| 1. |
- Andersson, Elin, et al.
(författare)
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Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study
- 2023
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Ingår i: Osteoarthritis and Cartilage Open. - 2665-9131. ; 5:3
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Tidskriftsartikel (refereegranskat)abstract
- 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 = 25) and knee-injury (n = 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.
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| 2. |
- Andersson, Elin, et al.
(författare)
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Quantification of chondroitin sulfates and hyaluronan in synovial fluid using high performance liquid chromatography
- 2022
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Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 30:Suppl 1, s. 106-106
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Konferensbidrag (refereegranskat)abstract
- Purpose: Extracellular proteins such as aggrecan may be primed with specific glycan-patterns which result in their degradation, and hence may play a role in the pathogenesis of osteoarthritis (OA). To be able to use glycans as molecular biomarkers, the method of analysis of these molecules needs to be validated. The primary aim of this study was to validate quantitative high performance liquid chromatography (HPLC) of chondroitin sulfate (CS) and hyaluronan (HA) in synovial fluid samples. The secondary aim was to examine the glycan-pattern in different subject groups, and the correlation between age and the concentration of specific glycans.Methods: OA (n=25, age=36-86 years, 40% women) and recent knee injury patients (0-5 days from injury; n=13, age=36-64 years, 46% women) were selected from a cross-sectional convenience cohort. Individual synovial fluid samples, a synovial fluid pool (SF-control; n=7) and a CS quality control sample (CS-QC; Sigma #C2905) were digested with chondroitinase ABC overnight. Samples and glycan standards (CS [n=8] and HA [n=1] standards from Iduron) were labelled with 2-aminoacridone (AMAC) and analyzed using a quantitative HPLC assay. Aggrecan from synovial fluid samples (n=6) was purified using density centrifugation (D1 mini-prep). Sulfated glycosaminoglycans (sGAG) were quantified using Alcian blue precipitation. Since the CS, HA and sGAG data were not normally distributed, non-parametric analyses for group comparisons were done. P-values less than 0.05 were considered statistically significant.Results: Synovial fluid samples were digested with varying concentrations of chondroitinase ABC and analyzed with the HPLC assay; 5 mU chondroitinase ABC per μg sGAG gave the highest CS- and HA-signals and were chosen for the rest of the study (data not shown).The CS profiles in synovial fluid and on aggrecan purified from corresponding synovial fluids were assessed from six knee injury patients. Of the six CS-markers that were detected, uronic acid (UA)-N-acetylgalactosamine (GalNAc) was only present in aggrecan samples, while UA2S-GalNAc and UA2S-GalNAc6S were found only in the synovial fluids. Similar proportions of UA-GalNAc4S and UA-GalNAc6S were found in synovial fluids and aggrecan samples (Figure 1), and these CS-glycans accounted for 95% of all glycans in the SF-control sample (Table 1).The technical performance of CS- and HA-markers using HPLC-assay were evaluated (Table 1). Of the nine markers, five were present in the majority of the synovial fluid samples (N=20-38) and included in the investigation of the technical performance. The mean intra coefficient of variation (CV) for the synovial fluid samples was between 1.2 and 12.9%. For the SF-control sample, the mean intra CV was 3.3-12.1% and the inter CV was 11.0-18.5%. For the CS-QC sample, the mean intra CV was 2.5-9.5% and the mean inter CV was 3.3-31.7%. For the glycan standards, the mean intra CV was 0.2-7.0%. With dilution of the SF-control sample up to 1:10, the dilution recovery rate for the five CS- and HA-markers was mainly between 75 and 125%.The synovial fluid concentration of biomarkers UA-GalNAc6S and UA2s-GalNAc6s and sGAG were approximately 2 to 3 times higher for the recent injury group compared to the age-matched OA group, while the HA levels were 3.7 times lower for the recent injury group (data not shown). No difference in biomarker concentrations were found between the sexes in any of the patient groups (data not shown). For correlation assessments, the two patient groups were merged (total N=38, assessment N = between 20 and 38). Synovial fluid concentrations of HA and UA-GalNAc4S,6S correlated positively with age (rS=0.420 and 0.532, respectively) while UA-GalNAc6S and sGAG correlated negatively with age (rS=-0.333 and -0.528, respectively). HA correlated negatively with UA-GalNAc6S (rS=-0.462) and sGAG (rS=-0.472) and positively with UA-GalNAc4S,6S (rS=0.868).Conclusions: The technical performance of the HPLC-assay indicates that the method is suitable for analyzing CS and HA markers in synovial fluid samples. Our results suggest that: the vast majority of CS in synovial fluid derives from aggrecan, the glycan pattern differs between OA and knee injured subjects and that the concentrations of some of the CS-markers seem to be associated with HA and age.
