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Formation of Iduronic Acid during Chondroitin/Dermatan Sulfate Biosynthesis

Tykesson, Emil LU (2016)
Abstract (Swedish)
All animals and some bacteria can synthesize linear polysaccharides with a backbone of repeating disaccharide
units, called glycosaminoglycans (GAGs). The GAGs are either attached to a protein core, as in proteoglycans (PGs), or exist as free polymer chains, as in hyaluronan. One of the most common types of GAGs is chondroitin/dermatan sulfate (CS/DS), where the repeating disaccharide backbone consists of the two epimeric carbohydrates glucuronic and iduronic acid (GlcA/IdoA), linked to N-acetylgalactosamine (GalNAc). The GAG polymer is linked to a core protein and can be sulfated at up to three positions in each disaccharide unit. The GAGs can bind cytokines and growth factors and are directly involved in receptor interactions. For... (More)
All animals and some bacteria can synthesize linear polysaccharides with a backbone of repeating disaccharide
units, called glycosaminoglycans (GAGs). The GAGs are either attached to a protein core, as in proteoglycans (PGs), or exist as free polymer chains, as in hyaluronan. One of the most common types of GAGs is chondroitin/dermatan sulfate (CS/DS), where the repeating disaccharide backbone consists of the two epimeric carbohydrates glucuronic and iduronic acid (GlcA/IdoA), linked to N-acetylgalactosamine (GalNAc). The GAG polymer is linked to a core protein and can be sulfated at up to three positions in each disaccharide unit. The GAGs can bind cytokines and growth factors and are directly involved in receptor interactions. For example, the presence and structure of CS/DS is important for migration and invasion of cancer cells, development of atherosclerosis, neuronal outgrowth, and malaria infection.
The aim of this work was to understand the role and mode of action of three important enzymes involved in the biosynthesis of CS/DS; namely dermatan sulfate epimerase 1 and 2 (DS-epi1 and 2) and dermatan 4-O- sulfotransferase 1 (D4ST1). The major findings are summarized below.
Mice deficient in DS-epi1 and 2 were carefully characterized in terms of their phenotypes and biochemical GAG composition. The resulting mice were completely devoid of iduronic acid, and the resulting CS chains were structurally different from the wild type chains. Consequently, a vast majority of the DKO mice died perinatally, with widely variable phenotypes at birth or late embryonic stages. Together, our results indicate an important role of dermatan sulfate in embryonic development and perinatal survival.
Further, we introduced a new technique to study the activity and mode of action of DS-epi1, where we combined a mass spectrometric analysis of heavy-atom labeled oligosaccharides with in silico simulations. Using an assay buffer prepared with heavy water (D2O) we analyzed the site-specific incorporation of deuterium into oligosaccharides of different lengths by collision-induced dissociation mass spectrometry (MS). The results from the MS experiments were then correlated to enzyme-substrate models prepared in silico, and we presented a model for the in vitro mode of action of DS-epi1.
We also reported findings regarding the possible interaction between DS-epi1 and D4ST1. We could show that DS-epi1 yields only a few iduronic acid residues at a slow speed, whereas the co-incubation with D4ST1 increased the speed of epimerization five-fold. The enzymes were cross-linked and then subjected to gel electrophoresis, where larger complexes were observed. MS showed that the complexes contained both DS-epi1 and D4ST1, suggesting that DS-epi1 and D4ST1 interact during the formation of CS/DS.
