| 1. |
- Vajro, Pietro, et al.
(författare)
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Three unreported cases of TMEM199-CDG, a rare genetic liver disease with abnormal glycosylation
- 2018
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Ingår i: Orphanet Journal of Rare Diseases. - : Springer Science and Business Media LLC. - 1750-1172. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: TMEM199 deficiency was recently shown in four patients to cause liver disease with steatosis, elevated serum transaminases, cholesterol and alkaline phosphatase and abnormal protein glycosylation. There is no information on the long-term outcome in this disorder. Results: We here present three novel patients with TMEM199-CDG. All three patients carried the same set of mutations (c.13-14delTT (p.Ser4Serfs∗30) and c.92G > C (p.Arg31Pro), despite only two were related (siblings). One mutation (c.92G > C) was described previously whereas the other was deemed pathogenic due to its early frameshift. Western Blot analysis confirmed a reduced level of TMEM199 protein in patient fibroblasts and all patients showed a similar glycosylation defect. The patients presented with a very similar clinical and biochemical phenotype to the initial publication, confirming that TMEM199-CDG is a non-encephalopathic liver disorder. Two of the patients were clinically assessed over two decades without deterioration. Conclusion: A rising number of disorders affecting Golgi homeostasis have been published over the last few years. A hallmark finding is deficiency in protein glycosylation, both in N- and O-linked types. Most of these disorders have signs of both liver and brain involvement. However, the present and the four previously reported patients do not show encephalopathy but a chronic, non-progressive (over decades) liver disease with hypertransaminasemia and steatosis. This information is crucial for the patient/families and clinician at diagnosis, as it distinguishes it from other Golgi homeostasis disorders, in having a much more favorable course.
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| 2. |
- Acosta, Helena, et al.
(författare)
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The serpin PN1 is a feedback regulator of FGF signaling in germ layer and primary axis formation.
- 2015
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Ingår i: Development: For advances in developmental biology and stem cells. - : The Company of Biologists. - 1477-9129. ; 142:6, s. 1146-1158
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Tidskriftsartikel (refereegranskat)abstract
- Germ layer formation and primary axis development rely on Fibroblast growth factors (FGFs). In Xenopus, the secreted serine protease HtrA1 induces mesoderm and posterior trunk/tail structures by facilitating the spread of FGF signals. Here, we show that the serpin Protease nexin-1 (PN1) is transcriptionally activated by FGF signals, suppresses mesoderm and promotes head development in mRNA-injected embryos. An antisense morpholino oligonucleotide against PN1 has the opposite effect and inhibits ectodermal fate. However, ectoderm and anterior head structures can be restored in PN1-depleted embryos when HtrA1 and FGF receptor activities are diminished, indicating that FGF signals negatively regulate their formation. We show that PN1 binds to and inhibits HtrA1, prevents degradation of the proteoglycan Syndecan 4 and restricts paracrine FGF/Erk signaling. Our data suggest that PN1 is a negative-feedback regulator of FGF signaling and has important roles in ectoderm and head development.
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| 3. |
- Akatsu, Chizuru, et al.
(författare)
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Dermatan sulfate epimerase 2 is the predominant isozyme in the formation of the chondroitin sulfate/dermatan sulfate hybrid structure in postnatal developing mouse brain
- 2011
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 21:5, s. 565-574
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Tidskriftsartikel (refereegranskat)abstract
- Chondroitin sulfate (CS) and dermatan sulfate (DS) are expressed in significant amounts in the brain and play important roles in the development of the central nervous system in mammals. CS and DS structures are often found in a single CS/DS hybrid chain. The L-iduronic acid (IdoA)-containing domain, which defines a DS-type domain, appears key to the biological functions of the CS/DS hybrid chain. In this study, to clarify the distribution of the DS-type structure in the brain during development, the expression patterns of DS epimerase 1 (DS-epi1) and DS-epi2, both of which convert D-glucuronic acid into IdoA, were investigated by in situ hybridization. DS-epi2 was ubiquitously expressed in the developing brain after birth, whereas the expression of DS-epi1 was faint and obscure at all developmental stages. Quantitative real-time polymerase chain reaction revealed the expression of DS-epi2 to be higher than that of DS-epi1 throughout development, suggesting that DS-epi2 but not DS-epi1 is mostly expressed in the brain and plays key roles in the epimerization of CS/DS during its biosynthesis. Moreover, an analysis of the disaccharides of CS/DS demonstrated significant amounts of IdoA-containing iD units [IdoA(2S)-GalNAc(6S)] and iB units [IdoA(2S)-GalNAc(4S)], where 2S, 4S and 6S stand for 2-O-, 4-O- and 6-O-sulfate, respectively, in every region of the brain examined. The proportion of these units in cerebellar CS/DS was greatly altered during postnatal development. These results suggest that the IdoA-containing structures in the developing brain are mainly produced by the actions of DS-epi2 and play crucial roles in postnatal development.
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| 4. |
- Barrueta Tenhunen, Annelie, et al.
