Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid - A technical performance study.

Objective: To validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples. Design: Synovial fluid from osteoarthritis (OA, n â€‹= â€‹25) and knee-injury (n â€‹= â€‹13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry. Results: Unsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3-12% and 11-19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low. Conclusions: The HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.

Production and HPLC-Based Disaccharide Analysis of Xyloside-Primed Glycosaminoglycans.

Although glycosaminoglycans (GAGs) are known to be involved in a variety of physiological and pathological processes, knowledge about their expression by cells or tissues, the GAGome, is limited. Xylosides can be used to induce the formation of GAGs without the presence of a proteoglycan core protein. The administration of xylosides to living cells tends to result in a considerable amplification in GAG production, and the xylosides can, therefore, be used as analytical tools to study the GAG produced by a certain cell type. One of the most common ways to analyze the GAGs structurally is by disaccharide analysis, which involves depolymerization of the GAGs into disaccharides, fluorescent labeling of the disaccharides with 2-aminoacridone, and quantification using high-pressure liquid chromatography (HPLC). Here, we describe the procedure of producing xyloside-primed GAGs and how to study them structurally by disaccharide analysis.

Assays for Evaluation of Substrates for and Inhibitors of ß-1,4-Galactosyltransferase 7.

ß-1,4-Galactosyltransferase 7 (ß4GalT7) is a key enzyme in the synthesis of two classes of glycosaminoglycans (GAG), i.e., heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS). GAG chains are linear polysaccharides of alternating hexuronic acid and N-acetylhexosamine residues, commonly linked to core proteins to form proteoglycans with important roles in the regulation of a range of biological processes. The biosynthesis of GAGs is initiated by xylosylation of a serine residue of the core protein followed by galactosylation, catalyzed by ß4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl ß-D-xylopyranoside. We have cloned and expressed ß4GalT7, and designed a cell-free assay to measure the activity of this enzyme. The assay employs a 96-well plate format for high throughput. In this chapter, we describe the cloning, expression, and purification of ß4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of ß4GalT7.

Azide-Functionalized Naphthoxyloside as a Tool for Glycosaminoglycan Investigations.

We present a xylosylated naphthoxyloside carrying a terminal azide functionality that can be used for conjugation using click chemistry. We show that this naphthoxyloside serves as a substrate for ß4GalT7 and induces the formation of soluble glycosaminoglycan (GAG) chains with physiologically relevant lengths and sulfation patterns. Finally, we demonstrate its usefulness by conjugation to the Alexa Fluor 647 and TAMRA fluorophores and coupling to a surface plasmon resonance chip for interaction studies with the hepatocyte growth factor known to interact with the GAG heparan sulfate.

Inhibition of iduronic acid biosynthesis by ebselen reduces glycosaminoglycan accumulation in mucopolysaccharidosis type I fibroblasts.

Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.

Harnessing the ECM Microenvironment to Ameliorate Mesenchymal Stromal Cell-Based Therapy in Chronic Lung Diseases.

It is known that the cell environment such as biomechanical properties and extracellular matrix (ECM) composition dictate cell behaviour including migration, proliferation, and differentiation. Important constituents of the microenvironment, including ECM molecules such as proteoglycans and glycosaminoglycans (GAGs), determine events in both embryogenesis and repair of the adult lung. Mesenchymal stromal/stem cells (MSC) have been shown to have immunomodulatory properties and may be potent actors regulating tissue remodelling and regenerative cell responses upon lung injury. Using MSC in cell-based therapy holds promise for treatment of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). However, so far clinical trials with MSCs in COPD have not had a significant impact on disease amelioration nor on IPF, where low cell survival rate and pulmonary retention time are major hurdles to overcome. Research shows that the microenvironment has a profound impact on transplanted MSCs. In our studies on acellular lung tissue slices (lung scaffolds) from IPF patients versus healthy individuals, we see a profound effect on cellular activity, where healthy cells cultured in diseased lung scaffolds adapt and produce proteins further promoting a diseased environment, whereas cells on healthy scaffolds sustain a healthy proteomic profile. Therefore, modulating the environmental context for cell-based therapy may be a potent way to improve treatment using MSCs. In this review, we will describe the importance of the microenvironment for cell-based therapy in chronic lung diseases, how MSC-ECM interactions can affect therapeutic output and describe current progress in the field of cell-based therapy.