Finally, we described a novel method for recombinant production of DS. Recombinantly expressed DS-epi1 and D4ST1 were used, together with a uronosyl 2-O-sulfotransferase and a bacterial polysaccharide, to produce a DS polymer composed of IdoA-2S-GalNAc-4S. These CS/DS polymers were capable of inactivation of thrombin with heparin cofactor II in the same order of magnitude as with heparin. (Less)
Abstract
All animals and some bacteria can synthesize linear polysaccharides with a backbone of repeating disaccharide
units, called glycosaminoglycans (GAGs). The GAGs are either attached to a protein core, as in proteoglycans (PGs), or exist as free polymer chains, as in hyaluronan. One of the most common types of GAGs is chondroitin/dermatan sulfate (CS/DS), where the repeating disaccharide backbone consists of the two epimeric carbohydrates glucuronic and iduronic acid (GlcA/IdoA), linked to N-acetylgalactosamine (GalNAc). The GAG polymer is linked to a core protein and can be sulfated at up to three positions in each disaccharide unit. The GAGs can bind cytokines and growth factors and are directly involved in receptor interactions. For... (More)
All animals and some bacteria can synthesize linear polysaccharides with a backbone of repeating disaccharide
units, called glycosaminoglycans (GAGs). The GAGs are either attached to a protein core, as in proteoglycans (PGs), or exist as free polymer chains, as in hyaluronan. One of the most common types of GAGs is chondroitin/dermatan sulfate (CS/DS), where the repeating disaccharide backbone consists of the two epimeric carbohydrates glucuronic and iduronic acid (GlcA/IdoA), linked to N-acetylgalactosamine (GalNAc). The GAG polymer is linked to a core protein and can be sulfated at up to three positions in each disaccharide unit. The GAGs can bind cytokines and growth factors and are directly involved in receptor interactions. For example, the presence and structure of CS/DS is important for migration and invasion of cancer cells, development of atherosclerosis, neuronal outgrowth, and malaria infection.
The aim of this work was to understand the role and mode of action of three important enzymes involved in the biosynthesis of CS/DS; namely dermatan sulfate epimerase 1 and 2 (DS-epi1 and 2) and dermatan 4-O- sulfotransferase 1 (D4ST1). The major findings are summarized below.
Mice deficient in DS-epi1 and 2 were carefully characterized in terms of their phenotypes and biochemical GAG composition. The resulting mice were completely devoid of iduronic acid, and the resulting CS chains were structurally different from the wild type chains. Consequently, a vast majority of the DKO mice died perinatally, with widely variable phenotypes at birth or late embryonic stages. Together, our results indicate an important role of dermatan sulfate in embryonic development and perinatal survival.
Further, we introduced a new technique to study the activity and mode of action of DS-epi1, where we combined a mass spectrometric analysis of heavy-atom labeled oligosaccharides with in silico simulations. Using an assay buffer prepared with heavy water (D2O) we analyzed the site-specific incorporation of deuterium into oligosaccharides of different lengths by collision-induced dissociation mass spectrometry (MS). The results from the MS experiments were then correlated to enzyme-substrate models prepared in silico, and we presented a model for the in vitro mode of action of DS-epi1.
We also reported findings regarding the possible interaction between DS-epi1 and D4ST1. We could show that DS-epi1 yields only a few iduronic acid residues at a slow speed, whereas the co-incubation with D4ST1 increased the speed of epimerization five-fold. The enzymes were cross-linked and then subjected to gel electrophoresis, where larger complexes were observed. MS showed that the complexes contained both DS-epi1 and D4ST1, suggesting that DS-epi1 and D4ST1 interact during the formation of CS/DS.
Finally, we described a novel method for recombinant production of DS. Recombinantly expressed DS-epi1 and D4ST1 were used, together with a uronosyl 2-O-sulfotransferase and a bacterial polysaccharide, to produce a DS polymer composed of IdoA-2S-GalNAc-4S. These CS/DS polymers were capable of inactivation of thrombin with heparin cofactor II in the same order of magnitude as with heparin. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor Liu, Jian, University of North Carolina at Chapel Hill, USA
organization
publishing date
type
Thesis
publication status
published
subject
keywords
GLYCOSAMINOGLYCAN, CHONDROITIN, DERMATAN, EPIMERASE, SULFOTRANSFERASE, GLYCOSAMINOGLYCAN, CHONDROITIN SULFATE, DERMATAN SULFATE, EPIMERASE, SULFOTRANSFERASE
pages
66 pages
publisher
Lund University, Faculty of Medicine
defense location
Belfragesalen, BMC D15, Klinikgatan 32, Lund
defense date
2016-05-25 09:00
ISBN
978-91-7619-294-8
language
English
LU publication?