(författare)
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Fluid restrictive resuscitation with high molecular weight hyaluronan infusion in early peritonitis sepsis
- 2023
-
Ingår i: Intensive Care Medicine Experimental. - : Springer Nature. - 2197-425X. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Sepsis is a condition with high morbidity and mortality. Prompt recognition and initiation of treatment is essential. Despite forming an integral part of sepsis management, fluid resuscitation may also lead to volume overload, which in turn is associated with increased mortality. The optimal fluid strategy in sepsis resuscitation is yet to be defined. Hyaluronan, an endogenous glycosaminoglycan with high affinity to water is an important constituent of the endothelial glycocalyx. We hypothesized that exogenously administered hyaluronan would counteract intravascular volume depletion and contribute to endothelial glycocalyx integrity in a fluid restrictive model of peritonitis. In a prospective, blinded model of porcine peritonitis sepsis, we randomized animals to intervention with hyaluronan (n = 8) or 0.9% saline (n = 8). The animals received an infusion of 0.1% hyaluronan 6 ml/kg/h, or the same volume of saline, during the first 2 h of peritonitis. Stroke volume variation and hemoconcentration were comparable in the two groups throughout the experiment. Cardiac output was higher in the intervention group during the infusion of hyaluronan (3.2 ± 0.5 l/min in intervention group vs 2.7 ± 0.2 l/min in the control group) (p = 0.039). The increase in lactate was more pronounced in the intervention group (3.2 ± 1.0 mmol/l in the intervention group and 1.7 ± 0.7 mmol/l in the control group) at the end of the experiment (p < 0.001). Concentrations of surrogate markers of glycocalyx damage; syndecan 1 (0.6 ± 0.2 ng/ml vs 0.5 ± 0.2 ng/ml, p = 0.292), heparan sulphate (1.23 ± 0.2 vs 1.4 ± 0.3 ng/ml, p = 0.211) and vascular adhesion protein 1 (7.0 ± 4.1 vs 8.2 ± 2.3 ng/ml, p = 0.492) were comparable in the two groups at the end of the experiment. In conclusion, hyaluronan did not counteract intravascular volume depletion in early peritonitis sepsis. However, this finding is hampered by the short observation period and a beneficial effect of HMW-HA in peritonitis sepsis cannot be discarded based on the results of the present study.
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| 5. |
- Bartolini, Barbara, et al.
(författare)
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Iduronic Acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells.
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
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Tidskriftsartikel (refereegranskat)abstract
- Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1-/- aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.
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| 6. |
- Bartolini, Barbara, et al.
(författare)
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Mouse development is not obviously affected by the absence of dermatan sulfate epimerase 2 in spite of a modified brain dermatan sulfate composition.
- 2012
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:7, s. 1007-1016
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate epimerase 2 (DS-epi2), together with its homologue DS-epi1, transform glucuronic acid into iduronic acid in dermatan sulfate polysaccharide chains. Iduronic acid gives dermatan sulfate increased chain flexibility and promotes protein binding. DS-epi2 is ubiquitously expressed and is the predominant epimerase in brain. Here we report the generation and initial characterization of DS-epi2 null mice. DS-epi2 deficient mice showed no anatomical, histological or morphological abnormalities. The body weights and lengths of mutated and wild-type littermates were indistinguishable. They were fertile and had a normal lifespan. Chondroitin/dermatan sulfate (CS/DS) isolated from newborn mutated mouse brains had a 38% reduction in iduronic acid compared to wild type littermates and compositional analysis revealed a decrease of 4-O-sulfate and an increase of 6-O-sulfate containing structures. Despite the reduction in iduronic acid, adult DS-epi2-/- brain showed normal extracellular matrix features by immunohistological stainings. We conclude that DS-epi1 compensates in vivo for the loss of DS-epi2.. These results extend previous findings of functional redundancy of brain extracellular matrix components.
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| 7. |
- Dick, Gunnar, et al.
(författare)
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PAPST1 regulates sulfation of heparan sulfate proteoglycans in epithelial MDCK II cells.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:1, s. 30-41
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycan (PG) sulfation depends on activated nucleotide sulfate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Transporters in the Golgi membrane translocate PAPS from the cytoplasm into the organelle lumen where PG sulfation occurs. Silencing of PAPS transporter (PAPST) 1 in epithelial MDCK cells reduced PAPS uptake into Golgi vesicles. Surprisingly, at the same time sulfation of heparan sulfate (HS) was stimulated. The effect was pathway specific in polarized epithelial cells. Basolaterally secreted PGs displayed an altered HS sulfation pattern and increased growth factor binding capacity. In contrast, the sulfation pattern of apically secreted PGs was unchanged while the secretion was reduced. Regulation of PAPST1 allows epithelial cells to prioritize between PG sulfation in the apical and basolateral secretory routes at the level of the Golgi apparatus. This provides sulfation patterns that ensure PG functions at the extracellular level, such as growth factor binding.
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| 8. |
- Doherty, Gareth G., et al.