The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms.

Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of N-glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.

Chemoenzymatic Synthesis of Glycopeptides Bearing Galactose-Xylose Disaccharide from the Proteoglycan Linkage Region.

Proteoglycans have important biological activities. To improve the overall synthetic efficiency, a new chemoenzymatic route has been established for the proteoglycan linkage region bearing a galactose-xylose disaccharide. The xylosylated glycopeptides were synthesized via solid phase synthesis, which was followed by the addition of the galactose unit by the galactosyl transferase ß4GalT7. This work leads to a better understanding of the acceptor preference of ß4GalT7 and opens the door for expeditious synthesis of the proteoglycan linkage region.

Fluorescently labeled xylosides offer insight into the biosynthetic pathways of glycosaminoglycans.

Five novel xylosides tagged with the fluorescent probe Pacific Blue™ were synthesized and found to act as substrates for ß4GalT7, a bottleneck enzyme in the biosynthetic pathways leading to glycosaminoglycans. By confocal microscopy of A549 cells, we showed that the xylosides were taken up by the cells, but did not enter the Golgi apparatus where most of the glycosaminoglycan biosynthesis occurs. Instead, after a possible double galactosylation by ß4GalT7 and ß3GalT6, the biosynthesis was terminated. We hypothesize this is due to the charge of the fluorescent probe, which is required for fluorescent ability and stability under physiological conditions.

Glycosaminoglycans: A Link Between Development and Regeneration in the Lung.

What can we learn from embryogenesis to increase our understanding of how regeneration of damaged adult lung tissue could be induced in serious lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and asthma? The local tissue niche determines events in both embryogenesis and repair of the adult lung. Important constituents of the niche are extracellular matrix (ECM) molecules, including proteoglycans and glycosaminoglycans (GAGs). GAGs, strategically located in the pericellular and extracellular space, bind developmentally active growth factors (GFs) and morphogens such as fibroblast growth factors (FGFs), transforming growth factor-ß (TGF-ß), and bone morphogenetic proteins (BMPs) aside from cytokines. These interactions affect activities in many cells, including stem cells, important in development and tissue regeneration. Moreover, it is becoming clear that the "inherent code," such as sulfation of disaccharides of GAGs, is a strong determinant of cellular outcome. Sulfation patterns, deacetylations, and epimerizations of GAG chains function as tuning forks in gradient formation of morphogens, growth factors, and cytokines. Learning to tune these fine instruments, that is, interactions between GFs, chemokines, and cytokines with the specific disaccharide code of GAGs in the adult lung, could become the key to unlock inherent regenerative forces to override pathological remodeling. This review aims to provide an overview of the role GAGs play during development and similar events in regenerative efforts in the adult lung.

Recombinant dermatan sulfate is a potent activator of heparin cofactor II-dependent inhibition of thrombin.

The glycosaminoglycan dermatan sulfate (DS) is a well-known activator of heparin cofactor II-dependent inactivation of thrombin. In contrast to heparin, dermatan sulfate has never been prepared recombinantly from material of non-animal origin. Here we report on the enzymatic synthesis of structurally well-defined DS with high anticoagulant activity. Using a microbial K4 polysaccharide and the recombinant enzymes DS-epimerase 1, dermatan 4-O-sulfotransferase 1, uronyl 2-O-sulfotransferase and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, several new glycostructures have been prepared, such as a homogenously sulfated IdoA-GalNAc-4S polymer and its 2-O-, 6-O- and 2,6-O-sulfated derivatives. Importantly, the recombinant highly 2,4-O-sulfated DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. The potential of this method can be extended to preparation of specific structures that are of importance for binding and activation of cytokines, and control of inflammation and metastasis, involving extravasation and migration.

