<p>Heparan sulfate proteoglycans are proteins with long, unbranched heparan sulfate (HS) polysaccharide chains attached to them. They are found on cell surfaces and in basement membranes where they exert their action by interacting with a wide range of enzymes and signaling molecules and are thereby involved in a range of various processes both during embryonic development and in adult physiology.</p><p>A great part of the biological functionality of proteoglycans can be directly related to the polysaccharide part. HS chains display very variable sulfation patterns where highly sulfated regions are responsible for a large part of the biological activity. The biosynthesis of HS is a complex process in which a number of enzymes are involved. Better comprehension of how this process is regulated could reveal clues to how formation of HS sulfation patterns occurs, and thereby how HS functionality is controlled.</p><p>This thesis is focusing on regulation of one of the enzymes responsible for HS sulfation, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), in an attempt to understand these mechanisms better. Different aspects of NDST regulation were studied in three projects:</p><p>I) “Heparin/heparan sulfate biosynthesis: Processive formation of N-sulfated domains”, where the sulfate donor PAPS is shown to influence the manner in which NDST modifies the substrate, affecting the domain structure of the polysaccharide.</p><p>II) “Heparan sulfate biosynthesis: Characterization of an NDST1 splice variant”, where a splice variant of NDST1 which appears to influence NDST1 protein levels and affect HS structure is described.</p><p>III) “Heparan sulfate biosynthesis in zebrafish: Five NDST genes with distinct expression patterns during embryonic development”, in which five zebrafish NDSTs were cloned and shown to be expressed in a temporally and spatially regulated manner.</p>

<p>Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is <i>N</i>-sulfated by the enzyme <i>N</i>-deacetylase <i>N</i>-sulfotransferase (NDST). <i>N</i>-Sulfation occurs in domains where further modifications (including <i>O</i>-sulfations) take place, giving the chain a complex sulfation pattern.</p><p>In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that <i>N</i>-deacetylation is the rate-limiting step in HS <i>N</i>-sulfation and that two different NDST molecules can work on the same GlcN unit.</p><p>By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for <i>N</i>-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on <i>N</i>-deacetylase activity. </p><p>We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. </p><p>Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones.</p><p>.</p>

Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is N-sulfated by the enzyme N-deacetylase N-sulfotransferase (NDST). N-Sulfation occurs in domains where further modifications (including O-sulfations) take place, giving the chain a complex sulfation pattern. In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that N-deacetylation is the rate-limiting step in HS N-sulfation and that two different NDST molecules can work on the same GlcN unit. By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for N-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on N-deacetylase activity. We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones. .

Heparan sulfate proteoglycans are proteins with long, unbranched heparan sulfate (HS) polysaccharide chains attached to them. They are found on cell surfaces and in basement membranes where they exert their action by interacting with a wide range of enzymes and signaling molecules and are thereby involved in a range of various processes both during embryonic development and in adult physiology. A great part of the biological functionality of proteoglycans can be directly related to the polysaccharide part. HS chains display very variable sulfation patterns where highly sulfated regions are responsible for a large part of the biological activity. The biosynthesis of HS is a complex process in which a number of enzymes are involved. Better comprehension of how this process is regulated could reveal clues to how formation of HS sulfation patterns occurs, and thereby how HS functionality is controlled. This thesis is focusing on regulation of one of the enzymes responsible for HS sulfation, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), in an attempt to understand these mechanisms better. Different aspects of NDST regulation were studied in three projects: I) “Heparin/heparan sulfate biosynthesis: Processive formation of N-sulfated domains”, where the sulfate donor PAPS is shown to influence the manner in which NDST modifies the substrate, affecting the domain structure of the polysaccharide. II) “Heparan sulfate biosynthesis: Characterization of an NDST1 splice variant”, where a splice variant of NDST1 which appears to influence NDST1 protein levels and affect HS structure is described. III) “Heparan sulfate biosynthesis in zebrafish: Five NDST genes with distinct expression patterns during embryonic development”, in which five zebrafish NDSTs were cloned and shown to be expressed in a temporally and spatially regulated manner.