(författare)
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Synthesis of Uronic Acid 1-Azasugars as Putative Inhibitors of α-Iduronidase, β-Glucuronidase and Heparanase
- 2023
-
Ingår i: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 24:4
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Tidskriftsartikel (refereegranskat)abstract
- 1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited β-glucuronidase (β-GLU). The d-GlcA-configured 9 was a potent inhibitor of β-GLU and a moderate inhibitor of the endo-β-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.
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| 9. |
- Ghiselli, Giancarlo, et al.
(författare)
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Drugs affecting glycosaminoglycan metabolism
- 2016
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Ingår i: Drug Discovery Today. - : Elsevier BV. - 1359-6446. ; 21:7, s. 1162-1169
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Forskningsöversikt (refereegranskat)abstract
- Glycosaminoglycans (GAGs) are charged polysaccharides ubiquitously present at the cell surface and in the extracellular matrix. GAGs are crucial for cellular homeostasis, and their metabolism is altered during pathological processes. However, little consideration has been given to the regulation of the GAG milieu through pharmacological interventions. In this review, we provide a classification of small molecules affecting GAG metabolism based on their mechanism of action. Furthermore, we present evidence to show that clinically approved drugs affect GAG metabolism and that this could contribute to their therapeutic benefit.
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| 10. |
- Gouignard, Nadège, et al.
(författare)
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Gene expression of the two developmentally regulated dermatan sulfate epimerases in the Xenopus embryo
- 2018
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 13:1
-
Tidskriftsartikel (refereegranskat)abstract
- Chondroitin sulfate (CS)/dermatan sulfate (DS) proteoglycans are abundant on the cell surface and in the extracellular matrix and have important functions in matrix structure, cell-matrix interaction and signaling. The DS epimerases 1 and 2, encoded by Dse and Dsel, respectively, convert CS to a CS/DS hybrid chain, which is structurally and conformationally richer than CS, favouring interaction with matrix proteins and growth factors. We recently showed that Xenopus Dse is essential for the migration of neural crest cells by allowing cell surface CS/DS proteoglycans to adhere to fibronectin. Here we investigate the expression of Dse and Dsel in Xenopus embryos. We show that both genes are maternally expressed and exhibit partially overlapping activity in the eyes, brain, trigeminal ganglia, neural crest, adenohypophysis, sclerotome, and dorsal endoderm. Dse is specifically expressed in the epidermis, anterior surface ectoderm, spinal nerves, notochord and dermatome, whereas Dsel mRNA alone is transcribed in the spinal cord, epibranchial ganglia, prechordal mesendoderm and myotome. The expression of the two genes coincides with sites of cell differentiation in the epidermis and neural tissue. Several expression domains can be linked to previously reported phenotypes of knockout mice and clinical manifestations, such as the Musculocontractural Ehlers-Danlos syndrome and psychiatric disorders.
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| 11. |
- Gouignard, Nadège, et al.
(författare)
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Musculocontractural Ehlers-Danlos syndrome and neurocristopathies : Dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin
- 2016
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Ingår i: Disease Models and Mechanisms. - : The Company of Biologists. - 1754-8403 .- 1754-8411. ; 9:6, s. 607-620
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Tidskriftsartikel (refereegranskat)abstract
- Of all live births with congenital anomalies, approximately one-third exhibit deformities ofthe head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers-Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused bydysfunction ofdermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 inconnective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1isimportant for the generationofisolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial-mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo. Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and metastasis in neuroblastoma and malignant melanoma suggest an association between DS and NC-derived cancers.
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| 12. |
- Gustafsson, Renata, et al.
(författare)
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Dermatan sulfate epimerase 1 deficient mice as a model for human abdominal wall defects.
- 2014
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Ingår i: Birth Defects Research. Part A: Clinical and Molecular Teratology. - : Wiley. - 1542-0760 .- 1542-0752. ; 100:9, s. 712-720
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate (DS) is a highly sulfated polysaccharide with a variety of biological functions in extracellular matrix organization and processes such as tumorigenesis and wound healing. A distinct feature of DS is the presence of iduronic acid, produced by the two enzymes, DS-epimerase 1 and 2, which are encoded by Dse and Dsel, respectively.
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| 13. |
- Hou, Shirui, et al.
(författare)
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The secreted serine protease xHtrA1 stimulates long-range FGF signaling in the early Xenopus embryo
- 2007
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Ingår i: Developmental Cell. - : Elsevier BV. - 1534-5807. ; 13:2, s. 226-241
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Tidskriftsartikel (refereegranskat)abstract
- We found that the secreted serine protease xHtrA1, expressed in the early embryo and transcriptionally activated by FGF signals, promotes posterior development in mRNA-injected Xenopus embryos. xHtrA1 mRNA led to the induction of secondary tail-like structures, expansion of mesoderm, and formation of ectopic neurons in an FGF-dependent manner. An antisense morpholino oligonucleotide or a neutralizing antibody against xHtrA1 had the opposite effects. xHtrA1 activates FGF/ ERK signaling and the transcription of FGF genes. We show that Xenopus Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1 and that heparan sulfate and dermatan sulfate trigger posteriorization, mesoderm induction, and neuronal differentiation via the FGIF signaling pathway. The results are consistent with a mechanism by which xHtrA1, through cleaving proteoglycans, releases cellsurface-bound FGF ligands and stimulates long-range FGF signaling.