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| 3. |
- Andersson, Elin, et al.
(författare)
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Quantification Of Glycosaminoglycans In Knee Synovial Fluid From Different Patient Groups And Knee-Healthy Subjects Using High Performance Liquid Chromatography
- 2023
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Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 31:Suppl 1, s. 109-109
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Konferensbidrag (refereegranskat)abstract
- Purpose: The glycosaminoglycans chondroitin sulfate (CS) and hyaluronic acid (HA) are important for the normal function of articular cartilage, and changes of their sulfation and concentration may play a role in the pathogenesis of osteoarthritis (OA). Therefore, these glycans may be clinically useful as biomarkers in OA management. The purpose of this study was to analyze the CS and HA pattern in knee synovial fluid from different subject groups.Methods: OA patients (n=20, age=34-75 years, 45% women), recently knee-injured patients (0-77 days from injury, n=46, age 15-64 years, 15% women), previously knee-injured patients (88 days-21 years from injury, n=30, age 25-65 years, 17% women) and knee-healthy subjects (n=22, age 17-48 years, 23% women) were selected from a cross-sectional convenience cohort. CS and HA in individual synovial fluid samples, a synovial fluid quality control sample (SF-QC; a pool of synovial fluids) and a CS quality control sample (CS-QC) were digested with chondroitinase ABC and glucose oxidase overnight. Samples and glycan standards (CS [n=6] and HA [n=1] standards) were labelled with 2-aminoacridone (AMAC) and analyzed using a quantitative high performance liquid chromatography (HPLC) assay. In total, 118 synovial fluid samples were run, whereof 34 in duplicates. SF-QC, CS-QC and standards were run in duplicates.Since the CS and HA data were not normally distributed, non-parametric analyses for group comparisons were done (Student’s T-test for age analysis, Chi-square test for sex analysis and Mann-Whitney U test for CS and HA analysis). The significance level was set at pResults: HPLC assay validation: The intra experiment coefficient of variation (CV) for the synovial fluid samples (n=36; including SF-QC) was 0.03-36.9% (median 5.4%) for CS and 0.4-44.9% (median 7.6%) for HA; intra CV for the CS-QC sample was 0.01-3.7%, and for the standards it was 0.2-6.7% for CS and 1.9-7.1% for HA. The inter experiment CV for SF-QC (n=5 experiments) was 9.8-17.5% for CS and 15.1% for HA; the inter CV for CS-QC (n=4 experiments) was 3.4-6.1%, and for the standards (n=5 experiments) it was 0.01-0.07% for CS and 0.1% for HA. Glucose oxidase was added to remove glucose that otherwise co-elutes with non-sulfated CS; it did not affect the CS and HA standards (data not shown). Group comparisons: Comparisons were made between the knee-healthy control group and the OA, recent injury and previous injury groups respectively, as well as between the recent injury and previous injury groups. There was no difference in sex between either of the groups (p=0.074-0.866). There was no difference in age between the knee-healthy group and the recent injury group (p=0.330), but the knee-healthy group was younger than the previous injury group and the OA group (pConclusions: Our data suggests that the groups with knee pathologies have higher concentrations of some CS variants and HA than the knee-healthy group. We also see a trend of higher levels of CS variants in the recent knee injury group compared to the previous knee injury group. This indicates that there is both an acute and chronic increase in the concentrations of CS variants and HA in synovial fluid following knee injury and/or cartilage damage.
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| 4. |
- Andersson Sjöland, Annika, et al.
