Dehydroabietinal (DA), an abietane diterpenoid, was previously demonstrated to be a potent activator of systemic acquired resistance (SAR). DA also promotes flowering time in Arabidopsis thaliana by repressing expression of the flowering repressor FLOWERING LOCUS C (FLC) while simultaneously upregulating expression of FLOWERING LOCUS D (FLD), FLOWERING LOCUS VE (FVE) and RELATIVE OF EARLY FLOWERING 6 (REF6), a set of flowering time promoters. To further understand the mechanism underlying signaling by abietane diterpenoids, Arabidopsis mutants exhibiting reduced responsiveness to abietane diterpenoids were identified. One such mutant plant, ems2/7, exhibited SAR-deficiency and delayed flowering, which were found to be associated with two independent, but linked loci. The gene responsible for the SAR defect in ems2/7 was identified as DEFECTIVE IN SYSTEMIC DEFENSE INDUCED BY ABIETANE DITERPENOID 1 (DSA1). Similar to the missense mutant dsa1-1 identified in the mutant screen, the T-DNA insertion bearing null allele dsa1-2 exhibited SAR deficiency that could be complemented by a genomic copy of DSA1. The gene responsible for the delayed flowering phenotype of ems2/7 remains to be identified. DSA1 encodes a protein that is homologous to human protein O-fucosyltransferase 2. DSA1 is required for long-distance transport of the SAR signal. It is hypothesized that DSA1 is involved in the O-fucosylation-facilitated channeling through the ER/Golgi network of a protein involved in long distance SAR signaling. In a yeast two-hybrid screen, all the DSA1-interacting proteins identified are chloroplast-localized proteins, thus raising the interesting possibility of ER interaction with chloroplast and its potential role in SAR signaling.

No description available.

The correct and timely delivery of immune cells is critical for protection against foreign antigen. In order for cells to access most organs, there are requirements that must be met to facilitate exit from the blood into extravasculature. The initial requirement is selectin-selectin ligand interactions that mediate tethering and rolling to allow shear resistance. For proper selectin-selectin ligand interaction, glycoproteins must be modified by fucosyltransferases –IV and –VII, which adds fucose to an acceptor substrate to form the sialyl-LewisX moiety. Using fucosyltransferase –IV and –VII double knockout (FtDKO) mice, we made several novel observations. Our first observation showed increased numbers of naïve T cells in non-lymphoid organs. To support this observation, we blocked chemokine-mediated entry into lymph nodes (LNs) with pertussis toxin and L-selectin mediated entry with anti-CD62L antibody in WT mice. We also treated WT mice with the S1P1 agonist, FTY720, to retain lymphocytes in LNs. Our results suggested that when access to LN is perturbed, lymphocytes accumulate in non-lymphoid organs. Our second observation showed an enrichment of effector/memory T cells in FtDKO LNs. To determine if effector/memory CD8 T cells were retained in LNs, we transferred naïve and memory CD8 T cells into WT mice then treated the recipient mice with anti-CD62L. We found that LN exit rates of naïve and memory CD8 T cells were similar, but slowed as T cell density decreased. To understand if memory CD8 T cells were using selectin ligand independent mechanisms, we transferred naïve and memory CD8 T cells into WT or FtDKO mice. We found reduced numbers of memory CD8 T cells in LNs, however, their frequency was increased. We explored this result by transferring CFSE labeled memory CD8 T cells. We found that memory CD8 T cells divide more in FtDKO mice compared to WT. These experiments suggested that selectin ligand deficiencies cause increased frequency of effector/memory T cells in LNs due to low density and increased emptiness induced proliferation. Taken together, these findings reveal how selectin ligand deficiencies contribute to T cell accumulation in non-lymphoid organs and elucidate mechanisms of retention in LNs.