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| 14. |
- Jia, Juan, et al.
(författare)
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Lack of L-iduronic acid in heparan sulfate affects interaction with growth factors and cell signaling
- 2009
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 284:23, s. 15942-15950
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Tidskriftsartikel (refereegranskat)abstract
- Glucuronyl C5-epimerase (Hsepi) catalyzes the conversion of D-glucuronic acid to L-iduronic acid in heparan sulfate (HS) biosynthesis. Disruption of the Hsepi gene in mouse yielded a lethal phenotype with selective organ defects, but had remarkably little effect on other organ systems. We have approached the underlying mechanisms by examining the course and effects of FGF2 signaling in a mouse embryonic fibroblast (MEF) cell line derived from the Hsepi-/- mouse. The HS produced by these cells is devoid of IdoA residues, but shows upregulated N- and 6-O-sulfation compared to wildtype (WT) MEF HS. In medium fortified with 10% FCS the Hsepi-/- MEFs proliferated and migrated similar to WT cells. Under starvation conditions both cell types showed attenuated proliferation and migration, that could be restored by addition of FGF2 to WT cells whereas Hsepi-/- cells were resistant. Moreover, ERK phosphorylation following FGF2 stimulation was delayed in Hsepi-/- compared to WT cells. Assessment of HS-growth factor interaction by nitrocellulose filter trapping revealed strikingly aberrant binding property of FGF2 and glia-derived neurotropic factor (GDNF) to Hsepi-/- but not to WT HS. GDNF has a key role in kidney development, defective in Hsepi-/- mice. By contrast, Hsepi-/- and WT HS interacted similarly and in conventional mode with FGF10. These findings correlate defective function of growth factors with their mode of HS interaction, and may help explain the partly modest organ phenotypes observed after genetic ablation of selected enzymes in HS biosynthesis.
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| 15. |
- Kim, Yoo-Mi, et al.
(författare)
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Determination of Autosomal Dominant or Recessive Methionine Adenosyltransferase I/III Deficiencies Based on Clinical and Molecular Studies.
- 2016
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Ingår i: Molecular Medicine. - : Springer Science and Business Media LLC. - 1528-3658 .- 1076-1551. ; 22, s. 147-155
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Tidskriftsartikel (refereegranskat)abstract
- Methionine adenosyltransferase (MAT) I/III deficiency can be inherited as autosomal dominant (AD) or as recessive (AR) traits in which mono- or bi-allelic MAT1A mutations have been identified, respectively. Although most patients have benign clinical outcomes, some with the AR form have neurological deficits. Here we describe 16 Korean patients with MAT I/III deficiency from 15 unrelated families identified by newborn screening. Ten probands had the AD MAT I/III deficiency, while six had AR MAT I/III deficiency. Plasma methionine (145.7 μmol/L vs. 733.2 μmol/L, P < 0.05) and homocysteine levels (12.3 μmol/L vs. 18.6 μmol/L, P < 0.05) were lower in the AD type than in AR type. In addition to the only reported AD MAT1A mutation, p.Arg264His, we identified two novel AD mutations, p.Arg249Gln and p.Gly280Arg. In the AR type, four previously reported and two novel mutations, p.Arg163Trp and p.Tyr335*, were identified. No exonic deletions were found by quantitative genomic PCR. Three-dimensional structural prediction programs indicated that the AD type mutations were located on the dimer interface or in the substrate binding site, hindering MAT I/III dimerization or substrate binding, respectively, whereas the AR mutations were distant from the interface or substrate binding site. These results indicate that the AD or AR MAT I/III deficiency is correlated with clinical findings, substrate levels, and structural features of the mutant proteins, which is important for the neurological management and genetic counseling of the patients.
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| 16. |
- Maccarana, Marco, et al.
(författare)
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Asporin-deficient mice have tougher skin and altered skin glycosaminoglycan content and structure
- 2017
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:8
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Tidskriftsartikel (refereegranskat)abstract
- The main structural component of connective tissues is fibrillar, cross-linked collagen whose fibrillogenesis can be modulated by Small Leucine-Rich Proteins/Proteoglycans (SLRPs). Not all SLRPs’ effects on collagen and extracellular matrix in vivo have been elucidated; one of the less investigated SLRPs is asporin. Here we describe the successful generation of an Aspn-/- mouse model and the investigation of the Aspn-/- skin phenotype. Functionally, Aspn-/- mice had an increased skin mechanical toughness, although there were no structural changes present on histology or immunohistochemistry. Electron microscopy analyses showed 7% thinner collagen fibrils in Aspn-/- mice (not statistically significant). Several matrix genes were upregulated, including collagens (Col1a1, Col1a2, Col3a1), matrix metalloproteinases (Mmp2, Mmp3) and lysyl oxidases (Lox, Loxl2), while lysyl hydroxylase (Plod2) was downregulated. Intriguingly no differences were observed in collagen protein content or in collagen cross-linking-related lysine oxidation or hydroxylation. The glycosaminoglycan content and structure in Aspn-/- skin was profoundly altered: chondroitin/dermatan sulfate was more than doubled and had an altered composition, while heparan sulfate was halved and had a decreased sulfation. Also, decorin and biglycan were doubled in Aspn-/- skin. Overall, asporin deficiency changes skin glycosaminoglycan composition, and decorin and biglycan content, which may explain the changes in skin mechanical properties.
