulfation. The mRNA expression levels of major enzymes involved in the synthesis of the "E"-disaccharide unit showed a decrease during diabetes. The changes in CS/DS had implications on ligand-binding properties when tested in vitro for binding to major extracellular matrix (ECM) components such as type IV collagen, laminin and fibronectin. Thus, this study provides insights into the structure-function relationship of CS/DS in the kidney during diabetes and alterations of which could aggravate conditions such as diabetic nephropathy by virtue of them being a part of ECM components.     

Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates

Circumsporozoite (CS) proteins, which densely coat malaria (Plasmodia) sporozoites, contain an amino acid sequence that is homologous to segments in other proteins which bind specifically to sulfated glycoconjugates. The presence of this homology suggests that sporozoites and CS proteins may also bind sulfated glycoconjugates. To test this hypothesis, recombinant P. yoelii CS protein was examined for binding to sulfated glycoconjugate-Sepharoses. CS protein bound avidly to heparin-, fucoidan-, and dextran sulfate-Sepharose, but bound comparatively poorly to chondroitin sulfate A- or C-Sepharose. CS protein also bound with significantly lower affinity to a heparan sulfate biosynthesis-deficient mutant cell line compared with the wild-type line, consistent with the possibility that the protein also binds to sulfated glycoconjugates on the surfaces of cells. This possibility is consistent with the observation that CS protein binding to hepatocytes, cells invaded by sporozoites during the primary stage of malaria infection, was inhibited by fucoidan, pentosan polysulfate, and heparin. The effects of sulfated glycoconjugates on sporozoite infectivity were also determined. P. berghei sporozoites bound specifically to sulfatide (galactosyl[3-sulfate]beta 1-1ceramide), but not to comparable levels of cholesterol-3-sulfate, or several examples of neutral glycosphingolipids, gangliosides, or phospholipids. Sporozoite invasion into hepatocytes was inhibited by fucoidan, heparin, and dextran sulfate, paralleling the observed binding of CS protein to the corresponding Sepharose derivatives. These sulfated glycoconjugates blocked invasion by inhibiting an event occurring within 3 h of combining sporozoites and hepatocytes. Sporozoite infectivity in mice was significantly inhibited by dextran sulfate 500,000 and fucoidan. Taken together, these data indicate that CS proteins bind selectively to certain sulfated glycoconjugates, that sporozoite infectivity can be inhibited by such compounds, and that invasion of host hepatocytes by sporozoites may involve interactions with these types of compounds.     

Enhancement of tenogenic differentiation of rat tendon-derived stem cells by biglycan

Biglycan (BGN) has been identified as one of the critical components of the tendon-derived stem cells (TDSCs) niche and may be related to tendon formation. However, so far, no study has demonstrated whether the soluble BGN could induce the tenogenic differentiation of TDSCs in vitro. The aim of this study was to investigate the effect of BGN on the tenogenic differentiation of TDSCs. The proliferation and tenogenic differentiation of TDSCs exposed to different concentrations of BGN (0, 50, 100, and 500 ng/ml) were determined by the live/dead cell staining assay, CCK-8 assay, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. The BGN signaling pathway of TDSCs (with and without 50 ng/ml of BGN) was determined by western blot analysis and qRT-PCR analysis. At a concentration of 50 ng/ml, BGN increased the expression of the tenogenic markers THBS-4 and TNMD at both the messenger RNA (mRNA) and protein levels. Meanwhile, 50 ng/ml of BGN inhibited the expression of the chondrogenic and osteogenic markers SOX9, ACN, and RUNX2 at both the mRNA and protein levels. Moreover, BGN (50 ng/ml) affected the expression of the components of the extracellular matrix of TDSCs. Additionally, BGN activated the Smad1/5/8 pathway as indicated by an increase in phosphorylation and demonstrated by inhibition experiments. Upregulation in the gene expression of BMP-associated receptors (BMPRII, ActR-IIa, and BMPR-Ib) and Smad pathway components (Smad4 and 8) was observed. Taken together, BGN regulates tenogenic differentiation of TDSCs via BMP7/Smad1/5/8 pathway and this regulation may provide a basic insight into treating tendon injury.     

