<p>The aim of the present study was to investigate the biosynthesis of the ATP synthase in various tissues, and to test hypotheses about possible models of activation of several mitochondrial proteins, the ATP/ADP translocase and UCPs, that could utilize the proton gradient, thus bypassing the ATP synthase. </p><p>We have examined the role of the expression of the P1 isoform of the c-F_o subunit in the biogenesis of ATP synthase in brown adipose tissue. Our findings point to a role for the c-F_o subunit in defining the final content of the ATP synthase in brown adipose tissue.</p><p>We have analyzed sequences in the 3’UTR of the β subunit F₁-ATPase mRNA that are important for formation of RNA-protein complexes. We could detect protein complexes that bind to two different sequence regions of the 3’UTR, one being the poly(A) tail and an adjacent region), and the other being a sequence stretch at the 3’ end of the 3’UTR able to form a stem-loop structure, which is evolutionarily conserved throughout mammalian species. </p><p>We investigated a role of the ATP/ADP carrier (ANT) in fatty acid-induced uncoupling in brown-fat mitochondria. We conclude that the ANT cannot substitute for UCP1 in fatty acid uncoupling in brown-fat mitochondria from mice lacking UCP1. We propose that the two ANT isoforms mediate proton translocation under different conditions.</p><p>We have investigated a role of UCP1 in defence against oxidative stress. We found that products of oxidative stress (4-HNE) could neither reactivate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1 in brown-fat mitochondria from UCP1(+/+) and UCP1(-/-) mice. We conclude that UCP1 is not involved in defence against oxidative stress. </p><p>We evaluated possible uncoupling activity of UCP3 in skeletal muscle from warm- and cold-acclimated UCP1(+/+) and UCP1(-/-) mice. We conclude that no evidence exists for a higher UCP3-mediated uncoupling activity; a high UCP3 content in cold-acclimated UCP1(-/-) mice could possibly be linked to improved fatty acid oxidative capacity.</p>

<p>Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures<i> </i>and growing<i> </i>plants<i>. </i>The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated.</p><p>A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb.</p><p>Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis.</p><p>Expression of the bacterial UGDH in <i>E. coli</i> resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. </p><p>Overexpression of UGD1, one of four <i>A. thaliana</i> UGDH genes, in <i>A. thaliana,</i> resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.</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>

<p>Heparan sulfates (HSs) are linear, negatively charged polysaccharides composed of alternating hexuronic acid (glucuronic acid or iduronic acid) and glucosamine residues that can be substituted to varying degrees with sulfate groups. HS, localized in the extracellular matrix and on the surface of most cells, interacts with a large number of proteins. The actions of HS largely depend on the amount and distribution of its sulfate groups, that provide binding sites for proteins. </p><p>This thesis focuses on the regulation of the structural diversity in HS, in particular the regulation of its 6-<i>O</i>-sulfation patterns that are generated by the combined action of 6-<i>O</i>-sulfotransferases (6OSTs) during biosynthesis, and 6-<i>O</i>-endosulfatases (Sulfs) after completed biosynthesis. In addition, a new model organism is introduced that offers good prospects for investigating the evolutional aspects of HS structural heterogeneity.</p><p>Our studies showed that the three mouse 6OSTs (6OST1-3) exhibit similar substrate specificities <i>in vitro</i>, with minor differences in target preferences. Overexpression of the 6OSTs in cells resulted in increased 6-<i>O</i>-sulfation of both <i>N</i>-sulfated and <i>N</i>-acetylated glucosamine residues. The changes were independent of enzyme isoform but positively correlated to the level of enzyme expressed.</p><p>Quail Sulf1 and Sulf2 enzymes were shown to be cell surface HS 6-<i>O-</i>endosulfatases with preference towards a subset of trisulfated disaccharides within HS chains. The Sulfs contain a “hydrophilic domain” that was shown to be essential for binding of HS, anchorage to the cell surface and endosulfatase activity. QSulf1 was also shown to promote Wnt-Frizzled signaling in cells. </p><p>An HS-like polysaccharide was isolated from the sea anemone <i>Nematostella vectensis</i> and characterized, and all the enzyme families involved in HS biosynthesis and modification in mammalian model systems were also identified. Our results suggest that <i>Nematostella</i> may be a useful tool for understanding the role of evolution in generating HS structural diversity.</p>