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| 17. |
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| 18. |
- Maccarana, Marco, et al.
(författare)
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Biosynthesis of dermatan sulfate: Chondroitin glucuronate C5-epimerase is identical to SART2.
- 2006
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 281:17, s. 11560-11568
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Tidskriftsartikel (refereegranskat)abstract
- We identified the gene encoding chondroitin-glucuronate C5-epimerase (EC 5.1.3.19 [EC] ) that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulfate biosynthesis. The enzyme was solubilized from bovine spleen, and an ~43,000-fold purified preparation containing a major 89-kDa candidate component was subjected to mass spectrometry analysis of tryptic peptides. SART2 (squamous cell carcinoma antigen recognized by T cell 2), a protein with unknown function highly expressed in cancer cells and tissues, was identified by 18 peptides covering 26% of the sequence. Transient expression of cDNA resulted in a 22-fold increase in epimerase activity in 293HEK cell lysate. Moreover, overexpressing cells produced dermatan sulfate chains with 20% of iduronic acid-containing disaccharide units, as compared with 5% for mock-transfected cells. The iduronic acid residues were preferentially clustered in blocks, as in naturally occurring dermatan sulfate. Given the discovered identity, we propose to rename SART2 (Nakao, M., Shichijo, S., Imaizumi, T., Inoue, Y., Matsunaga, K., Yamada, A., Kikuchi, M., Tsuda, N., Ohta, K., Takamori, S., Yamana, H., Fujita, H., and Itoh, K. (2000) J. Immunol. 164, 2565–2574) with a functional designation, chondroitin-glucuronate C5-epimerase (or DS epimerase). DS epimerase activity is ubiquitously present in normal tissues, although with marked quantitative differences. It is highly homologous to part of the NCAG1 protein, encoded by the C18orf4 gene, genetically linked to bipolar disorder. NCAG1 also contains a putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis. The functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid-dependent interactions with growth factors, selectins, cytokines, or coagulation inhibitors.
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| 19. |
- Maccarana, Marco, et al.
(författare)
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Dermatan Sulfate Epimerase 1-Deficient Mice have Reduced Content and Changed Distribution of Iduronic acids in Dermatan Sulfate and an Altered Collagen Structure in Skin.
- 2009
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Ingår i: Molecular and Cellular Biology. - 0270-7306. ; 29, s. 5517-5528
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate epimerase 1 (DS-epi1) and 2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease of iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than wild-type littermates, but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect the collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.
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| 20. |
- Maccarana, Marco, et al.
(författare)
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Implications of Heparanase on Heparin Synthesis and Metabolism in Mast Cells
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:9
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Tidskriftsartikel (refereegranskat)abstract
- Heparin is a polysaccharide expressed in animal connective tissue-type mast cells. Owing to the special pentasaccharide sequence, heparin specifically binds to antithrombin (AT) and increases the inhibitory activity of AT towards coagulation enzymes. Heparin isolated from porcine intestinal mucosa has an average molecular weight of 15 kDa, while heparins recovered from rat skin and the peritoneal cavity were 60-100 kDa and can be fragmented by the endo-glucuronidase heparanase in vitro. In this study, we have examined heparin isolated from in vitro matured fetal skin mast cells (FSMC) and peritoneal cavity mast cells (PCMC) collected from wildtype (WT), heparanase knockout (Hpa-KO), and heparanase overexpressing (Hpa-tg) mice. The metabolically S-35-labeled heparin products from the mast cells of WT, Hpa-KO, and Hpa-tg mice were compared and analyzed for molecular size and AT-binding activity. The results show that PCMC produced heparins with a size similar to heparin from porcine intestinal mast cells, whilst FSMC produced much longer chains. As expected, heparanase overexpression resulted in the generation of smaller fragments in both cell types, while heparins recovered from heparanase knockout cells were slightly longer than heparin from WT cells. Unexpectedly, we found that heparanase expression affected the production of total glycosaminoglycans (GAGs) and the proportion between heparin and other GAGs but essentially had no effect on heparin catabolism.
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| 21. |
- 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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| 22. |
- Maccarana, Marco, et al.
(författare)
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Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts
- 2024
-
Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 34:6
-
Tidskriftsartikel (refereegranskat)abstract
- Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%–86% of DS-epi1 activity at 10 μM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients.