Delivery of dermatan sulfate from polyelectrolyte complex-containing alginate composite microspheres for tissue regeneration

Dermatan sulfate (DS) is a glycosaminoglycan (GAG) with a great potential as a new therapeutic agent in tissue engineering. The aim of the present study was to investigate the formation of polyelectrolyte complexes (PECs) between chitosan and dermatan sulfate (CS/DS) and delivery of DS from PEC-containing alginate/chitosan/dermatan sulfate (Alg/CS/DS) microspheres for application in tissue regeneration. The CS/DS complexes were initially formed at different conditions including varying CS/DS ratio (positive/negative charge ratio), buffer, and pH. The obtained CS/DS complexes exhibited stronger electrostatic interaction, smaller complex size, and more stable colloidal structure when chitosan was in large excess (CS/DS 3:1) and prepared at pH 3.5 as compared to pH 5 using acetate buffer. The CS/DS complexes were subsequently incorporated into an alginate matrix by spray drying to form Alg/CS/DS composite microspheres with a DS encapsulation efficiency of 90-95%. The excessive CS induced a higher level of sustained DS release into Tris buffer (pH 7.4) from the microspheres formulated at pH 3.5; however, the amount of CS did not have a significant effect on the release from the microspheres formulated at pH 5. Significant cell proliferation was stimulated by the DS released from the microspheres in vitro. The present results provide a promising drug delivery strategy using PECs for sustained release of DS from microspheres intended for site-specific drug delivery and ultimately for use in tissue engineering.     

Transforming growth factor-beta (TGF-beta) type I receptor/ALK5-dependent activation of the GADD45beta gene mediates the induction of biglycan expression by TGF-beta

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.     

Glycoform quantification of chondroitin/dermatan sulfate using a liquid chromatography-tandem mass spectrometry platform

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, N-acetylgalactosamine disaccharide units {[HexAbeta/alpha(1-3)GalNAcbeta(1-4)](n)()}. CS chains are polydisperse with respect to chain length, sulfate content, and glucuronic acid epimerization content, resulting in a distribution of glycoforms for a chain bound to any given serine residue. Usually, CS glycoforms exist, differing in sulfation position and uronic acid epimerization. This work introduces a novel LC-MS/MS platform for the quantification of mixtures of CS oligosaccharides. The CS polysaccharides were partially depolymerized and labeled with either the light (d(0)) or heavy (d(4)) form of 2-anthranilic acid (2-AA). Excess reagent was removed, and mixtures of the CS standard (d(0)) and unknown (d(4)) were made. The CS mixture was subjected to size exclusion chromatography (SEC) with on-line electrospray ionization mass spectrometric detection in the negative ion mode. Tandem mass spectra were acquired, and quantification of unknown samples within the mixture was made using relative ion abundances of specific diagnostic ions. The high accuracy and precision of the glycomics platform were demonstrated using glycoform mixtures made from standard CS preparations. The CS glycoform analysis method was then applied to cartilage extract, versican, and several dermatan sulfate preparations. This work presents the first application of a glycomics platform for the quantification of CS oligosaccharide mixtures for obtaining specific information about the positions of GalNAc sulfation and uronic acid epimerization.     

The glycosaminoglycan attachment regions of human aggrecan

Aggrecan possesses both chondroitin sulfate (CS) and keratan sulfate (KS) chains attached to its core protein, which reside mainly in the central region of the molecule termed the glycosaminoglycan-attachment region. This region is further subdivided into the KS-rich domain and two adjacent CS-rich domains (CS1 and CS2). The CS1 domain of the human is unique in exhibiting length polymorphism due to a variable number of tandem amino acid repeats. The focus of this work was to determine how length polymorphism affects the structure of the CS1 domain and whether CS and KS chains can coexist in the different glycosaminoglycan-attachment domains. The CS1 domain possesses several amino acid repeat sequences that divide it into three subdomains. Variation in repeat number may occur in any of these domains, with the consequence that CS1 domains of the same length may possess different amino acid sequences. There was no evidence to support the presence of KS in either the CS1 or the CS2 domains nor the presence of CS in the KS-rich domain. The structure of the CS chains was shown to vary between the CS1 and CS2 domains, particularly in the adult, with variation occurring in chain length and the sulfation of the non-reducing terminal N-acetyl galactosamine residue. CS chains in the adult CS2 domain were shorter than those in the CS1 domain and possessed disulfated terminal residues in addition to monosulfated residues. There was, however, no change in the sulfation pattern of the disaccharide repeats in the CS chains from the two domains.     