Amino acid biosynthesis is an essential process in living organisms. Certain amino acids can be synthesized by some organisms but not by others. L-Lysine is one of the essential amino acids that bacteria can synthesize but humans cannot. This is somewhat inconvenient for humans as much of their L-lysine must come from their diet. However, the lack of the lysine biosynthetic pathway in humans makes the bacterial enzymes within the pathway attractive drug targets. Recently, a novel lysine biosynthetic pathway was discovered in plants, Chlamydiae and some archaea. It is called the diaminopimelate aminotransferase (DAP-AT) pathway. In this pathway, LL-DAP-AT plays a key role by directly converting L-tetrahydrodipicolinate to LL-DAP in a single step. This is a quite interesting characteristic of LL-DAP-AT as the above conversion takes three sequential enzymatic steps in the previously known lysine biosynthetic pathways. Due to its absence in humans, LL-DAP-AT would be an attractive target for the development of novel antibiotics. In order to understand the catalytic mechanism and substrate recognition of LL-DAP-AT, the structural characterization of LL-DAP-AT is of paramount importance. In this thesis, the overall architecture of LL-DAP-AT and its substrate recognition mechanism revealed by the crystal structures of LL-DAP-AT from Arabidopsis thaliana and Chlamydia trachomatis will be discussed. The crystal structure of the native LL-DAP-AT from A. thaliana (AtDAP-AT) presented in this thesis is the first structure of LL-DAP-AT to be determined. This structure revealed that LL-DAP-AT forms a functional homodimer and belongs to the type I fold family of PLP dependent aminotransferases. The subsequent determination of the substrate-bound AtDAP-AT structure showed how the two substrates, (LL-DAP and L-Glu) significantly different in size, are recognized by the same set of residues without significant conformational changes in the backbone structure. In addition, the LL-DAP-bound AtDAP-AT structure shows that the C-amino group of LL-DAP is recognized stereospecifically by the active site residues that are unique to the family of LL-DAP-AT enzymes. Lastly, the chlamydial LL-DAP-AT presented in this thesis shows a new open conformation for LL-DAP-AT. The implications of the conformational flexibility of CtDAP-AT on the differences in substrate specificities among LL-DAP-AT are discussed.

Dans le cadre de ce travail, nous avons poursuivi l’étude de la biosynthèse d’alcaloïdes défensifs des coccinelles. Trois espèces ont été plus particulièrement étudiées : Adalia bipunctata (qui produit l’adaline [32]), Coccinella septempunctata (contenant la coccinelline [29]) et Harmonia axyridis (produisant l’harmonine [34]). Afin d’identifier le (ou les) acide(s) gras précurseur(s) de ces alcaloïdes, nous avons dans un premier temps synthétisé des acides gras spécifiquement marqués. Nous avons ainsi préparé les acides [14-3H]myristique, [16-3H]palmitique, [18-3H]stéarique, [18-13C]stéarique et [11,11,12,12,13,13,14,14,15,15,16, 16,17,17,18,18,18-2H]stéarique. Les différents acides gras marqués au tritium sur le méthyle terminal ont ensuite été incorporés successivement chez les trois espèces de coccinelles mentionnées ci-dessus, en utilisant la technique d’incorporation in vitro mise au point par Laurent et al. ( ) Les incorporations chez Adalia bipunctata ont montré que l’acide myristique est incorporé préférentiellement dans l’adaline. Chez Coccinella septempunctata par contre, l’acide stéarique est incorporé dans la coccinelline environ 25 fois plus efficacement que les acides myristique et palmitique. Enfin, les incorporations chez Harmonia axyridis ont établi que l’acide stéarique est le précurseur de l’harmonine. De plus, grâce à l’incorporation de l’acide [11,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18,18-2H]stéarique, le mécanisme de formation de l’amine secondaire a été précisé. / In spite of their red-orange colors, which could increase risks of predation, Coccinellidae are rarely exploited as food sources by predators. Many of them owe their protection, at least in part, to the presence of repellents and, in some cases, toxic alkaloids in the hemolymph emitted during a process called "reflex bleeding". Previous studies have shown that the biosynthesis of these alkaloids is related to fatty acid metabolism. In our doctoral thesis, we wanted to clarify what are the fatty acids precursors of adaline (Adalia bipunctata), coccinelline (Coccinella septempunctata) and harmonine (Harmonia axyridis), with the use of various techniques of labelling (3H, D, 13C, etc.).