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| 23. |
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| 24. |
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| 25. |
- Malmström, Anders, et al.
(författare)
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Iduronic Acid in Chondroitin/Dermatan Sulfate: Biosynthesis and Biological Function
- 2012
-
Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 60:12, s. 916-925
-
Tidskriftsartikel (refereegranskat)abstract
- The ability of chondroitin/dermatan sulfate (CS/DS) to convey biological information is enriched by the presence of iduronic acid. DS-epimerases 1 and 2 (DS-epi1 and 2), in conjunction with DS-4-O-sulfotransferase 1, are the enzymes responsible for iduronic acid biosynthesis and will be the major focus of this review. CS/DS proteoglycans (CS/DS-PGs) are ubiquitously found in connective tissues, basement membranes, and cell surfaces or are stored intracellularly. Such wide distribution reflects the variety of biological roles in which they are involved, from extracellular matrix organization to regulation of processes such as proliferation, migration, adhesion, and differentiation. They play roles in inflammation, angiogenesis, coagulation, immunity, and wound healing. Such versatility is achieved thanks to their variable composition, both in terms of protein core and the fine structure of the CS/DS chains. Excellent reviews have been published on the collective and individual functions of each CS/DS-PG. This short review presents the biosynthesis and functions of iduronic acid-containing structures, also as revealed by the analysis of the DS-epi1- and 2-deficient mouse models. (J Histochem Cytochem 60: 916-925, 2012)
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| 26. |
- Nadafi, Reza, et al.
(författare)
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Dendritic cell migration to skin-draining lymph nodes is controlled by dermatan sulfate and determines adaptive immunity magnitude
- 2018
-
Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9:FEB
-
Tidskriftsartikel (refereegranskat)abstract
- For full activation of naïve adaptive lymphocytes in skin-draining lymph nodes (LNs), presentation of peptide:MHC complexes by LN-resident and skin-derived dendritic cells (DCs) that encountered antigens (Ags) is an absolute prerequisite. To get to the nearest draining LN upon intradermal immunization, DCs need to migrate from the infection site to the afferent lymphatics, which can only be reached by traversing a collagen-dense network located in the dermis of the skin through the activity of proteolytic enzymes. Here, we show that mice with altered collagen fibrillogenesis resulting in thicker collagen fibers in the skin display a reduced DC migration to the draining LN upon immune challenge. Consequently, the initiation of the cellular and humoral immune response was diminished. Ag-specific CD8+ and CD4+ T cells as well as Ag-specific germinal center B cells and serum immunoglobulin levels were significantly decreased. Hence, we postulate that alterations to the production of extracellular matrix, as seen in various connective tissue disorders, may in the end affect the qualitative outcome of adaptive immunity.
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| 27. |
- 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
-
Ingår i: Molecular Cancer Therapeutics. - 1535-7163 .- 1538-8514. ; 15:10, s. 2455-2464
-
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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| 28. |
- Olsson, P. Olof, et al.
(författare)
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Fibromodulin deficiency reduces collagen structural network but not glycosaminoglycan content in a syngeneic model of colon carcinoma.
- 2017
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:8
-
Tidskriftsartikel (refereegranskat)abstract
- Tumor barrier function in carcinoma represents a major challenge to treatment and is therefore an attractive target for increasing drug delivery. Variables related to tumor barrier include aberrant blood vessels, high interstitial fluid pressure, and the composition and structure of the extracellular matrix. One of the proteins associated with dense extracellular matrices is fibromodulin, a collagen fibrillogenesis modulator expressed in tumor stroma but scarce in normal loose connective tissues. Here, we investigated the effects of fibromodulin on stroma ECM in a syngeneic murine colon carcinoma model. We show that fibromodulin deficiency decreased collagen fibril thickness but glycosaminoglycan content and composition were unchanged. Furthermore, vascular density, pericyte coverage and macrophage amount were unaffected. Fibromodulin can therefore be a unique effector of dense collagen matrix assembly in tumor stroma and, without affecting other major matrix components or the cellular composition, can function as a main agent in tumor barrier function.
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| 29. |
- Pacheco, Benny, et al.
(författare)
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Dermatan 4-O-sulfotransferase 1 is pivotal in the formation of iduronic acid blocks in dermatan sulfate.
