The FRIABLE1 (FRB1) gene is identified to be a novel glycosyltransferase involved in cell adhesion, based on reverse genetics and immunocytochemistry studies. A total of 31 FRB1 paralogues were found in Arabidopsis thaliana using a bioinformatics approach. The following expression analysis has revealed 6 FRB1 paralogues to be pollen-specific. One pollen-specific FRB1 paralogue, At1g14970, exhibits longer silique lengths when exposed to higher than normal temperature at 28oC in its T-DNA insertional knockout when compared to Columbia wildtype plants. This may be due to the loss of temperature sensing and the continuous stimulated pollen tube cell wall growth or the up-regulation of genes that encode other glycosyltransferases. Thus, the identification of FRB1 paralogues and homologues in both rice and poplar may have tremendous potential to increase their yield in global warming for agricultural and industrial benefits.

Glycosylation is one of the most abundant post-translational modifications of proteins. Glycoproteins participate in virtually all aspects of cellular functions. ST6Gal I is a glycosyltransferase highly expressed by B and T cells. Here, we interrogated the role of ST6Gal I in viral specific B and T cell immune responses, as well as examined how loss of this enzyme impacted viral pathogenesis. First, to understand how loss of ST6Gal I expression impacted viral specific humoral responses, we infected ST6Gal I-/- mice with influenza virus. We discovered that loss of ST6Gal I expression results in both reduced influenza specific antibodies levels and decreased viral-specific antibody secreting cells numbers. Following influenza infection, mice that received ST6Gal I-/- B cells showed reduced influenza-specific IgM responses compared to mice that received wild-type B cells. These experiments demonstrated that the expression of ST6Gal I by B cells is required for optimal viral-specific humoral response. We further examined how loss of ST6Gal I expression impacted the anti-influenza IgA response. We observed that immune ST6Gal I-/- mice displayed higher viral specific IgA levels and altered sialylation of IgG and IgA, which have been implicated in a human disease, IgA nephropathy. Moreover, ST6Gal I-/- mice exhibited increased immunoglobulin deposition in kidney glomeruli following influenza infection. These data suggest that ST6Gal I deficiency, together with influenza infection, may result in the initiation of a kidney disease. Finally, we examined how ST6Gal I expression regulated CD8 T cell responses. We discovered that ST6Gal I is differentially expressed during CD8 T cell activation. To understand its relevance, we infected ST6Gal I-/- mice and demonstrated that the early expansion of effector T cells was impaired in a cell intrinsic manner. Moreover, in the absence of ST6Gal I, the differentiation of CD8 T cells skewed towards memory precursor cells, whereas terminal effector cell expansion was impaired. Mechanistically, we identified delayed surface expression of IL-2Ralpha on ST6Gal I-/- CD8 T cells due to impaired IL-2/IL-2R signaling. These studies implicate that ST6Gal I expression enhances early proliferation of terminal effector CD8 T cells by promoting the rapid surface expression of IL2Ralpha during acute viral infection. 

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-99) Also available in print.

A ciclodextrina glicosiltransferase (CGTase, EC 2.4.1.19) faz parte da família das α-amilases e se destaca por ser a única enzima capaz de produzir ciclodextrinas (CDs). Esses oligossacarídeos cíclicos possuem a capacidade de formar complexos de inclusão com uma variedade de moléculas, alterando suas características como, por exemplo, solubilidade, volatilidade e estabilidade. Desta forma, CDs tem encontrado aplicação nas mais diversas áreas. Na indústria de alimentos, se destacam por serem potenciais estabilizantes naturais. Buscando alternativas viáveis para produção destas ciclodextrinas, neste trabalho, a enzima CGTase foi imobilizada covalentemente em esferas de quitosana e posteriormente utilizada em um reator enzimático para uso contínuo. O rendimento da imobilização foi de aproximadamente 100 %, com uma carga de 20 mg de enzima por grama de suporte seco. O processo de imobilização foi capaz de manter o comportamento da enzima frente à variação de pH e temperatura de reação, apresentando pH ótimo em 5,0 e a faixa de temperatura ótima de 70 a 95 ºC, para ambos. A estabilidade conferida ao catalisador imobilizado possibilitou sua reutilização, 61 % da sua atividade inicial foi mantida após 100 ciclos de reação. Durante utilização contínua, realizada em um reator de leito fixo, analisou-se a influência da taxa de fluxo e da concentração do substrato na geração de β-CD. A máxima produção (1,32 g / L) foi alcançada utilizando-se 4 % de amido solúvel em uma taxa de fluxo de 3 mL / min. Além disso, o biocatalisador apresentou uma ótima estabilidade operacional a 60 °C, mantendo 100 % da atividade inicial após 100 h de uso contínuo. Estes resultados demonstram que o desempenho do reator é diretamente afetado pelos parâmetros analisados e que a produção pode ser otimizada por regulação simples na velocidade de fluxo, ou pela concentração do substrato; e sugerem a possibilidade de utilizar este biocatalisador imobilizado na produção contínua de CDs. / Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is member of the family α-amylase and is known for being the only enzyme able to produce cyclodextrins (CDs). These cyclic oligosaccharides have the ability to form inclusion complexes with a variety of molecules, changing its characteristics, for example, solubility, volatility and stability. Therefore, CDs have found application in several fields. In the food industry stand out for being potential natural stabilizers. Seeking to alternatives for producing these cyclodextrins, in this work, the CGTase enzyme was immobilized covalently on chitosan beads and subsequently used in enzymatic reactor for continuous use. The immobilization yield was high, reaching about 100 %, representing a load of 20 mg enzyme per gram of dry support. The immobilization process was capable of maintaining the behavior of the enzyme to the variation of pH and temperature of reaction, with pH optimum at 5.0 and the optimal temperature range of 70 - 95 ° C, for both. The stability afforded to the immobilized catalyst made possible its reuse, maintaining 61 % of its initial activity after 100 cycles of reaction. During its continuous use, in a packed bed reactor, we analyzed the influence of flow rate and concentration of the substrate in the generation of β-CD. The maximum yield (1.32 g / L) was achieved using 4 % soluble starch at a flow rate of 3 mL / min. In addition, the biocatalyst showed a great operational stability at 60 ° C, maintaining 100 % of initial activity after 100 h of continuous use. These results demonstrate that the performance is directly affected by the parameters analyzed and that the production can be optimized by simple adjustment in flow rate through the reactor, or the substrate concentration used and suggests the possibility of using this biocatalyst immobilized to the continuous production of CDs.