(författare)
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Versican in inflammation and tissue remodelling: the impact on lung disorders.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:3, s. 243-251
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Forskningsöversikt (refereegranskat)abstract
- Versican is a proteoglycan that has many different roles in tissue homeostasis and inflammation. The biochemical structure is comprised of four different types of the core protein with attached glycosaminoglycans that can be sulphated to various extents and has the capacity to regulate differentiation of different cell types, migration, cell adhesion, proliferation, tissue stabilization and inflammation. Versican's regulatory properties are of importance during both homeostasis and changes that lead to disease progression. The glycosaminoglycans that are attached to the core protein are of the chondroitin sulfate/dermatan sulfate type and are known to be important in inflammation through interactions with cytokines and growth factors. For a more complex understanding of versican it is of importance to study the tissue niche, where the wound healing process in both healthy and diseased conditions take place. In previous studies our group has identified changes in the amount of the multifaceted versican in chronic lung disorders such as asthma, chronic obstructive pulmonary disease and bronchiolitis obliterans syndrome, which could be a result of pathologic, transforming growth factor β driven, on-going remodelling processes. Reversely, the context of versican in its niche is of great importance since versican has been reported to have a beneficial role in other contexts e.g. emphysema. Here we explore the vast mechanisms of versican in healthy lung and in lung disorders.
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| 5. |
- Dowood, Rua Kareem, et al.
(författare)
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Determination of 3′-phosphoadenosine-5′-phosphosulfate in cells and Golgi fractions using hydrophilic interaction liquid chromatography–mass spectrometry
- 2016
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1470, s. 70-75
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Tidskriftsartikel (refereegranskat)abstract
- 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 10 min 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.
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| 6. |
- Elowsson Rendin, Linda, et al.
(författare)
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Harnessing the ECM Microenvironment to Ameliorate Mesenchymal Stromal Cell-Based Therapy in Chronic Lung Diseases
- 2021
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Ingår i: Frontiers in Pharmacology. - : Frontiers Media SA. - 1663-9812. ; 12
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 7. |
- Gao, Jia, et al.
(författare)
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Chemoenzymatic Synthesis of Glycopeptides Bearing Galactose-Xylose Disaccharide from the Proteoglycan Linkage Region
- 2021
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Ingår i: Organic Letters. - : American Chemical Society (ACS). - 1523-7060 .- 1523-7052. ; 23:5, s. 1738-1741
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Tidskriftsartikel (refereegranskat)abstract
- 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 β4GalT7. This work leads to a better understanding of the acceptor preference of β4GalT7 and opens the door for expeditious synthesis of the proteoglycan linkage region.
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| 8. |
- Hasan, Mahmudul, et al.
(författare)
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The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms
- 2021
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 12:5, s. 1869-1885
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Maccarana, Marco, et al.
(författare)
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Inhibition of iduronic acid biosynthesis by ebselen reduces glycosaminoglycan accumulation in mucopolysaccharidosis type I fibroblasts
- 2021
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Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 31:10, s. 1319-1329
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Mastio, Roberto, et al.
(författare)
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Fluorescently labeled xylosides offer insight into the biosynthetic pathways of glycosaminoglycans
- 2021
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Ingår i: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 11:60, s. 38283-38292
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Tidskriftsartikel (refereegranskat)abstract
- Five novel xylosides tagged with the fluorescent probe Pacific Blue™ were synthesized and found to act as substrates for β4GalT7, 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 β4GalT7 and β3GalT6, 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.
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| 11. |
- Olsson, Olof, et al.
(författare)
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The Tyrosine Kinase Inhibitor Imatinib Augments Extracellular Fluid Exchange and Reduces Average Collagen Fibril Diameter in Experimental Carcinoma
- 2016
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Ingår i: Molecular Cancer Therapeutics. - 1535-7163 .- 1538-8514. ; 15:10, s. 2455-2464
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 12. |
- Persson, Andrea, et al.
(författare)
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Production and HPLC-Based Disaccharide Analysis of Xyloside-Primed Glycosaminoglycans
- 2022
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Ingår i: Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Balagurunathan K., Nakato H., Desai U., Saijoh Y. (eds). - New York, NY : Springer. - 1064-3745 .- 1940-6029. - 9781071613986 ; , s. 173-182
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- 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. © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
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| 13. |
- Persson, Andrea, et al.