- 2009
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 19, s. 1197-1203
-
Tidskriftsartikel (refereegranskat)abstract
- Chondroitin/dermatan sulfate is a highly complex linear polysaccharide ubiquitously found in the extracellular matrix and at the cell surface. Several of its functions, such as binding to growth factors, are mediated by domains composed of alternating iduronic acid and 4-O-sulfated N-acetylgalactosamine residues, named 4-O-sulfated iduronic acid blocks. These domains are generated by the action of two DS-epimerases, which convert D-glucuronic acid into its epimer L-iduronic acid, in close connection with 4-O-sulfation. In this study, dermatan sulfate structure was evaluated after downregulating or increasing dermatan 4-O-sulfotransferase 1 (D4ST-1) expression. SiRNA-mediated downregulation of D4ST-1 in primary human lung fibroblasts led to a drastic specific reduction of iduronic acid blocks. No change of epimerase activity was found, indicating that the influence of D4ST-1 on epimerization is not due to an altered expression level of the DS-epimerases. Analysis of the dermatan sulfate chains showed that D4ST-1 is essential for the biosynthesis of the disulfated structure iduronic acid-2-O-sulfate-N-acetylgalactosamine-4-O-sulfate, thus confirmed to be strictly connected with the iduronic acid blocks. Also the biologically important residue hexuronic acid-N-acetylgalactosamine-4,6-O-disulfate considerably decreased after D4ST-1 downregulation. In conclusion, D4ST-1 is a key enzyme and is indispensable in the formation of important functional domains in dermatan sulfate and cannot be compensated by other 4-O-sulfotransferases.
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| 30. |
- Pacheco, Benny, et al.
(författare)
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Identification of the active site of DS-epimerase 1 and requirement of N-glycosylation for enzyme function.
- 2009
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 284:3, s. 1741-1747
-
Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate, which are formed by the action of two DS-epimerases, have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1, His205, Tyr261 and His450, by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues, which resulted in complete loss of epimerase activity. Based on these data, and the close relationship between lyase and epimerase reactions, we propose a model where His450 functions as a general base abstracting the C5-proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5-carbon by His205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction generating iduronic acid. In addition, we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.
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| 31. |
- Pacheco, Benny, et al.
(författare)
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Two dermatan sulfate epimerases form iduronic acid domains in dermatan sulfate.
- 2009
-
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 284, s. 9788-9795
-
Tidskriftsartikel (refereegranskat)abstract
- A second dermatan sulfate epimerase (DS-epi2) was identified as a homolog of the first epimerase (DS-epi1), which was previously described by our group. DS-epi2 is 1,222 a.a. long and has a ~700-a.a. N-terminal epimerase domain that is highly conserved between the two enzymes. In addition, the C-terminal portion is predicted to be an O-sulfotransferase domain. In this study we found that DS-epi2 has epimerase activity, which involves conversion of D-glucuronic acid to L-iduronic acid (EC 5.1.3.19), but no O-sulfotransferase activity was detected. In dermatan sulfate, iduronic acid residues are either clustered together in blocks or alternating with glucuronic acid, forming hybrid structures. By using an siRNA approach, we found that DS-epi2 and DS-epi1 are both involved in the biosynthesis of the iduronic acid blocks in fibroblasts and that DS-epi2 can also synthesize the hybrid structures. Both iduronic acid-containing domains have been shown to bind to several growth factors, many of which have biological roles in brain development. DS-epi2 has been genetically linked to bipolar disorder, which suggests that the dermatan sulfate domains generated by a defective enzyme may be involved in the etiology of the disease.
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| 32. |
- Pera, Edgar, et al.
(författare)
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Aberrant neural crest development causes craniofacial and other malformations in an animal model of Musculocontractural Ehlers-Danlos syndrome.
- 2016
-
Ingår i: Journal of Rare Diseases Research & Treatment. - 2572-9411. ; 1:3, s. 74-77
-
Forskningsöversikt (refereegranskat)abstract
- Musculocontractural Ehlers-Danlos syndrome (MC-EDS) is a rare recessive disorder that is characterized by connective tissue fragility, distinct craniofacial features and congenital malformations. MC-EDS patients have defects in the enzymes dermatan sulfate epimerase-1 and dermatan 4-O-sulfotransferase-1, which are involved in the biosynthesis of iduronic acid in the chondroitin sulfate/dermatan sulfate (CS/DS) chains of proteoglycans (PGs). While the connective tissue defect is a result of disturbed collagen fibril assembly based on a decreased iduronic acid content of interacting CS/DS-PGs, the cause of the developmental malformations in MC-EDS is not well understood. This review focuses on a new role of CS/DS-PGs in the development of multipotent and highly migratory neural crest (NC) cells in the Xenopus embryo model of MC-EDS. Single iduronic acid residues in CS/DS-PGs are involved in the formation of NC-derived craniofacial structures by facilitating the migration and adhesion of NC cells to fibronectin. Our results suggest a defect in NC development as cause of the craniofacial and other congenital anomalies in MC-EDS patients, which might contribute to an improved diagnosis and etiology-based therapy.
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| 33. |
- 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
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:10
-
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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| 34. |
- Thelin, Martin, et al.
(författare)
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Biological Functions of Iduronic Acid in Chondroitin/Dermatan Sulfate.
- 2013
-
Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 280:10, s. 2431-2446
-
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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| 35. |
- Thelin, Martin, et al.
(författare)
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Dermatan sulfate is involved in the tumorigenic properties of Esophagus Squamous Cell Carcinoma.