A ciclodextrina glicosiltransferase (CGTase, EC 2.4.1.19) faz parte da família das α-amilases e se destaca por ser a única enzima capaz de produzir ciclodextrinas (CDs). Esses oligossacarídeos cíclicos possuem a capacidade de formar complexos de inclusão com uma variedade de moléculas, alterando suas características como, por exemplo, solubilidade, volatilidade e estabilidade. Desta forma, CDs tem encontrado aplicação nas mais diversas áreas. Na indústria de alimentos, se destacam por serem potenciais estabilizantes naturais. Buscando alternativas viáveis para produção destas ciclodextrinas, neste trabalho, a enzima CGTase foi imobilizada covalentemente em esferas de quitosana e posteriormente utilizada em um reator enzimático para uso contínuo. O rendimento da imobilização foi de aproximadamente 100 %, com uma carga de 20 mg de enzima por grama de suporte seco. O processo de imobilização foi capaz de manter o comportamento da enzima frente à variação de pH e temperatura de reação, apresentando pH ótimo em 5,0 e a faixa de temperatura ótima de 70 a 95 ºC, para ambos. A estabilidade conferida ao catalisador imobilizado possibilitou sua reutilização, 61 % da sua atividade inicial foi mantida após 100 ciclos de reação. Durante utilização contínua, realizada em um reator de leito fixo, analisou-se a influência da taxa de fluxo e da concentração do substrato na geração de β-CD. A máxima produção (1,32 g / L) foi alcançada utilizando-se 4 % de amido solúvel em uma taxa de fluxo de 3 mL / min. Além disso, o biocatalisador apresentou uma ótima estabilidade operacional a 60 °C, mantendo 100 % da atividade inicial após 100 h de uso contínuo. Estes resultados demonstram que o desempenho do reator é diretamente afetado pelos parâmetros analisados e que a produção pode ser otimizada por regulação simples na velocidade de fluxo, ou pela concentração do substrato; e sugerem a possibilidade de utilizar este biocatalisador imobilizado na produção contínua de CDs. / Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is member of the family α-amylase and is known for being the only enzyme able to produce cyclodextrins (CDs). These cyclic oligosaccharides have the ability to form inclusion complexes with a variety of molecules, changing its characteristics, for example, solubility, volatility and stability. Therefore, CDs have found application in several fields. In the food industry stand out for being potential natural stabilizers. Seeking to alternatives for producing these cyclodextrins, in this work, the CGTase enzyme was immobilized covalently on chitosan beads and subsequently used in enzymatic reactor for continuous use. The immobilization yield was high, reaching about 100 %, representing a load of 20 mg enzyme per gram of dry support. The immobilization process was capable of maintaining the behavior of the enzyme to the variation of pH and temperature of reaction, with pH optimum at 5.0 and the optimal temperature range of 70 - 95 ° C, for both. The stability afforded to the immobilized catalyst made possible its reuse, maintaining 61 % of its initial activity after 100 cycles of reaction. During its continuous use, in a packed bed reactor, we analyzed the influence of flow rate and concentration of the substrate in the generation of β-CD. The maximum yield (1.32 g / L) was achieved using 4 % soluble starch at a flow rate of 3 mL / min. In addition, the biocatalyst showed a great operational stability at 60 ° C, maintaining 100 % of initial activity after 100 h of continuous use. These results demonstrate that the performance is directly affected by the parameters analyzed and that the production can be optimized by simple adjustment in flow rate through the reactor, or the substrate concentration used and suggests the possibility of using this biocatalyst immobilized to the continuous production of CDs.

