Sowohl in Süßwasserseen als auch in marinen Gewässern kommt es immer wieder zu Massenentwicklungen von Cyanobakterien, sogenannten “Blüten”. In Seen werden diese oftmals von Cyanobakterien der Gattung Microcystis dominiert, deren Arten häufig Toxine bilden. Die verbreitesten dieser Toxine sind die leberschädigen Microcystine, die eine Klasse nichtribosomal synthetisierter Peptide darstellen. Nachdem die toxische Wirkung der Microcystine bisher als deren Hauptfunktion angesehen wurde, deuten neuere Forschungsergebnisse darauf hin, dass Microcystine eine andere Primärfunktion für die Produzenten besitzen. Im Rahmen dieser Studie wurde Microvirin (Mvn), ein putatives Lektin aus Microcystis aeruginosa PCC 7806, von dem angnommen wurde, dass es funktional mit Microcystin assoziiert ist, charakterisiert. Zunächst konnte gezeigt werden, dass Mvn tatsächlich zuckerbindende Aktivität besitzt und spezifisch Mannan, ein Oligosaccharid aus Mannoseuntereinheiten, erkennt. Bindestudien zeigten, dass Zucker dieses Typs auf der Zelloberfläche von M. aeruginosa PCC 7806 lokalisiert sind und eine Bindestelle für das sekretierte Mvn darstellen. Mit Hilfe fluoreszenzmikroskopiebasierender Methoden wurde gezeigt, dass sowohl Mvn als auch das korrespondierende Mannanoligosaccharid stammspezifisch sind. Weiterhin konnte durch PCR gezeigt werden, dass das mvn-Gen in allen getesteten Microcystis-Stämmen vorkommt, die auch Gene für die Microcystinbiosynthese besitzen. Eine direkte Interaktion von Microcystin und Mvn konnte in vitro bestätigt werden. Microcystin bindet dabei über seinen N-Methyl-Dehydroalaninrest kovalent an die reduzierten Cysteinreste des Proteins. Ein Einfluss auf die Oligomerisierung des Proteins wurde festgestellt. Microcystin bindet an Cysteinreste von Proteinen, und es konnte gezeigt werden, dass dies besonders unter oxidativen Stressbedingungen geschieht. Die Daten liefern somit weitere Indizien für eine Rolle von Microcystin in der Stressadaptation. / Cyanobacteria frequently appear as so-called “water-blooms” during summer months. Cyanobacteria of the genus Microcystis, whose species often dominate freshwater lakes, produce toxins that represent a potential threat for humans and animals. The most prominent toxins are the non-ribosomally synthesised hepatotoxic microcystins. Toxicity has been considered the main function of these peptides, but recent studies propose different primary functions of microcystins for their producers. The involvement of microcystins in the response to oxidative stress was proposed recently. Within this study the putative lectin microvirin (Mvn), which was suggested to be functionally related to microcystin, was characterised. Initially it was shown that Mvn does indeed possess a carbohydrate binding activity, and specificity for mannan, an oligosaccharide made of mannose subunits, was proven. Binding studies using fluorescence-labelled Mvn and antibodies identified carbohydrates of this type at the cell surface of M. aeruginosa being a binding site for the secreted Mvn. Fluorescence microscopy techniques were employed to show that Mvn as well as the corresponding mannan oligosaccharide are strain-specific. Additionally it was shown by PCR that the mvn gene is present in all tested Microcystis strains possessing microcystin biosynthesis genes. A direct interaction of microcystin and Mvn was confirmed in vitro. Microcystin covalently binds to the reduced cysteine residues of the protein via its N-methyl-dehydroalanine moiety. An impact on the oligomerisation state of Mvn was observed. Microcystin seems to bind cysteine residues in an unspecific manner in vivo, and it was shown that this occurs especially under conditions of oxidative stress such as iron depletion and exposition to high light. Hence, the data provide further evidence for an involvement of microcystins in stress adaptation.