This study introduces a method for multiparallel analysis of small organic compounds in the unicellular green alga Chlamydomonas reinhardtii, one of the premier model organisms in cell biology. The comprehensive study of the changes of metabolite composition, or metabolomics, in response to environmental, genetic or developmental signals is an important complement of other functional genomic techniques in the effort to develop an understanding of how genes, proteins and metabolites are all integrated into a seamless and dynamic network to sustain cellular functions. The sample preparation protocol was optimized to quickly inactivate enzymatic activity, achieve maximum extraction capacity and process large sample quantities. As a result of the rapid sampling, extraction and analysis by gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF) more than 800 analytes from a single sample can be measured, of which over a 100 could be positively identified. As part of the analysis of GC-TOF raw data, aliquot ratio analysis to systematically remove artifact signals and tools for the use of principal component analysis (PCA) on metabolomic datasets are proposed. Cells subjected to nitrogen (N), phosphorus (P), sulfur (S) or iron (Fe) depleted growth conditions develop highly distinctive metabolite profiles with metabolites implicated in many different processes being affected in their concentration during adaptation to nutrient deprivation. Metabolite profiling allowed characterization of both specific and general responses to nutrient deprivation at the metabolite level. Modulation of the substrates for N-assimilation and the oxidative pentose phosphate pathway indicated a priority for maintaining the capability for immediate activation of N assimilation even under conditions of decreased metabolic activity and arrested growth, while the rise in 4-hydroxyproline in S deprived cells could be related to enhanced degradation of proteins of the cell wall. The adaptation to sulfur deficiency was analyzed with greater temporal resolution and responses of wild-type cells were compared with mutant cells deficient in SAC1, an important regulator of the sulfur deficiency response. Whereas concurrent metabolite depletion and accumulation occurs during adaptation to S deprivation in wild-type cells, the sac1 mutant strain is characterized by a massive incapability to sustain many processes that normally lead to transient or permanent accumulation of the levels of certain metabolites or recovery of metabolite levels after initial down-regulation. For most of the steps in arginine biosynthesis in Chlamydomonas mutants have been isolated that are deficient in the respective enzyme activities. Three strains deficient in the activities of N-acetylglutamate-5-phosphate reductase (arg1), N2 acetylornithine-aminotransferase (arg9), and argininosuccinate lyase (arg2), respectively, were analyzed with regard to activation of endogenous arginine biosynthesis after withdrawal of externally supplied arginine. Enzymatic blocks in the arginine biosynthetic pathway could be characterized by precursor accumulation, like the amassment of argininosuccinate in arg2 cells, and depletion of intermediates occurring downstream of the enzymatic block, e.g. N2-acetylornithine, ornithine, and argininosuccinate depletion in arg9 cells. The unexpected finding of substantial levels of the arginine pathway intermediates N-acetylornithine, citrulline, and argininosuccinate downstream the enzymatic block in arg1 cells provided an explanation for the residual growth capacity of these cells in the absence of external arginine sources. The presence of these compounds, together with the unusual accumulation of N-Acetylglutamate, the first intermediate that commits the glutamate backbone to ornithine and arginine biosynthesis, in arg1 cells suggests that alternative pathways, possibly involving the activity of ornithine aminotransferase, may be active when the default reaction sequence to produce ornithine via acetylation of glutamate is disabled. / Entwicklung und Anwendung von Methoden zur multiparallelen Analyse von Metaboliten in der einzelligen Grünalge Chlamydomonas reinhardtii, einem der wichtigsten