The correct and timely delivery of immune cells is critical for protection against foreign antigen. In order for cells to access most organs, there are requirements that must be met to facilitate exit from the blood into extravasculature. The initial requirement is selectin-selectin ligand interactions that mediate tethering and rolling to allow shear resistance. For proper selectin-selectin ligand interaction, glycoproteins must be modified by fucosyltransferases –IV and –VII, which adds fucose to an acceptor substrate to form the sialyl-LewisX moiety. Using fucosyltransferase –IV and –VII double knockout (FtDKO) mice, we made several novel observations. Our first observation showed increased numbers of naïve T cells in non-lymphoid organs. To support this observation, we blocked chemokine-mediated entry into lymph nodes (LNs) with pertussis toxin and L-selectin mediated entry with anti-CD62L antibody in WT mice. We also treated WT mice with the S1P1 agonist, FTY720, to retain lymphocytes in LNs. Our results suggested that when access to LN is perturbed, lymphocytes accumulate in non-lymphoid organs. Our second observation showed an enrichment of effector/memory T cells in FtDKO LNs. To determine if effector/memory CD8 T cells were retained in LNs, we transferred naïve and memory CD8 T cells into WT mice then treated the recipient mice with anti-CD62L. We found that LN exit rates of naïve and memory CD8 T cells were similar, but slowed as T cell density decreased. To understand if memory CD8 T cells were using selectin ligand independent mechanisms, we transferred naïve and memory CD8 T cells into WT or FtDKO mice. We found reduced numbers of memory CD8 T cells in LNs, however, their frequency was increased. We explored this result by transferring CFSE labeled memory CD8 T cells. We found that memory CD8 T cells divide more in FtDKO mice compared to WT. These experiments suggested that selectin ligand deficiencies cause increased frequency of effector/memory T cells in LNs due to low density and increased emptiness induced proliferation. Taken together, these findings reveal how selectin ligand deficiencies contribute to T cell accumulation in non-lymphoid organs and elucidate mechanisms of retention in LNs.

Techn. Universiẗat, Diss., 2005--Darmstadt.

Les fucosyltransférases sont les enzymes responsables du transfert d'un groupement fucose à partir du GDP-fucose sur des accepteurs variés (oligosaccharides, protéines,...). Chez l'homme, les rôles de ces glycosyltransférases sont importants dans un grand nombre de processus biologiques et pathologiques. De nombreuses fucosyltransférases existent chez les végétaux. Notamment FuT1, qui transfert un fucose en 1,2 sur un résidu galactose du xyloglucane : l'une des hémicelluloses majeures de la paroi des dicotylédones. Ce polysaccharide ramifié est très étudié en raison de ses applications actuelles et potentielles dans différents secteurs de l'industrie : textile, alimentation, pharmaceutique, etc. Les objectifs de ce doctorat ont été d'obtenir des informations biochimiques et structurales sur la fucosyltransférase AtFuT1 de la plante modèle Arabidopsis thaliana. Pour cela, une forme recombinante soluble de l'enzyme a été produite avec le système baculovirus /cellules d'insectes. De manière à obtenir suffisamment de protéines pour les études structurales, une méthode de culture en suspension des cellules a été mise en place au laboratoire. Un protocole de purification en deux étapes, impliquant une chromatographie par affinité puis par exclusion de taille, a permis d'obtenir à partir d'un litre de culture cellulaire entre un à deux mg de protéine pure et homogène. Ce résultat a permis de mieux comprendre le comportement de l'enzyme vis-à-vis de ses substrats (GDP-fucose et xyloglucane), d'obtenir des cristaux de la protéine et de résoudre la structure 3D d'AtFuT1 en complexe avec le GDP et un oligosaccharide dérivé du xyloglucane, à une résolution de 2,2 Å. La forme AtFut1 produite se comporte en solution et dans le cristal sous forme d'un dimère non covalent. La protéine adopte un repliement qui est un variant du type GT-B classique. Parallèlement à ces travaux, des tests d'activité glycosyltransférase ont été mis au point permettant le criblage de nombreuses conditions de réactions. L'ensemble des méthodes et techniques développées constitue une base utile, devant faciliter la caractérisation d'autres glycosyltransférases. / Fucosyltransferases are enzymes that transfer a fucose residue from GDP-fucose on varied acceptors (oligosaccharides, proteins). In Human, these glycosyltransferases are involved in many biological and pathological processes. Numerous fucosyltransferase exist in the plant kingdom. Among them, FuT1 transfers a fucose linked in 1,2 onto a galactose of xyloglucan: a major hemicellulose of dicots cell wall. This branched polysaccharide is intensively studied because of its current and potential industrial applications in textile, food, pharmaceuticals, etc. The main objective of this PhD program was to obtain biochemical and structural information on the fucosyltransferase AtFuT1 from the model plant Arabidopsis thaliana. A recombinant form of this protein has therefore been produced, using the baculovirus/insect cell system. In order to get sufficient amount of protein for structural studies, a suspension cell culture method has been set-up in the lab. A two-step purification protocol, involving affinity and size exclusion chromatography was established. The active, and highly pure recovered protein was used to determine the biochemical properties of the protein towards its substrates (GDP-fucose and xyloglucan), to get protein crystals and hence to solve its 3D structure in complex with GDP and a xyloglucan derived oligosaccharide (2.2 Å resolution). AtFuT1 behaves in solution and in crystallo as a non-covalent dimer. The protein adopts a variant of the classical GTB fold. In addition, novel glycosyltransferase assay have been designed allowing the screening of numerous reaction conditions. Methods and techniques that were developed during this study should be a useful base for the characterization of other glycosyltransferase.

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