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Das Lektin aus <i>Pisum sativum</i>, der Gartenerbse, ist Teil der Familie der Leguminosenlektine. Diese Proteine haben untereinander eine hohe Sequenzhomologie, und die Struktur ihrer Monomere, ein all-ß-Motiv, ist hoch konserviert. Dagegen gibt es innerhalb der Familie eine große Vielfalt an unterschiedlichen Quartärstrukturen, die Gegenstand kristallographischer und theoretischer Arbeiten waren. Das Erbsenlektin ist ein dimeres Leguminosenlektin mit einer Besonderheit in seiner Struktur: Nach der Faltung in der Zelle wird aus einem Loop eine kurze Aminosäuresequenz herausgeschnitten, so dass sich in jeder Untereinheit zwei unabhängige Polypeptidketten befinden. Beide Ketten sind aber stark miteinander verschränkt und bilden eine gemeinsame strukturelle Domäne. Wie alle Lektine bindet Erbsenlektin komplexe Oligosaccharide, doch sind seine physiologische Rolle und der natürliche Ligand unbekannt. In dieser Arbeit wurden Versuche zur Entwicklung eines Funktionstests für Erbsenlektin durchgeführt und seine Faltung, Stabilität und Monomer-Dimer-Gleichgewicht charakterisiert. Um die spezifische Rolle der Prozessierung für Stabilität und Faltung zu untersuchen, wurde ein unprozessiertes Konstrukt in <i>E. coli</i> exprimiert und mit der prozessierten Form verglichen. <br /> <br /> Beide Proteine zeigen die gleiche kinetische Stabilität gegenüber chemischer Denaturierung. Sie denaturieren extrem langsam, weil nur die isolierten Untereinheiten entfalten können und das Monomer-Dimer-Gleichgewicht bei mittleren Konzentrationen an Denaturierungsmittel auf der Seite der Dimere liegt. Durch die extrem langsame Entfaltung zeigen beide Proteine eine apparente Hysterese im Gleichgewichtsübergang, und es ist nicht möglich, die thermodynamische Stabilität zu bestimmen. Die Stabilität und die Geschwindigkeit der Assoziation und Dissoziation in die prozessierten bzw. nichtprozessierten Untereinheiten sind für beide Proteine gleich. Darüber hinaus konnte gezeigt werden, dass auch unter nicht-denaturierenden Bedingungen die Untereinheiten zwischen den Dimeren ausgetauscht werden.<br /> <br /> Die Renaturierung der unprozessierten Variante ist unter stark nativen Bedingungen zu 100 % möglich. Das prozessierte Protein dagegen renaturiert nur zu etwa 50 %, und durch die Prozessierung ist die Faltung stark verlangsamt, der Faltungsprozess ist erst nach mehreren Tagen abgeschlossen. Im Laufe der Renaturierung wird ein Intermediat populiert, in dem die längere der beiden Polypeptidketten ein Homodimer mit nativähnlicher Untereinheitenkontaktfläche bildet. Der geschwindigkeitsbestimmende Schritt der Renaturierung ist die Assoziation der entfalteten kürzeren Kette mit diesem Dimer. / The lectin from <i>Pisum sativum</i> (garden pea) is a member of the family of legume lectins. These proteins share a high sequence homology, and the structure of their monomers, an all-ß-motif, is highly conserved. Their quaternary structures, however, show a great diversity which has been subject to cristallographic and theoretical studies. Pea lectin is a dimeric legume lectin with a special structural feature: After folding is completed in the cell, a short amino acid sequence is cut out of a loop, resulting in two independent polypeptide chains in each subunit. Both chains are closely intertwined and form one contiguous structural domain. Like all lectins, pea lectin binds to complex oligosaccharides, but its physiological role and its natural ligand are unknown. In this study, experiments to establish a functional assay for pea lectin have been conducted, and its folding, stability and monomer-dimer-equilibrium have been characterized. To investigate the specific role of the processing for stability and folding, an unprocessed construct was expressed in <i>E. coli</i> and compared to the processed form.<br /> <br /> Both proteins have the same kinetic stability against chemical denaturant. They denature extremely slowly, because only the isolated subunits can unfold, and the monomer-dimer-equilibrium favors the dimer at moderate concentrations of denaturant. Due to the slow unfolding, both proteins exhibit an apparent hysteresis in the denaturation transition. Therefore it has not been possible to determine their thermodynamic stability. For both proteins, the stability and the rates of association and dissociation into processed or unprocessed subunits, respectively, are equal. Furthermore it could be shown that even under non-denaturing conditions the subunits are exchanged between dimers.<br /> <br /> Renaturation of the unprocessed variants is possible under strongly native conditions with 100 % yield. The processed protein, however, can be renatured with yields of about 50 %, and its refolding is strongly decelerated. The folding process is finished only after several days. During renaturation, an intermediate is populated, in which the longer of the two polypeptide chains forms a homodimer with a native-like subunit interface. The rate limiting step of renaturation is the association of the unfolded short chain with this dimer.