- 2012
-
Ingår i: Cancer Research. - 1538-7445. ; 72:8, s. 1943-1952
-
Tidskriftsartikel (refereegranskat)abstract
- Extracellular matrix, either produced by cancer cells or by cancer-associated fibroblasts, influences angiogenesis, invasion and metastasis. Chondroitin/dermatan sulfate (CS/DS) proteoglycans, which occur both in the matrix and at the cell surface, play important roles in these processes. The unique feature that distinguishes DS from CS is the presence of iduronic acid (IdoA) in DS. Here, we report that CS/DS is increased five-fold in human biopsies of esophagus squamous cell carcinoma (ESCC), an aggressive tumor with poor prognosis, as compared with normal tissue. The main IdoA-producing enzyme, DS epimerase 1 (DS-epi1), together with the 6-O- and 4-O-sulfotransferases, were highly up-regulated in ESCC biopsies. Importantly, CS/DS structure in patient tumors was significantly altered compared with normal tissue, as determined by sensitive mass spectrometry. To further understand the roles of IdoA in tumor development, DS-epi1 expression, and consequently IdoA content, wasdownregulated in ESCC cells. IdoA-deficient cells exhibited decreased migration and invasion capabilities in vitro, which was associated with reduced cellular binding of hepatocyte growth factor, inhibition of pERK-1/2 signaling, and de-regulated actin cytoskeleton dynamics and focal adhesion formation. Our findings demonstrate that IdoA in DS influences tumorigenesis by affecting cancer cell behavior. Therefore, down-regulation of IdoA by DS-epi1 inhibitors may represent a new anti-cancer therapy.
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| 36. |
- Tiedemann, K, et al.
(författare)
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Regulation of the chondroitin/dermatan fine structure by transforming growth factor-beta 1 through effects on polymer-modifying enzymes
- 2005
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 15:12, s. 1277-1285
-
Tidskriftsartikel (refereegranskat)abstract
- The chondroitin/dermatan sulfate proteoglycans (CS/DSPGs), biglycan, decorin, and versican play several important roles in extracellular matrix influencing matrix organization, cell proliferation, and recruitment. Moreover, they bind and regulate growth factors in the extracellular matrix. We have previously shown that cultured human lung fibroblasts treated with transforming growth factor-beta (TGF-beta) alone or in combination with epidermal growth factor and platelet-derived growth factor, increase the production of these PGs. In this report, we describe that the structure of their galactosaminoglycan side chains is altered, albeit there is no alteration of polysaccharide length. The findings showed that iduronic acid content is reduced by 50% in decorin and biglycan, whereas 4-O-sulfation is increased 2-fold in versican. To unravel the mechanism behind these changes, the activities of chondroitin C-5 epimerase and of O-sulfotransferases in cellular fractions prepared from fibroblasts were quantitated, and transcript levels of the relevant sulfotransferases were measured by real time polymerase chain reaction (RT-PCR). The C-5 epimerase activity was reduced by 25% in TGF-beta 1 treated cells and 50% in fibroblasts treated with the growth factor combination. No change in activity in dermatan 4-O sulfotransferase was observed, and only a minor decrease in dermatan 4-O-sulfotransferase-1 (D4ST-1) mRNA was observed. On the other hand, chondroitin 4-O sulfotransferase activity increased 2-fold upon TGF-beta 1 treatment and 3-fold upon treatment with the growth factor combination. This is in agreement with a 2-fold up-regulation of chondroitin-4-O-sulfotransferase 1 (C4ST-1) mRNA, and no changes in chondroitin-4-O-sulfotransferase 2 (C4ST-2) mRNA. Thus, cellular activity and transcript level correlated well with the changes in the structure of the dermatan/chondroitin sulfate chains.
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| 37. |
- 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
-
Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6539 .- 2041-6520. ; 7:2, s. 1447-1456
-
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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| 38. |
- 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
-
Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 293:35, s. 13725-13735
-
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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| 39. |
- Tykesson, Emil, et al.
(författare)
-
Recombinant dermatan sulfate is a potent activator of heparin cofactor II-dependent inhibition of thrombin
- 2019
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 29:6, s. 446-451
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 40. |
- Westergren-Thorsson, Gunilla, et al.
(författare)
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Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis
- 2017
-
Ingår i: International Journal of Biochemistry & Cell Biology. - : Elsevier BV. - 1357-2725 .- 1878-5875. ; 83, s. 27-38
-
Tidskriftsartikel (refereegranskat)abstract
- 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. (C) 2016 The Authors. Published by Elsevier Ltd.
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| 41. |
- Zhang, Tianji, et al.
(författare)
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Interacting polymer-modification enzymes in heparan sulfate biosynthesis
- 2023
-
Ingår i: Carbohydrate Polymers. - : Elsevier. - 0144-8617 .- 1879-1344. ; 299
-
Tidskriftsartikel (refereegranskat)abstract
- Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcA beta 1-4GlcNSO3 alpha 1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotrans-ferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluores-cence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a func-tional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O -sulfated glucosamine residues. The inability to achieve simultaneous 2-O-and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme in-teractions in heparan sulfate biosynthesis.
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