(författare)
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Xyloside-primed chondroitin sulfate/dermatan sulfate from breast carcinoma cells with a defined disaccharide composition has cytotoxic effects in vitro
- 2016
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 291:28, s. 14871-14882
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Tidskriftsartikel (refereegranskat)abstract
- We have previously reported that the xyloside 2-(6-hydroxynaphthyl) β-D-xylopyranoside (XylNapOH), in contrast to 2-naphthyl β-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 (CS/DS) 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 (HS) GAGs. In contrast, neither the CS/DS nor the HS 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 considerably 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 CS/DS.
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| 14. |
- Siegbahn, Anna, et al.
(författare)
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Exploration of the active site of beta 4GalT7 : modifications of the aglycon of aromatic xylosides
- 2015
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 13:11, s. 3351-3362
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase beta 4GalT7. Some beta-D-xylosides, such as 2-naphthyl beta-D-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for beta 4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for beta 4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with beta-glycosidic linkages function as good substrates for beta 4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by beta 4GalT7 is dependent on subtle differences in orientation of the xylose moiety.
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| 15. |
- Siegbahn, Anna, et al.
(författare)
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Exploration of the active site of β4GalT7: modifications of the aglycon of aromatic xylosides.
- 2015
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Ingår i: Organic and Biomolecular Chemistry. - : Royal Society of Chemistry (RSC). - 1477-0539 .- 1477-0520. ; 13:11, s. 3351-3362
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase β4GalT7. Some β-d-xylosides, such as 2-naphthyl β-d-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for β4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for β4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with β-glycosidic linkages function as good substrates for β4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by β4GalT7 is dependent on subtle differences in orientation of the xylose moiety.
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| 16. |
- Siegbahn, Anna, et al.
(författare)
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Rules for priming and inhibition of glycosaminoglycan biosynthesis; probing the beta 4GalT7 active site
- 2014
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 5:9, s. 3501-3508
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Tidskriftsartikel (refereegranskat)abstract
- beta-1,4-Gatactosyltransferase 7 (beta 4GalT7) is an essential enzyme in the biosynthesis of glycosaminoglycan (GAG) chains of proteoglycans (PGs). Mammalian cells produce PGs, which are involved in biological processes such as cell growth and differentiation. The PGs consist of a core protein, with one or several GAG chains attached. Both the structure of the PGs and the GAG chains, and the expression of the enzymes involved in their biosynthesis and degradation, vary between normal cells and tumor cells. The biosynthesis of GAG chains is initiated by xylosylation of a serine residue of the core protein, followed by galactosylation by beta 4GalT7. The biosynthesis can also be initiated by exogenously added beta-D-xylopyranosides with hydrophobic aglycons, which thus can act as acceptor substrates for beta 4GalT7. To determine the structural requirements for beta 4GalT7 activity, we have cloned and expressed the enzyme and designed a focused library of 2-naphthyl beta-D-xylopyranosides with modifications of the xylose moiety. Based on enzymatic studies, that is galactosylation and its inhibition, conformational analysis and molecular modeling using the crystal structure, we propose that the binding pocket of beta 4GalT7 is very narrow, with a precise set of important hydrogen bonds. Xylose appears to be the optimal acceptor substrate for galactosylation by beta 4GalT7. However, we show that modifications of the xylose moiety of the beta-D-xylopyranosides can render inhibitors of galactosylation. Such compounds will be valuable tools for the exploration of GAG and PG biosynthesis and a starting point for development of anti-tumor agents.
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| 17. |
- Stachtea, Xanthi, et al.
(författare)
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Dermatan Sulfate-Free Mice Display Embryological Defects and Are Neonatal Lethal Despite Normal Lymphoid and Non-Lymphoid Organogenesis.
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:10
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Tidskriftsartikel (refereegranskat)abstract
- The epimerization of glucuronic acid into iduronic acid adds structural variability to chondroitin/dermatan sulfate polysaccharides. Iduronic acid-containing domains play essential roles in processes such as coagulation, chemokine and morphogen modulation, collagen maturation, and neurite sprouting. Therefore, we generated and characterized, for the first time, mice deficient in dermatan sulfate epimerase 1 and 2, two enzymes uniquely involved in dermatan sulfate biosynthesis. The resulting mice, termed DKO mice, were completely devoid of iduronic acid, and the resulting chondroitin sulfate chains were structurally different from the wild type chains, from which a different protein binding specificity can be expected. As a consequence, a vast majority of the DKO mice died perinatally, with greatly variable phenotypes at birth or late embryological stages such as umbilical hernia, exencephaly and a kinked tail. However, a minority of embryos were histologically unaffected, with apparently normal lung and bone/cartilage features. Interestingly, the binding of the chemokine CXCL13, an important modulator of lymphoid organogenesis, to mouse DKO embryonic fibroblasts was impaired. Nevertheless, the development of the secondary lymphoid organs, including the lymph nodes and spleen, was normal. Altogether, our results indicate an important role of dermatan sulfate in embryological development and perinatal survival.