Inhibitory Effects of Extracellular Matrix Modulator(s) on Lipopolysaccharide (LPS)- and Acidosis-Damaged Glia Containing Cerebellar Granule Cell Cultures

The inhibitory effects of a novel chondroitin sulfate compound on lipopolysaccharide (LPS)- and acidosis-induced neuronal dysfunctions were examined. Cell viabilities in cultured neurons and/or astrocyte-rich cerebellar granule cells were measured by the calcein-AM method. Ten and 20 microg, as a final dosage, of LPS damaged less than 20% cells during a-2 h incubation. More than 5000 ng/ml of chondroitin sulphate-dipalmitoylphosphatidylethanolamine (CS-PE), but not chondroitin sulfate (CS) treatment, significantly inhibited such damage. Twenty microg of LPS damaged more than 40% cells during 24 h incubation, and these cell damages were significantly inhibited by less than 1000 ng/ml of CS-PE. Moreover, treatments with between 5 and 500 ng/ml CS-PE, but not CS, significantly reduced the number of acidosis-damaged cells in a dose-dependent manner. The current results indicate that modulator(s) of ECM and its derivative containing covalently linked dipalmitoylphosphatidylethanolamine show neuroprotective effects under conditions of brain inflammation.     

Structural effects of a covalent linkage between antithrombin and heparin: covalent N-terminus attachment of heparin enhances the maintenance of antithrombin's activated state

We have produced a molecule comprising of permanently-activated covalently linked antithrombin and heparin (ATH). This study was designed to elucidate the covalent linkage point(s) for heparin on antithrombin and conformational properties of the ATH molecule. ATH was produced using Schiff base/Amadori rearrangement by incubating antithrombin with unfractionated heparin for 14 d at 40 degrees C. ATH was then digested using Proteinase K, and the heparin-peptide was reacted with NaIO4/NaBH4/mild acid to degrade the heparin moiety. Sequencing of the remaining peptide was performed by Edman degradation with linkage point confirmation by LC-MS. The degree of insertion of the reactive center loop (RCL) of antithrombin into the A-sheet of ATH was examined using synthesized antithrombin RCL peptides. Binding between the peptides and ATH, and the formation of ATH in the presence of the peptides were tested. CD was used to further examine the secondary and tertiary structures of ATH. The results suggest that heparin is conjugated to the amino terminal of antithrombin in the majority of ATH molecules, proximal to the previously determined heparin binding domain of antithrombin. From the linkage data, a model is proposed for the structure of ATH. Studies using the RCL peptides and CD analysis of ATH support this model.     

Mechanism of inhibition of the prothrombinase complex by a covalent antithrombin-heparin complex

Factor-Xa assembly into the prothrombinase complex decreases its availability for inhibition by antithrombin + unfractionated heparin (AT + UFH). We have developed a novel covalent antithrombin-heparin complex (ATH), with enhanced anticoagulant actions compared with AT + UFH. The present study was performed to extend understanding of the anticoagulant mechanisms of ATH by determining its inhibition of Xa within the critical prothrombinase. Discontinuous inhibition assays were performed to determine final k(2) values for inhibition of Xa. Fluorescent microscopy was conducted to evaluate inhibitor-prothrombinase interactions. The k(2) for inhibition of prothrombinase versus free Xa by AT + UFH was lower, whereas for ATH were much higher. Relative to intact prothrombinase, rates for Xa inhibition by AT + UFH in complexes devoid of prothrombin/vesicles/factor-Va were higher. For ATH, exclusion of prothrombin decreased k(2), removal of vesicles increased k(2) and exclusion of factor-Va gave no effect. While UFH may displace Xa from prothrombinase, Xa is detained within prothrombinase during ATH reactions. We confirm prothrombinase hinders inhibitory action of AT + UFH, whereas ATH is less affected with prothrombin being a key component in the complex responsible for the opposing effects. Overall, the results suggest that covalent linkage between AT-heparin assists access and neutralization of complexed Xa, with concomitant inhibition of prothrombinase function compared with conventional non-conjugated heparin.     

Thermal properties and flammability of polylactide nanocomposites with aluminum trihydrate and organoclay

Biodegradable polylactide (PLA) nanocomposites with aluminum trihydrate (ATH) and modified montmorillonite (MMT) were prepared via direct melt compounding using a twin-screw micro extruder. The exfoliated and intercalated structures of clay in the matrix were observed by TEM and XRD. The thermal oxidative degradation temperature and activation energy of the PLA/ATH/MMT nanocomposite determined by thermogravimetric analysis are higher than that without addition of ATH and organoclay. The incorporation of layered silicates into the PLA/ATH composite results in further stabilization throughout the degradation step. The V-0 rating (UL94 V) of the PLA nanocomposite has been achieved, and the melt dripping was reduced during combustion. Results showed that high loading of the conventional flame retardant ATH yielded brittle PLA composites; however, replacing a portion of the ATH with modified MMT in the PLA matrix improved this result.     