Dermatan sulfate epimerase 1 and dermatan 4-O-sulfotransferase 1 form complexes that generate long epimerized 4-O-sulfated blocks.

During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid-containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid-forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.

Synthesis of Double-Modified Xyloside Analogues for Probing the ß4GalT7 Active Site.

Monosubstituted naphthoxylosides have been shown to function as substrates for, and inhibitors of, the enzyme ß4GalT7, a key enzyme in the biosynthetic pathway leading to glycosaminoglycans and proteoglycans. In this article, we explore the synthesis of 16 xyloside analogues, modified at two different positions, as well as their function as inhibitors of and/or substrates for the enzyme. Seemingly simple compounds turned out to require complex synthetic pathways. A meta-analysis of the synthetic work shows that, regardless of the abundance of methods available for carbohydrate synthesis, even simple modifications can turn out to be problematic, and double modifications present additional challenges due to conformational, steric, and stereoelectronic effects.

Naphthyl Thio- and Carba-xylopyranosides for Exploration of the Active Site of ß-1,4-Galactosyltransferase 7 (ß4GalT7).

Xyloside analogues with substitution of the endocyclic oxygen atom by sulfur or carbon were investigated as substrates for ß-1,4-galactosyltransferase 7 (ß4GalT7), a key enzyme in the biosynthesis of glycosaminoglycan chains. The analogues with an endocyclic sulfur atom proved to be excellent substrates for ß4GalT7, and were galactosylated approximately fifteen times more efficiently than the corresponding xyloside. The 5a-carba-ß-xylopyranoside in the d-configuration proved to be a good substrate for ß4GalT7, whereas the enantiomer in the l-configuration showed no activity. Further investigations by X-ray crystallography, NMR spectroscopy, and molecular modeling provided a rationale for the pronounced activity of the sulfur analogues. Favorable π-π interactions between the 2-naphthyl moiety and a tyrosine side chain of the enzyme were observed for the thio analogues, which open up for the design of efficient GAG primers and inhibitors.

Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.

Determination of 3'-phosphoadenosine-5'-phosphosulfate in cells and Golgi fractions using hydrophilic interaction liquid chromatography-mass spectrometry.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is a key player in the sulfation of biomolecules, but methods for selective measurements are lacking. A liquid chromatography-mass spectrometry (LC-MS) approach for measuring PAPS was developed. A central feature of the method was employing hydrophilic interaction liquid chromatography (HILIC), which is highly suited for separating very polar/charged compounds, and is compatible with electrospray MS. Using simple instrumentation, the analysis time per sample was below 10min and the method was characterized by easy sample preparation. The method was used to monitor decreasing levels of PAPS as function of sodium chlorate treatment (an inhibitor of PAPS synthesis) in whole-cell lysates as well as Golgi-fractions. The method allowed PAPS to be chromatographically separated from ADP and ATP, which can interfere with measurements if a less resolving LC-MS method is used.

The Tyrosine Kinase Inhibitor Imatinib Augments Extracellular Fluid Exchange and Reduces Average Collagen Fibril Diameter in Experimental Carcinoma.

A typical obstacle to cancer therapy is the limited distribution of low molecular weight anticancer drugs within the carcinoma tissue. In experimental carcinoma, imatinib (STI571) increases efficacy of synchronized chemotherapy, reduces tumor interstitial fluid pressure, and increases interstitial fluid volume. STI571 also increases the water-perfusable fraction in metastases from human colorectal adenocarcinomas. Because the mechanism(s) behind these effects have not been fully elucidated, we investigated the hypothesis that STI571 alters specific properties of the stromal extracellular matrix. We analyzed STI571-treated human colorectal KAT-4/HT-29 experimental carcinomas, known to have a well-developed stromal compartment, for solute exchange and glycosaminoglycan content, as well as collagen content, structure, and synthesis. MRI of STI571-treated KAT-4/HT-29 experimental carcinomas showed a significantly increased efficacy in dynamic exchanges of solutes between tumor interstitium and blood. This effect was paralleled by a distinct change of the stromal collagen network architecture, manifested by a decreased average collagen fibril diameter, and increased collagen turnover. The glycosaminoglycan content was unchanged. Furthermore, the apparent effects on the stromal cellular composition were limited to a reduction in an NG2-positive stromal cell population. The current data support the hypothesis that the collagen network architecture influences the dynamic exchanges of solutes between blood and carcinoma tissue. It is conceivable that STI571 reprograms distinct nonvascular stromal cells to produce a looser extracellular matrix, ultimately improving transport characteristics for traditional chemotherapeutic agents. Mol Cancer Ther; 15(10); 2455-64. ©2016 AACR.

