| 1. |
- Malmström, Anders, et al.
(författare)
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Iduronic Acid in Chondroitin/Dermatan Sulfate: Biosynthesis and Biological Function
- 2012
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 60:12, s. 916-925
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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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| 2. |
- 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
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 19, s. 1197-1203
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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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| 3. |
- Pacheco, Benny
(författare)
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FROM GENE TO STRUCTURE: Formation of Iduronic Acid in Dermatan Sulfate by Two DS-epimerases
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During embryonic development and adult life a wide range of cell behaviors such as differentiation, proliferation and migration are in effect to maintain tissue integrity and function. An integral part of these dynamic processes is the interplay between the cells and their environment, i.e. the extracellular space. Complex polysaccharides, such as dermatan sulfate play a key role in these processes. Dermatan sulfate is a long linear polysaccharide of a repeating disaccharide unit consisting of N-acetylgalactosamine and glucuronic acid/iduronic acid. Formation of iduronic acid through epimerisation of glucuronic acid is a key modification in dermatan sulfate biosynthesis. In the present thesis two enzymes have been identified that catalyzes this reaction, i.e. DS-epimerase 1 and 2. By using fold-recognition modeling a structural model was obtained for DS-epimerase 1, which enabled characterization of the active site of the enzyme with mutational analysis. A feature of a dermatan sulfate chain is the presence of sequences of iduronic acid containing disaccharides separated by stretches of glucuronic acid containing disaccharides. The former are also commonly associated with sulfation in the 4-O-position of N-acetylgalactosamine suggesting that there is interplay between sulfation and epimerization. In this thesis it is shown that both epimerases are simultaneously needed together with the sulfotransferase D4ST-1 to generate these types of domains. Interestingly, 4-O-sulfated iduronic acid blocks constitute the binding site for several growth factors, including fibroblast growth factor 2. Through this binding, dermatan sulfate can sequester the growth factor in the extracellular matrix and regulate the availability of the growth factor at the cell surface. Dynamic changes in dermatan sulfate structure thus offers a way to fine-tune signaling. In this thesis evidence is presented that common growth factors such as transforming growth factor-β, platelet derived growth factor and epidermal growth factor have the ability to regulate both epimerase activity and gene expression of several sulfotransferases, including D4ST-1, which results in reduction of 4-O-sulfated iduronic acid blocks.
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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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