The aim of the study was to investigate the expression NDST transcripts in a wide variety of tissues using RNA-sequencing experimental data from five published studies, using two common in silico tools: the Tophat-Cufflink pipeline and the HTSeq-DEXSeq pipeline. We show that to detect NDST alternative transcripts, paired-end sequencing should be used with replicates of samples or conditions together with 100 base read length to allow for reliable detection of the low expressed transcripts in the NDST family.  As a demonstration project, we also characterized HS synthesized by the adrenal carcinoma (ACC) cell line H295R and determined expression of NDSTs in the cells and in ACC tumor samples. We could show that roughly 65% of newly synthesized proteoglycans isolated after metabolic 35S-sulfate labeling of the cells are made up of heparan sulfate (HS) with an average chain length of 45 kDa. The HS chains show a high frequency of N-sulfation and a high total degree of sulfation. Interestingly, disaccharide analysis demonstrated a three-time higher amount of stored chondroitin sulfate (CS) compared to HS in the ACC cell line.

<p>In the extracellular space, many specialized proteins are located to support cells and to mediate cell-cell signalling. One class of such molecules is heparan sulfate (HS) proteoglycans, which are proteins with different properties and locations but all of them decorated with long unbranched HS polysaccharide chains. During biosynthesis the HS chains are modified by sulfation and a C5-epimerase converts some glucuronic acid residues to iduronic acid. The patterns of the modifications vary distinctly between tissues and developing stages and give HS chains different affinity for biologically important proteins. Thus, the regulation of HS biosynthesis is likely to influence a wide variety of biological events.</p><p>This thesis focuses on the biosynthesis of HS in animals with targeted disruptions in genes important for HS production. The N-deacetylase N-sulfotransferase (NDST) is a key enzyme in HS biosynthesis, directing other modifications. We show that NDST isoforms have very different roles in HS biosynthesis. Inactivation of NDST1 affects HS biosynthesis in all tissues. In embryonic liver HS from NDST1-/- mice the N-sulfation was decresed with twothirds, while the absence of NDST2 did not affect HS structure. In the absence of NDST1 in the liver, however, NDST2 is active in HS N-sulfation. </p><p>In a study of embryonic stem cells lacking both NDST1 and NDST2, no N-sulfate groups could be detected. 6-O-sulfate groups were, however, still present at half of its normal level. This was an unexpected finding since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition.</p><p>By adapting an automated method for HS analysis to mammalian tissues, we could extend our analyses to more tissues and other transgene models. We also developed a protocol to create a sensitive “fingerprint” of HS structure. With these methods we could determine the individual HS structure of different mouse tissues. </p>

<p>Heparan sulfate (HS) is a ubiquitous polysaccharide on the cell surface and in the extracellular matrix. HS is an important actor in the regulation of cell signaling, especially in the developing embryo. In combination with cell culture and biochemical experiments, <i>in vivo</i> studies of genetically modified animals have pointed out the sulfation pattern of HS as highly important for binding of ligands, their receptors and other signaling modulators.</p><p>The sulfation pattern of an HS chain is gained by several modifying steps, performed by multiple enzymes during biosynthesis in the Golgi apparatus. By alterations of sulfation pattern, and the amount of sulfate groups, a cell can regulate the binding properties of its HS to different molecules. The most highly sulfated form of HS is called heparin, and can only be found intracellularly in mast cells.</p><p>This thesis describes the phenotypes and the alterations in HS/heparin biosynthesis of two genetically modified mouse strains deficient in N-deacetylase/N-sulfotransferase-1 (NDST1) and -2 (NDST2) respectively. We have found NDST1 to be important for correct sulfation of HS and that NDST2 is crucial in heparin biosynthesis. NDST2 deficient mice completely lack heparin and therefore have a severe mast cell phenotype. NDST1 deficient mice produce undersulfated HS and show several developmental disturbances. Some NDST1 embryos die in utero while the rest die neonatally due to breathing difficulties. Defect brain, eye and skeletal development has also been observed while some organs, such as the liver, appear to be largely unaffected. Several phenotypes are similar to defects seen in other mouse strains with impaired fibroblast growth factor and bone morphogenetic protein signaling, among others. This suggests the phenotypes of NDST1 deficient embryos to be of a multi factorial origin, in complete accordance to the many signaling pathways HS is suggested to modulate.</p>