Modellorganismen der Zellbiologie, sind Gegenstand dieser Arbeit. Metabolomanalyse, die umfassende Analyse von Veränderungen der Konzentrationen von Stoffwechselprodukten durch Umweltreize oder genetische und entwicklungsbedingte Signale, ist ein wichtiges Komplement anderer Genomanalysemethoden, um die Integration von Genen, Proteinen und Metaboliten in ein nahtloses und dynamisches Netzwerk zur Aufrechterhaltung der Lebensfunktionen eines Organismus zu verstehen. Die Methode wurde im Hinblick auf schnelle Inaktivierung enzymatischer Aktivität, Maximierung der Extraktionskapazität und Behandlung großer Probenmengen optimiert. Im Ergebnis der Probenaufarbeitung, Extraktion und Analyse mittels Gaschromatographie und Time-Of-Flight-Massenspektrometrie konnten mehr als 800 analytische Signale in Einzelproben dargestellt werden, von denen über 100 identifiziert werden konnten. Die Arbeit stellt methodische Innovationen zur systematischen Erkennung von Artefakten in GC-MS Chromatogrammen und Werkzeuge zur Anwendung der Hauptkomponentenanalyse auf Metabolom-Daten vor. Zellen unter Stickstoff- (N), Phosphor- (P), Schwefel- (S), oder Eisen- (Fe) Mangel zeigen deutliche Unterschiede in ihrer Metabolitenausstattung. Die Anpassung an die einzelnen Nährstoffmangelsituationen ist durch spezifische Änderungen einer Reihe von Metaboliten zentraler Prozesse des Primärstoffwechsels gekennzeichnet. Die Konzentrationsänderungen von Substraten für die Stickstoffassimilation und den oxidativen Pentosephosphatweg deuten darauf hin, dass die Fähigkeit zur schnellen Aktivierung der N-Assimilation auch unter Bedingungen herabgesetzter Stoffwechsel- und Wachstumsaktivität aufrechterhalten wird. Die Akkumulation von 4-Hydroxyprolin unter Schwefelmangel könnte im Zusammenhang stehen mit der Degradation von Proteinen der Chlamydomonas-Zellwand, deren wesentlicher Bestandteil hydroxyprolinreiche Glykoproteine sind und die unter Schwefelmangel aktiv umgebaut wird. Die Anpassung an Schwefelmangel wurde mit größerer zeitlicher Auflösung in Wildtyp-Zellen und Zellen des sac1-Stammes untersucht. SAC1 ist ein zentraler Regulator der Schwefelmangelantwort in Chlamydomonas. Zeitgleiche Ab- und Zunahme von Metaboliten ist ein charakteristisches Element der Anpassung an Schwefelmangel in Wildtypzellen. Die Reaktion von SAC1-Mutanten auf Schwefelmangel ist durch weit reichenden Verlust zur Steuerung von Prozessen gekennzeichnet, die normalerweise zur vorübergehenden oder dauerhaften Anreicherung bestimmter Metabolite führen. Die Verfügbarkeit von Chlamydomonas-Stämmen mit fehlender Enzymaktivität für fast jeden der Schritte der Argininbiosynthese eröffnet die Möglichkeit, das Potential der Metabolitenanalyse zur Untersuchung der Regulation der Aminosäurebiosynthese in photosynthetischen Eukaryoten zur Anwendung zu bringen. Drei Stämme, mit fehlender Aktivität für N-Acetylglutamat-5-phosphat Reduktase (arg1), N2 Acetylornithin-Aminotransferase (arg9) beziehungsweise Argininosuccinat Lyase (arg2) wurden in Bezug auf die Aktivierung ihrer endogenen Argininbiosynthese nach Entzug externer Argininquellen analysiert. Die einzelnen enzymatischen Blocks konnten durch Precursor-Anreicherung, wie die Anhäufung von Argininosuccinat in arg2-Zellen, und Erschöpfung von Intermediaten nachgelagerter Reaktionen, beispielsweise die deutliche Abnahme von N2-Acetylornithin, Ornithin und Argininosuccinat in arg9-Zellen charakterisiert werden. Das unerwartete Vorhandensein von zum Teil das Wildtyp-Niveau überschreitender Mengen von N2-Acetylornithin, Citrullin und Argininosuccinat, die Produkte bzw. Substrate dem enzymatischen Block nachgelagerter Reaktionen in arg1-Zellen sind, bot eine Erklärung für eine noch vorhandene Restkapazität zum Wachstum des arg1-Stamms auch ohne äußere Arginingabe. Der Nachweis dieser Verbindungen sowie die ungewöhnliche Anreicherung von N-Acetylglutamat, der ersten Verbindung, die das Glutamat-Gerüst für die Ornithin- und Argininsynthese bindet, in arg1-Zellen könnte auf alternative Reaktionen, möglicherweise unter Beteiligung von Ornithin-Aminotransferase, zur Synthese von Ornithin hindeuten, die in Erscheinung treten, wenn die Synthesekette nach Acetylierung von Glutamat blockiert ist.

