| 1. |
- Cluff, AH, et al.
(författare)
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Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue
- 2006
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Ingår i: Reproductive Biology and Endocrinology. - : Springer Science and Business Media LLC. - 1477-7827. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour. Methods: Uterine isthmic biopsies were collected from non-pregnant (n = 7), term pregnant women not in labour (n = 14), in normal labour (n = 7) and in prolonged labour (n = 7). mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy. Results: In women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p < 0.05). In term-pregnant tissue, the expression of syndecan 3 and connexin 43 did not differ significantly compared to nonpregnant and normal labour. The immunoreactivity of syndecan 3 was strong at normal labour, in contrast to prolonged labour, where both a weaker expression and an irregular distribution were detected. The immunoreactivity of connexin 43 increased until term and further stronger staining occurred at normal labour. At prolonged labour, the immunoreactivity was weaker and more unevenly distributed. At labour, a co-localization of syndecan 3 and connexin 43 could be demonstrated in the smooth muscle by confocal microscopy. Conclusion: The high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility.
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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 284:3, s. 1741-1747
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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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| 5. |
- Pacheco, Benny, et al.
(författare)
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Two dermatan sulfate epimerases form iduronic acid domains in dermatan sulfate.
- 2009
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 284, s. 9788-9795
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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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| 6. |
- ten Dam, Gerdy B., et al.
(författare)
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Dermatan sulfate domains defined by the novel antibody GD3A12, in normal tissues and ovarian adenocarcinomas
- 2009
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Ingår i: Histochemistry and Cell Biology. - : Springer Science and Business Media LLC. - 1432-119X .- 0948-6143. ; 132:1, s. 117-127
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate (DS) expression in normal tissue and ovarian cancer was investigated using the novel, phage display-derived antibody GD3A12 that was selected against embryonic glycosaminoglycans (GAGs). Antibody GD3A12 was especially reactive with DS rich in IdoA-GalNAc4S disaccharide units. IdoA residues are important for antibody recognition as DS polymers with low numbers of IdoA residues were less reactive, and expression of the DS epimerase in ovarian carcinoma cells was associated with expression of the GD3A12 epitope. Moreover, staining of antibody GD3A12 was abolished by chondroitinase-B lyase digestion. Expression of DS domains defined by antibody GD3A12 was confined to connective tissue of most organs examined and presented as a typical fibrillar-type of staining. Differential expression of the DS epitopes recognized by antibodies GD3A12 and LKN1 (4/2,4 di-O-sulfated DS) was best seen in thymus and spleen, indicating differential expression of various DS domains in these organs. In ovarian carcinomas strong DS expression was found in the stromal parts, and occasionally on tumor cells. Partial co-localization in ovarian carcinomas was observed with decorin, versican and type I collagen suggesting a uniform distribution of this specific DS epitope. This unique anti-DS antibody may be instrumental to investigate the function, expression, and localization of specific DS domains in health and disease.
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