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| 18. |
- Thelin, Martin, et al.
(författare)
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Biological Functions of Iduronic Acid in Chondroitin/Dermatan Sulfate.
- 2013
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 280:10, s. 2431-2446
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Forskningsöversikt (refereegranskat)abstract
- The presence of iduronic acid in chondroitin/dermatan sulfate changes the properties of the polysaccharides, as it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/dermatan sulfate influences multiple cellular properties such as migration, proliferation, differentiation, angiogenesis and regulation of cytokine/growth factor activities. During pathological conditions such as wound healing, inflammation and cancer iduronic acid has diverse regulatory functions. Iduronic acid is formed by the two epimerases DS-epimerase 1 and DS-epimerase 2 which have different tissue distribution and properties. The role of IdoA in chondroitin/dermatan sulfate is underlined by the vast changes of connective tissue features in patients with a new type of Ehler-Danlos syndrome, adducted thumb-clubfoot syndrome. Future direction of research is to understand the roles of the two epimerases and their interplay with sulfotransferases involved in CS/DS biosynthesis. Further, a better definition of chondroitin/dermatan sulfate functions using different knock-out models is needed. In this review, we focus on the two enzymes responsible for iduronic acid formation and the role of iduronic acid in health and disease. © 2013 The Authors Journal compilation © 2013 FEBS.
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| 19. |
- Thorsheim, Karin, et al.
(författare)
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Disubstituted naphthyl β-D-xylopyranosides : Synthesis, GAG priming, and histone acetyltransferase (HAT) inhibition
- 2016
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 33:2, s. 57-245
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Tidskriftsartikel (refereegranskat)abstract
- 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)β-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.
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| 20. |
- Thorsheim, Karin, et al.
(författare)
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Hydroxylated oxanes as xyloside analogs for determination of the minimal binding requirements of β4GalT7
- 2017
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Ingår i: Tetrahedron Letters. - : Elsevier BV. - 0040-4039. ; 58:35, s. 3466-3469
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Tidskriftsartikel (refereegranskat)abstract
- β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the biosynthesis of glycosaminoglycan (GAG) chains. Natural and synthetic xylosides can be used to both inhibit and prime GAG synthesis by acting as inhibitors or substrates for β4GalT7. In this report, we exploit hydroxylated oxanes as deoxygenated xyloside analogs to clarify the minimum protein-ligand interactions required for galactosylation and/or inhibition. Enantiomerically pure substances were synthesized using a chiral pool approach whereas the corresponding racemates were obtained from simple starting materials. The results of a β4GalT7 assay show that a single hydroxyl group on an oxane ring is insufficient to induce galactosylation or inhibition, which implies that at least two substituents, one of which being 3-OH, needs to be present.
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| 21. |
- Thorsheim, Karin, et al.
(författare)
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Naphthyl Thio- and Carba-xylopyranosides for Exploration of the Active Site of ß-1,4-Galactosyltransferase 7 (ß4GalT7)
- 2017
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Ingår i: Chemistry - A European Journal. - : Wiley. - 0947-6539 .- 1521-3765. ; 23:71, s. 18057-18065
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Tidskriftsartikel (refereegranskat)abstract
- Xyloside analogues with substitution of the endocyclic oxygen atom by sulfur or carbon were investigated as substrates for -1,4-galactosyltransferase7 (4GalT7), a key enzyme in the biosynthesis of glycosaminoglycan chains. The analogues with an endocyclic sulfur atom proved to be excellent substrates for 4GalT7, and were galactosylated approximately fifteen times more efficiently than the corresponding xyloside. The 5a-carba--xylopyranoside in the d-configuration proved to be a good substrate for 4GalT7, 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 - 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.