<p>Heparan sulfate (HS) proteoglycans consist of sulfated HS chains covalently bound to core proteins. They are ubiquitously expressed, on the cell surface and in the extracellular matrix, throughout the body. During biosynthesis the HS chain is modified to generate a highly variable pattern of sulfated residues, able to interact with a wide variety of ligands, such as growth factors, morphogens and extracellular matrix molecules. The presence of HS proteoglycans is crucial during various developmental processes as they are involved in generation of morphogen gradients and influence the function of several growth factor pathways essential for tissue assembly and differentiation.</p><p>In this thesis the phenotypes of two mouse strains, deficient in different isoforms of the HS biosynthetic enzyme N-deacetylase/N-sulfotransferase (NDST) have been analyzed. In addition, NDST deficient embryonic stem (ES) cells have been analyzed with regard to HS structure and differentiation capacity. Mice deficient in NDST1 die peri-natally. The embryos display an overall low-sulfated HS and several developmental defects, with a lung phenotype as the predominant cause of death. Mice deficient in NDST2 lack sulfated heparin in connective tissue type mast cells while HS structure is unaltered. These results indicate that NDST1 is the isoform mainly responsible for HS biosynthesis during development. However, NDST1/2 deficient embryos do not survive beyond E5.5 and have a greatly disturbed morphology, suggesting that NDST2 has an essential role during early embryonic development. HS synthesized by NDST1/2 deficient ES cells had a total lack of N-sulfate groups while, interestingly, about half of the 6-O-sulfate groups remained. This result was unexpected since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition. Further characterization of the NDST1/2 deficient ES cells during in vitro differentiation demonstrated that the expression pattern of markers for all three germ layers was disturbed. In addition, it was demonstrated that NDST1 is not needed for mast cell development, that lack of NDST2 results in abnormal mast cells and that no mast cells is formed from NDST1/2 deficient ES cells.</p>

In the extracellular space, many specialized proteins are located to support cells and to mediate cell-cell signalling. One class of such molecules is heparan sulfate (HS) proteoglycans, which are proteins with different properties and locations but all of them decorated with long unbranched HS polysaccharide chains. During biosynthesis the HS chains are modified by sulfation and a C5-epimerase converts some glucuronic acid residues to iduronic acid. The patterns of the modifications vary distinctly between tissues and developing stages and give HS chains different affinity for biologically important proteins. Thus, the regulation of HS biosynthesis is likely to influence a wide variety of biological events. This thesis focuses on the biosynthesis of HS in animals with targeted disruptions in genes important for HS production. The N-deacetylase N-sulfotransferase (NDST) is a key enzyme in HS biosynthesis, directing other modifications. We show that NDST isoforms have very different roles in HS biosynthesis. Inactivation of NDST1 affects HS biosynthesis in all tissues. In embryonic liver HS from NDST1-/- mice the N-sulfation was decresed with twothirds, while the absence of NDST2 did not affect HS structure. In the absence of NDST1 in the liver, however, NDST2 is active in HS N-sulfation. In a study of embryonic stem cells lacking both NDST1 and NDST2, no N-sulfate groups could be detected. 6-O-sulfate groups were, however, still present at half of its normal level. This was an unexpected finding since 6-O-sulfotransferases have been thought to be strictly dependent on N-sulfate groups for substrate recognition. By adapting an automated method for HS analysis to mammalian tissues, we could extend our analyses to more tissues and other transgene models. We also developed a protocol to create a sensitive “fingerprint” of HS structure. With these methods we could determine the individual HS structure of different mouse tissues.

Heparan sulfate (HS) is a ubiquitous polysaccharide on the cell surface and in the extracellular matrix. HS is an important actor in the regulation of cell signaling, especially in the developing embryo. In combination with cell culture and biochemical experiments, in vivo studies of genetically modified animals have pointed out the sulfation pattern of HS as highly important for binding of ligands, their receptors and other signaling modulators. The sulfation pattern of an HS chain is gained by several modifying steps, performed by multiple enzymes during biosynthesis in the Golgi apparatus. By alterations of sulfation pattern, and the amount of sulfate groups, a cell can regulate the binding properties of its HS to different molecules. The most highly sulfated form of HS is called heparin, and can only be found intracellularly in mast cells. This thesis describes the phenotypes and the alterations in HS/heparin biosynthesis of two genetically modified mouse strains deficient in N-deacetylase/N-sulfotransferase-1 (NDST1) and -2 (NDST2) respectively. We have found NDST1 to be important for correct sulfation of HS and that NDST2 is crucial in heparin biosynthesis. NDST2 deficient mice completely lack heparin and therefore have a severe mast cell phenotype. NDST1 deficient mice produce undersulfated HS and show several developmental disturbances. Some NDST1 embryos die in utero while the rest die neonatally due to breathing difficulties. Defect brain, eye and skeletal development has also been observed while some organs, such as the liver, appear to be largely unaffected. Several phenotypes are similar to defects seen in other mouse strains with impaired fibroblast growth factor and bone morphogenetic protein signaling, among others. This suggests the phenotypes of NDST1 deficient embryos to be of a multi factorial origin, in complete accordance to the many signaling pathways HS is suggested to modulate.