This work is centered on the study of the NKG2 C-type lectin-like receptors on NK and CD4+T cells. We provide evidence supporting that CD4+T cells specific for Human Cytomegalovirus (HCMV) may express different NK cell receptors, and demonstrate that the C-type lectin-like receptor NKG2D is expressed on cytotoxic CD4+T cells with an effector/memory phenotype, enhancing their TCR-dependent proliferation and cytokine production. A second part of the work is centered on the study of the CD94/NKG2 receptors on NK cells. We show that NKG2A can be induced on NKG2C+ NK cells upon activation with rIL-12 or when cocultured with HCMV-infected dendritic cells, and that NKG2A expression inhibits the response of NKG2C+NK clones against HLA-E-expressing targets, providing a potential regulatory feedback mechanism to control cell activation. Altogether, our results support that expression of NKG2 C-type lectin like receptors may be shaped during the course of viral infections, providing mechanisms to finely regulate both NK and CD4+T cell functions. / Aquesta tesi es centra en l'estudi dels receptors lectina de tipus C NKG2 en cèl·lules Natural Killer i T CD4+. Demostrem que les cèl·lules T CD4+ específiques pel Cytomegalovirus Humà poden expressar diferents receptors NK, i que el receptor lectina tipus C NKG2D s'expressa en cèl·lules citotòxiques i de memòria, potenciant la proliferació i secreció de citocines depenent del TCR. La segona part d'aquesta tesi es centra en l'estudi de l'expressió dels receptors CD94/NKG2 en cèl·lules NK. Mostrem com l'expressió de CD94/NKG2A s'indueix en cèl·lules CD94/NKG2C+ estimulades amb IL-12 o cultivades amb cèl·lules dendrítiques infectades pel Cytomegalovirus Humà, i que l'expressió de CD94/NKG2A inhibeix la resposta de clons NK CD94/NKG2C+ envers dianes HLA-E+, constituint un possible mecanisme de feedback negatiu per controlar l'activació cel·lular. En resum, els nostres resultats demostren que l'expressió dels receptors lectina tipus C NKG2 pot ser modificada durant les infeccions víriques consitutint un possible mecanisme per regular la resposta tant de cèl·lules NK com T CD4+.