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| 22. |
- Tykesson, Emil, et al.
(författare)
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Assays for Evaluation of Substrates for and Inhibitors of β-1,4-Galactosyltransferase 7
- 2022
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Ingår i: Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Balagurunathan K., Nakato H., Desai U., Saijoh Y. (eds). - New York, NY : Springer. - 1064-3745 .- 1940-6029. - 9781071613986 ; , s. 477-486
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- β-1,4-Galactosyltransferase 7 (β4GalT7) 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 β4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl β-D-xylopyranoside. We have cloned and expressed β4GalT7, 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 β4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of β4GalT7. © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
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| 23. |
- Tykesson, Emil, et al.
(författare)
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Deciphering the mode of action of the processive polysaccharide modifying enzyme dermatan sulfate epimerase 1 by hydrogen-deuterium exchange mass spectrometry
- 2016
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6539 .- 2041-6520. ; 7:2, s. 1447-1456
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 24. |
- Tykesson, Emil, et al.
(författare)
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Dermatan sulfate epimerase 1 and dermatan 4-O-sulfotransferase 1 form complexes that generate long epimerized 4-O-sulfated blocks
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 293:35, s. 13725-13735
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 25. |
- Tykesson, Emil
(författare)
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Formation of Iduronic Acid during Chondroitin/Dermatan Sulfate Biosynthesis
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All animals and some bacteria can synthesize linear polysaccharides with a backbone of repeating disaccharideunits, called glycosaminoglycans (GAGs). The GAGs are either attached to a protein core, as in proteoglycans (PGs), or exist as free polymer chains, as in hyaluronan. One of the most common types of GAGs is chondroitin/dermatan sulfate (CS/DS), where the repeating disaccharide backbone consists of the two epimeric carbohydrates glucuronic and iduronic acid (GlcA/IdoA), linked to N-acetylgalactosamine (GalNAc). The GAG polymer is linked to a core protein and can be sulfated at up to three positions in each disaccharide unit. The GAGs can bind cytokines and growth factors and are directly involved in receptor interactions. For example, the presence and structure of CS/DS is important for migration and invasion of cancer cells, development of atherosclerosis, neuronal outgrowth, and malaria infection.The aim of this work was to understand the role and mode of action of three important enzymes involved in the biosynthesis of CS/DS; namely dermatan sulfate epimerase 1 and 2 (DS-epi1 and 2) and dermatan 4-O- sulfotransferase 1 (D4ST1). The major findings are summarized below.Mice deficient in DS-epi1 and 2 were carefully characterized in terms of their phenotypes and biochemical GAG composition. The resulting mice were completely devoid of iduronic acid, and the resulting CS chains were structurally different from the wild type chains. Consequently, a vast majority of the DKO mice died perinatally, with widely variable phenotypes at birth or late embryonic stages. Together, our results indicate an important role of dermatan sulfate in embryonic development and perinatal survival.Further, we introduced a new technique to study the activity and mode of action of DS-epi1, where we combined a mass spectrometric analysis of heavy-atom labeled oligosaccharides with in silico simulations. Using an assay buffer prepared with heavy water (D2O) we analyzed the site-specific incorporation of deuterium into oligosaccharides of different lengths by collision-induced dissociation mass spectrometry (MS). The results from the MS experiments were then correlated to enzyme-substrate models prepared in silico, and we presented a model for the in vitro mode of action of DS-epi1.We also reported findings regarding the possible interaction between DS-epi1 and D4ST1. We could show that DS-epi1 yields only a few iduronic acid residues at a slow speed, whereas the co-incubation with D4ST1 increased the speed of epimerization five-fold. The enzymes were cross-linked and then subjected to gel electrophoresis, where larger complexes were observed. MS showed that the complexes contained both DS-epi1 and D4ST1, suggesting that DS-epi1 and D4ST1 interact during the formation of CS/DS.Finally, we described a novel method for recombinant production of DS. Recombinantly expressed DS-epi1 and D4ST1 were used, together with a uronosyl 2-O-sulfotransferase and a bacterial polysaccharide, to produce a DS polymer composed of IdoA-2S-GalNAc-4S. These CS/DS polymers were capable of inactivation of thrombin with heparin cofactor II in the same order of magnitude as with heparin.
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