Role of glycosaminoglycans of biglycan in BMP-2 signaling

Recently we have reported that biglycan (BGN) promotes osteoblast differentiation and that this function is due in part to its ability to positively modulate bone morphogenetic protein (BMP) functions. In this study we investigated the role of glycosaminoglycans (GAGs) of BGN in this function using in vitro and in vivo models. C2C12 myogenic cells were treated or untreated with BMP-2 alone or in combination with glycanated, partially glycanated or de-glycanated BGN, and the effects on BMP signaling and function were assessed by Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the effect of de-glycanation of BGN on BMP-2 induced osteogenesis was investigated employing a rat mandible defect model. The defects were filled with collagen scaffolds loaded with glycanated or de-glycanated BGN alone or in combination with a sub-optimal dose of BMP-2 (subBMP). In in vitro experiments, BMP signaling and function were the greatest when BMP-2 was combined with de-glycanated BGN among the groups tested. In the rat mandible experiments, μCT analyses revealed that the newly formed bone was significantly increased only when subBMP was combined with de-glycanated BGN. The data indicate that the GAG component of BGN functions as a suppressor for the BGN-assisted BMP function.     

Cellulose synthesis is coupled to cell cycle progression at G1 in the dinoflagellate Crypthecodinium cohnii

Cellulosic deposition in alveolar vesicles forms the "internal cell wall" in thecated dinoflagellates. The availability of synchronized single cells, the lack of secondary deposition, and the absence of cellulosic cell plates at division facilitate investigation of the possible roles of cellulose synthesis (CS) in the entire cell cycle. Flow cytograms of cellulosic contents revealed a stepwise process of CS in the dinoflagellate cell cycle, with the highest rate occurring at G(1). A cell cycle delay in G(1), but not G(2)/M, was observed after inhibition of CS. A cell cycle inhibitor of G(1)/S, but not G(2)/M, was able to delay cell cycle progression with a corresponding reduction of CS. The increase of cellulose content in the cell cycle corresponded well to the expected increase of surface area. No differences were observed in the cellulose to surface area ratio between normal and fast-growing G(1) cells, implicating the significance of surface area in linking CS to the coupling of cell growth with cell cycle progression. The coupling of CS to G(1) implicates a novel link between CS and cell cycle control, and we postulate that the coupling mechanism might integrate cell wall integrity to the cell size checkpoint.     

Oscillation and regulation of proline content by P5CS and ProDH gene expressions in the light/dark cycles in Arabidopsis thaliana L

The fluctuation of proline content, and protein and mRNA levels of delta1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH), both of which are involved in proline biosynthesis and degradation, in the shoots of Arabidopsis grown in light/dark cycles were demonstrated under salt-stressed and unstressed conditions. Proline content, as well as proteins and mRNAs of these enzymes, clearly oscillated in the light/dark cycles under the stressed and unstressed conditions. A reciprocal relationship between P5CS and ProDH was observed. Protein levels of P5CS and ProDH were well synchronized with their mRNA levels, although the fluctuation of protein levels was not as significant as that of their mRNA levels. Both mRNA and protein levels of the two enzymes as well as the proline content did not oscillate under the continuous light or the dark conditions. Thus, P5CS and ProDH gene expressions seemed to be involved in light irradiation. Moreover, relative water content (RWC) in the plants oscillated in the light/dark cycles. The fluctuations of proline content in shoot reversely responded to that of RWC. It is suggested that the expression of two genes responds sensitively to a subtle change of cellular water status, and accumulated proline keeps the osmotic balance between cells and the outer environment.     

Lysed and disrupted Bifidobacterium bifidum BGN4 cells promote anti-inflammatory activities in lipopolysaccharide-stimulated RAW 264.7 cells

Bifidobacterium bifidum BGN4 has been shown to improve the immune system by regulating interleukin (IL)-6 in RAW 264.7 macrophage cells. In this study, the dead cells of B. bifidum BGN4 were produced by enzymatic and physical processing to enhance the inhibition properties of pro-inflammatory cytokines using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Notably, the secretion levels of cytokines such as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α were decreased by the cell-wall disrupted extracts compared to heat-killed cells. The result suggests that the exposed interior-surface of B. bifidum BGN4 has a potential ability to regulate the immune-responses in the gastrointestinal tract due to major substances in inside-cell wall such as peptidoglycan and teichoic acids. In conclusion, the lysed and disrupted cells from the inside out of B. bifidum BGN4 have anti-inflammatory properties as paraprobiotic agents to control chronic inflammatory related-diseases.