Cystic fibrosis (CF) is a single gene Mendelian disorder characterized by pulmonary disease and pancreatic insufficiency. Pulmonary disease is the major cause of death in CF patients. Although some cystic fibrosis transmembrane conductance regulator (CFTR) genotypes are associated with less severe disease, patients possessing the same genotype show great variation in pulmonary disease severity and progression. Genes involved in modulating the inflammatory response and genes increasing susceptibility to infection are proposed as modifiers of pulmonary disease severity. Polymorphisms selected for based on evidence that they affect the function of the gene and prevalence of the putative risk allele: 1) antiprotease gene alpha-1-antitrypsin (alpha-1-AT), 2) innate immunity genes: mannose binding lectin (MBL2) (promoter [G→C] at -221 and codon 52 (Arg52Cys, D allele), 54 (Gly54Asp, B allele), and 57 (Gly57Glu, C allele), and pulmonary surfactant genes SPA-1 (Arg219Trp), SPA-2 (Thr9Asn, Lys223Gln) and SPD (Thr11Met), 3) antioxidant genes GSTM1 and T1 (gene deletion polymorphisms), GSTP1 (Ile105Val) and GCLC repeats, 4) mucin genes (MUC2 and MUC5B). Pulmonary disease progression and survival in patients with chronic Burkholderia cepacia complex (BCC) infection were also investigated controlling for genomovar and RAPD type of the organism. BCC infection was associated with more severe pulmonary disease progression and worse survival. Alpha-1-AT genotype was not a major contributor to variability of pulmonary disease severity, but the results suggest that alpha-1-AT plasma levels during pulmonary infections may be affected by poor nutritional status. We showed similar pulmonary disease progression and MBL2 genotype. Contrary to the previous literature, wild-type MBL2 genotype was associated with steeper decline in pulmonary disease over time following chronic infection with BCC, but genotype was not associated with increased susceptibility to BCC infection. We showed inconsistant results for the pulmonary surfactant gene polymorphisms, GSTM1, T1 and GSTP1 polymorphisms, and number of repeats for GCLC and MUC5B depending on the phenotype investigated. We conclude that some of the variability in pulmonary disease severity and progression in CF is explained by polymorphisms in secondary genes.

Cystic fibrosis (CF) is a single gene Mendelian disorder characterized by pulmonary disease and pancreatic insufficiency. Pulmonary disease is the major cause of death in CF patients. Although some cystic fibrosis transmembrane conductance regulator (CFTR) genotypes are associated with less severe disease, patients possessing the same genotype show great variation in pulmonary disease severity and progression. Genes involved in modulating the inflammatory response and genes increasing susceptibility to infection are proposed as modifiers of pulmonary disease severity. Polymorphisms selected for based on evidence that they affect the function of the gene and prevalence of the putative risk allele: 1) antiprotease gene alpha-1-antitrypsin (alpha-1-AT), 2) innate immunity genes: mannose binding lectin (MBL2) (promoter [G→C] at -221 and codon 52 (Arg52Cys, D allele), 54 (Gly54Asp, B allele), and 57 (Gly57Glu, C allele), and pulmonary surfactant genes SPA-1 (Arg219Trp), SPA-2 (Thr9Asn, Lys223Gln) and SPD (Thr11Met), 3) antioxidant genes GSTM1 and T1 (gene deletion polymorphisms), GSTP1 (Ile105Val) and GCLC repeats, 4) mucin genes (MUC2 and MUC5B). Pulmonary disease progression and survival in patients with chronic Burkholderia cepacia complex (BCC) infection were also investigated controlling for genomovar and RAPD type of the organism. BCC infection was associated with more severe pulmonary disease progression and worse survival. Alpha-1-AT genotype was not a major contributor to variability of pulmonary disease severity, but the results suggest that alpha-1-AT plasma levels during pulmonary infections may be affected by poor nutritional status. We showed similar pulmonary disease progression and MBL2 genotype. Contrary to the previous literature, wild-type MBL2 genotype was associated with steeper decline in pulmonary disease over time following chronic infection with BCC, but genotype was not associated with increased susceptibility to BCC infection. We showed inconsistant results for the pulmonary surfactant gene polymorphisms, GSTM1, T1 and GSTP1 polymorphisms, and number of repeats for GCLC and MUC5B depending on the phenotype investigated. We conclude that some of the variability in pulmonary disease severity and progression in CF is explained by polymorphisms in secondary genes.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.