Xyloside-primed Chondroitin Sulfate/Dermatan Sulfate from Breast Carcinoma Cells with a Defined Disaccharide Composition Has Cytotoxic Effects in Vitro.

We previously reported that the xyloside 2-(6-hydroxynaphthyl) ß-d-xylopyranoside (XylNapOH), in contrast to 2-naphthyl ß-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.

Disubstituted naphthyl ß-D-xylopyranosides: Synthesis, GAG priming, and histone acetyltransferase (HAT) inhibition.

Xylosides are a group of compounds that can induce glycosaminoglycan (GAG) chain synthesis independently of a proteoglycan core protein. We have previously shown that the xyloside 2-(6-hydroxynaphthyl)ß-D-xylopyranoside has a tumor-selective growth inhibitory effect both in vitro and in vivo, and that the effect in vitro was correlated to a reduction in histone H3 acetylation. In addition, GAG chains have previously been reported to inhibit histone acetyltransferases (HAT). To investigate if xylosides, or the corresponding xyloside-primed GAG chains, can be used as HAT inhibitors, we have synthesized a series of naphthoxylosides carrying structural motifs similar to the aromatic moieties of the known HAT inhibitors garcinol and curcumin, and studied their biological activities. Here, we show that the disubstituted naphthoxylosides induced GAG chain synthesis, and that the ones with at least one free phenolic group exhibited moderate HAT inhibition in vitro, without affecting histone H3 acetylation in cell culture. The xyloside-primed GAG chains, on the other hand, had no effect on HAT activity, possibly explaining why the effect of the xylosides on histone H3 acetylation was absent in cell culture as the xylosides were recruited for GAG chain synthesis. Further investigations are required to find xylosides that are effective HAT inhibitors or xylosides producing GAG chains with HAT inhibitory effects.

Deciphering the Mode of Action of the Processive Polysaccharide Modifying Enzyme Dermatan Sulfate Epimerase 1 by Hydrogen-Deuterium Exchange Mass Spectrometry.

Distinct from template-directed biosynthesis of nucleic acids and proteins, the enzymatic synthesis of heterogeneous polysaccharides is a complex process that is difficult to study using common analytical tools. Therefore, the mode of action and processivity of those enzymes are largely unknown. Dermatan sulfate epimerase 1 (DS-epi1) is the predominant enzyme during the formation of iduronic acid residues in the glycosaminoglycan dermatan sulfate. Using recombinant DS-epi1 as a model enzyme, we describe a tandem mass spectrometry-based method to study the mode of action of polysaccharide processing enzymes. The enzyme action on the substrate was monitored by hydrogen-deuterium exchange mass spectrometry and the sequence information was then fed into mathematical models with two different assumptions of the mode of action for the enzyme: processive reducing end to non-reducing end, and processive non-reducing end to reducing end. Model data was scored by correlation to experimental data and it was found that DS-epi1 attacks its substrate on a random position, followed by a processive mode of modification towards the non-reducing end and that the substrate affinity of the enzyme is negatively affected by each additional epimerization event. It could also be shown that the smallest active substrate was the reducing end uronic acid in a tetrasaccharide and that octasaccharides and longer oligosaccharides were optimal substrates. The method of using tandem mass spectrometry to generate sequence information of the complex enzymatic products in combination with in silico modeling can be potentially applied to study the mode of action of other enzymes involved in polysaccharide biosynthesis.