Polysaccharides are found in all forms of life and serve diverse purposes. They are enzymatically synthesised from activated monosaccharide precursors, nucleotide sugars. One such nucleotide sugar is UDP-glucuronic acid, which is formed from UDP-glucose by the UDP-glucose dehydrogenase (UGDH) enzyme. UGDH has been proposed to have a regulatory role in the biosynthesis of polysaccharides. The aim of the studies presented in this thesis was to investigate the role of UGDH in the polysaccharide biosynthesis in three different systems: human cell culture, bacterial cultures and growing plants. The effects of UGDH-overexpression on polysaccharide biosyntheses and, when achievable, on UDP-sugar levels, were investigated. A mammalian UGDH was cloned from a kidney cDNA library. Transient expression of the cloned enzyme in mammalian cells led to an increased UGDH-activity. Northern blotting analyses revealed a single transcript of 2.6 kb in adult mouse tissues whereas human tissues expressed a predominant transcript of 3.2 kb and a minor transcript of 2.6 kb. Overexpression of the bovine UGDH in mammalian cells induced increased synthesis of the glycosaminoglycans; heparan sulphate, chondroitin sulphate and hyaluronan, without changing their relative proportions. The effects on glycosaminoglycan synthesis caused by an increased demand of UDP-glucuronic acid were studied by overexpression of hyaluronan synthase (Has3), which requires UDP-glucuronic acid as substrate. Overexpression of Has3 and coexpression of Has3 and UGDH resulted in highly augmented production of hyaluronan without noticeably affecting heparan sulfate and chondroitin sulfate synthesis. Expression of the bacterial UGDH in E. coli resulted in increased formation of UDP-glucuronic acid, but, unexpectedly, also to synthesis of fewer K5 polysaccharide chains. Overexpression of UGD1, one of four A. thaliana UGDH genes, in A. thaliana, resulted in dwarfism. Analysis of the cell wall polysaccharides showed alteration in saccharide composition. Paradoxically, the UDP-sugars derived from UDP-glucuronic acid decreased in amount.

The aim of the present study was to investigate the biosynthesis of the ATP synthase in various tissues, and to test hypotheses about possible models of activation of several mitochondrial proteins, the ATP/ADP translocase and UCPs, that could utilize the proton gradient, thus bypassing the ATP synthase. We have examined the role of the expression of the P1 isoform of the c-Fo subunit in the biogenesis of ATP synthase in brown adipose tissue. Our findings point to a role for the c-Fo subunit in defining the final content of the ATP synthase in brown adipose tissue. We have analyzed sequences in the 3’UTR of the β subunit F1-ATPase mRNA that are important for formation of RNA-protein complexes. We could detect protein complexes that bind to two different sequence regions of the 3’UTR, one being the poly(A) tail and an adjacent region), and the other being a sequence stretch at the 3’ end of the 3’UTR able to form a stem-loop structure, which is evolutionarily conserved throughout mammalian species. We investigated a role of the ATP/ADP carrier (ANT) in fatty acid-induced uncoupling in brown-fat mitochondria. We conclude that the ANT cannot substitute for UCP1 in fatty acid uncoupling in brown-fat mitochondria from mice lacking UCP1. We propose that the two ANT isoforms mediate proton translocation under different conditions. We have investigated a role of UCP1 in defence against oxidative stress. We found that products of oxidative stress (4-HNE) could neither reactivate purine nucleotide-inhibited UCP1, nor induce additional activation of innately active UCP1 in brown-fat mitochondria from UCP1(+/+) and UCP1(-/-) mice. We conclude that UCP1 is not involved in defence against oxidative stress. We evaluated possible uncoupling activity of UCP3 in skeletal muscle from warm- and cold-acclimated UCP1(+/+) and UCP1(-/-) mice. We conclude that no evidence exists for a higher UCP3-mediated uncoupling activity; a high UCP3 content in cold-acclimated UCP1(-/-) mice could possibly be linked to improved fatty acid oxidative capacity.

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. .