Les composantes innée et acquise de l'immunité travaillent ensemble pour assurer une protection efficace de l'organisme. Les cellules dendritiques, cellules sentinelles de l’immunité capturent via des récepteurs de surface les agents pathogènes et les présentent aux lymphocytes T pour stimuler les réponses immunitaires adaptatives spécifiques. Une famille de ces récepteurs, nommée Récepteurs Lectines de type C (CLRs) ont un rôle important dans la reconnaissance de motifs oligosaccharides des pathogènes. Leurs fonctions sont parfois détournées par certains pathogènes à leur avantage et notamment le VIH. La reconnaissance du virus par DC-SIGN, une des CLRs, favorise la dissémination du virus. A l’inverse, la langerine, autre CLR, est considérée comme une barrière naturelle au VIH. Ainsi, DC-SIGN est devenue une cible thérapeutique prometteuse mais sa reconnaissance des ligands osidiques est largement partagée par la langerine.Ce travail vise à développer des antagonistes de DC-SIGN spécifiques et de hautes affinités permettant de rivaliser avec la présentation multivalente des glycosylations de gp120 du VIH avec DC-SIGN. Une approche rationnelle a été employée dans le développement de ligands glycomimétiques hautement sélectifs pour DC-SIGN à partir de l’étude du site de liaison des deux CLRs. Puis, des plates-formes de présentations de ces glycomimétiques, de valences et de géométries différentes, sont comparées par SPR. Les améliorations spectaculaires d'affinités parfois observées sont liées à différents mécanismes d’interactions multivalentes responsables d’un phénomène d’avidité.Sur une des architecture de présentation sélectionnée (RODs), un travail de caractérisation fine des mécanismes responsables de ce gain d’affinité et/ou d’avidité a été conduit par la combinaison de plusieurs techniques biophysiques (SPR, ITC, polarisation de fluorescence et AUC). L’influence de la topologie de cette structure sur les mécanismes d’interactions est ainsi mise en évidence. Par les travaux menés, plusieurs ligands multivalents ont montré des affinités sans précédent pour DC SIGN atteignant des affinités du nanomolaire et représentant les meilleurs inhibiteurs connus à ce jour.Associé au développement d’antagonistes multivalents, une CLR (DCIR) a été identifiée récemment comme impliquée dans la dissémination du VIH, comme DC¬SIGN. Dans une perspective future de développement de glycomimétique, des travaux ont été menés sur la caractérisation structurale et fonctionnelle de ce nouvel acteur dans la problématique VIH. / The innate and acquired immunity components work together to provide efficient protection of organisms. Dendritic cells, sentinel cells of the immunity, are able to capture pathogens through their receptors on the surface and they can present the antigens to lymphocytes T in order to stimulate specific adaptive immune responses. Among these receptors, there is a family named C-type lectin receptors (CLRs), which has an important role in the recognition of pathogenic oligosaccharide motifs. CLRs can be hijacked by many pathogens including HIV. DC-SIGN, one of the CLRs, interacts with the virus and promotes its dissemination. Unlike DC-SIGN, langerin, another CLR, has a protective role against the HIV infection. In this context, DC-SIGN became a promising therapeutic target but it shares ligand specificities with langerin.This work aims to develop highly specific antagonists against DC-SIGN in order to compete with the multivalent glycosylated gp120 protein of HIV. Using the study of the two lectins binding sites as starting point, a rational approach has been exploited to develop highly selective glycomimetics against DC SIGN. The SPR technique was used to investigate multivalent platforms with different valencies as well as ligand presentation in space. The amazing improvement of the affinity observed in some cases can be linked to different mechanisms of multivalent interactions, leading to an avidity phenomenon. On a selected scaffold (RODs), we characterized the different mechanisms responsible for the affinity and/or avidity gains, using a combination of different biophysical techniques (SPR, ITC, fluorescence polarization, AUC). In this work, we highlighted that the topology of this structure can influence the mechanisms of interactions. Overall, different multivalent ligands showed unique affinities for DC-SIGN, reaching the nanomolar affinity range, and they represent the best inhibitors to date.Finally, another CLR has been recently identified as one of the protein involved in the HIV infection as well as DC-SIGN. In a future perspective of glycomimetic development, structural and functional characterization has been done on this new actor involved in the HIV issue.