yes
id
d7cc80ec-d320-4ced-87ea-55b46f8f8608
date added to LUP
2016-05-02 11:28:14
date last changed
2016-09-19 08:45:20
@phdthesis{d7cc80ec-d320-4ced-87ea-55b46f8f8608,
  abstract     = {All animals and some bacteria can synthesize linear polysaccharides with a backbone of repeating disaccharide<br/>units, called glycosaminoglycans (GAGs). The GAGs are either attached to a protein core, as in proteoglycans (PGs), or exist as free polymer chains, as in hyaluronan. One of the most common types of GAGs is chondroitin/dermatan sulfate (CS/DS), where the repeating disaccharide backbone consists of the two epimeric carbohydrates glucuronic and iduronic acid (GlcA/IdoA), linked to N-acetylgalactosamine (GalNAc). The GAG polymer is linked to a core protein and can be sulfated at up to three positions in each disaccharide unit. The GAGs can bind cytokines and growth factors and are directly involved in receptor interactions. For example, the presence and structure of CS/DS is important for migration and invasion of cancer cells, development of atherosclerosis, neuronal outgrowth, and malaria infection.<br/>The aim of this work was to understand the role and mode of action of three important enzymes involved in the biosynthesis of CS/DS; namely dermatan sulfate epimerase 1 and 2 (DS-epi1 and 2) and dermatan 4-O- sulfotransferase 1 (D4ST1). The major findings are summarized below.<br/>Mice deficient in DS-epi1 and 2 were carefully characterized in terms of their phenotypes and biochemical GAG composition. The resulting mice were completely devoid of iduronic acid, and the resulting CS chains were structurally different from the wild type chains. Consequently, a vast majority of the DKO mice died perinatally, with widely variable phenotypes at birth or late embryonic stages. Together, our results indicate an important role of dermatan sulfate in embryonic development and perinatal survival.<br/>Further, we introduced a new technique to study the activity and mode of action of DS-epi1, where we combined a mass spectrometric analysis of heavy-atom labeled oligosaccharides with in silico simulations. Using an assay buffer prepared with heavy water (D2O) we analyzed the site-specific incorporation of deuterium into oligosaccharides of different lengths by collision-induced dissociation mass spectrometry (MS). The results from the MS experiments were then correlated to enzyme-substrate models prepared in silico, and we presented a model for the in vitro mode of action of DS-epi1.<br/>We also reported findings regarding the possible interaction between DS-epi1 and D4ST1. We could show that DS-epi1 yields only a few iduronic acid residues at a slow speed, whereas the co-incubation with D4ST1 increased the speed of epimerization five-fold. The enzymes were cross-linked and then subjected to gel electrophoresis, where larger complexes were observed. MS showed that the complexes contained both DS-epi1 and D4ST1, suggesting that DS-epi1 and D4ST1 interact during the formation of CS/DS.<br/>Finally, we described a novel method for recombinant production of DS. Recombinantly expressed DS-epi1 and D4ST1 were used, together with a uronosyl 2-O-sulfotransferase and a bacterial polysaccharide, to produce a DS polymer composed of IdoA-2S-GalNAc-4S. These CS/DS polymers were capable of inactivation of thrombin with heparin cofactor II in the same order of magnitude as with heparin.},
  author       = {Tykesson, Emil},
  isbn         = {978-91-7619-294-8},
  keyword      = {GLYCOSAMINOGLYCAN,CHONDROITIN,DERMATAN,EPIMERASE,SULFOTRANSFERASE,GLYCOSAMINOGLYCAN,CHONDROITIN SULFATE,DERMATAN SULFATE,EPIMERASE,SULFOTRANSFERASE},
  language     = {eng},
  pages        = {66},
  publisher    = {Lund University, Faculty of Medicine},
  school       = {Lund University},
  title        = {Formation of Iduronic Acid during Chondroitin/Dermatan Sulfate Biosynthesis},
  year         = {2016},
}