A ciclodextrina glicosiltransferase (CGTase, EC 2.4.1.19) faz parte da família das α-amilases e se destaca por ser a única enzima capaz de produzir ciclodextrinas (CDs). Esses oligossacarídeos cíclicos possuem a capacidade de formar complexos de inclusão com uma variedade de moléculas, alterando suas características como, por exemplo, solubilidade, volatilidade e estabilidade. Desta forma, CDs tem encontrado aplicação nas mais diversas áreas. Na indústria de alimentos, se destacam por serem potenciais estabilizantes naturais. Buscando alternativas viáveis para produção destas ciclodextrinas, neste trabalho, a enzima CGTase foi imobilizada covalentemente em esferas de quitosana e posteriormente utilizada em um reator enzimático para uso contínuo. O rendimento da imobilização foi de aproximadamente 100 %, com uma carga de 20 mg de enzima por grama de suporte seco. O processo de imobilização foi capaz de manter o comportamento da enzima frente à variação de pH e temperatura de reação, apresentando pH ótimo em 5,0 e a faixa de temperatura ótima de 70 a 95 ºC, para ambos. A estabilidade conferida ao catalisador imobilizado possibilitou sua reutilização, 61 % da sua atividade inicial foi mantida após 100 ciclos de reação. Durante utilização contínua, realizada em um reator de leito fixo, analisou-se a influência da taxa de fluxo e da concentração do substrato na geração de β-CD. A máxima produção (1,32 g / L) foi alcançada utilizando-se 4 % de amido solúvel em uma taxa de fluxo de 3 mL / min. Além disso, o biocatalisador apresentou uma ótima estabilidade operacional a 60 °C, mantendo 100 % da atividade inicial após 100 h de uso contínuo. Estes resultados demonstram que o desempenho do reator é diretamente afetado pelos parâmetros analisados e que a produção pode ser otimizada por regulação simples na velocidade de fluxo, ou pela concentração do substrato; e sugerem a possibilidade de utilizar este biocatalisador imobilizado na produção contínua de CDs. / Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is member of the family α-amylase and is known for being the only enzyme able to produce cyclodextrins (CDs). These cyclic oligosaccharides have the ability to form inclusion complexes with a variety of molecules, changing its characteristics, for example, solubility, volatility and stability. Therefore, CDs have found application in several fields. In the food industry stand out for being potential natural stabilizers. Seeking to alternatives for producing these cyclodextrins, in this work, the CGTase enzyme was immobilized covalently on chitosan beads and subsequently used in enzymatic reactor for continuous use. The immobilization yield was high, reaching about 100 %, representing a load of 20 mg enzyme per gram of dry support. The immobilization process was capable of maintaining the behavior of the enzyme to the variation of pH and temperature of reaction, with pH optimum at 5.0 and the optimal temperature range of 70 - 95 ° C, for both. The stability afforded to the immobilized catalyst made possible its reuse, maintaining 61 % of its initial activity after 100 cycles of reaction. During its continuous use, in a packed bed reactor, we analyzed the influence of flow rate and concentration of the substrate in the generation of β-CD. The maximum yield (1.32 g / L) was achieved using 4 % soluble starch at a flow rate of 3 mL / min. In addition, the biocatalyst showed a great operational stability at 60 ° C, maintaining 100 % of initial activity after 100 h of continuous use. These results demonstrate that the performance is directly affected by the parameters analyzed and that the production can be optimized by simple adjustment in flow rate through the reactor, or the substrate concentration used and suggests the possibility of using this biocatalyst immobilized to the continuous production of CDs.

Abstract Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen. Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types. Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product. The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.

Abstract Lysyl hydroxylase (LH, EC 1.14.11.4) catalyzes the post-translational hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. The hydroxylysyl residues participate in the formation of collagen cross-links, and some of the hydroxylysyl residues are further glycosylated. Three lysyl hydroxylase isoforms LH1, LH2 and LH3, encoded by three individual genes have been characterized and one isoform, LH3 is a multifunctional enzyme containing lysyl hydroxylase, collagen galactosyltransferase (GT, E.C. 2.4.1.50) and glucosyltransferase (GGT, E.C. 2.4.1.66) activities in vitro. In this thesis the genes for the mouse lysyl hydroxylases were each mapped to a different chromosome. In addition, the roles of the lysyl hydroxylase isoforms were characterized in mice by studying their expression during development and the distribution of LH2 and LH3 in adult mice. The results revealed a widespread expression of the mouse lysyl hydroxylases during embryonic development whereas LH2 and LH3 showed tissue- or cell-specific expression patterns in the adult. Alternative splicing of the gene for LH2 also showed developmental and tissue-specific regulation. The different functions of LH3 were studied in vivo by generating three different LH3 manipulated mouse lines. Analysis of the mouse lines revealed that LH3 has lysyl hydroxylase and glucosyltransferase activities in vivo, and that, in particular, the glucosyltransferase activity of LH3 is essential for normal development. The loss of glucosyltransferase activity caused disruption of basement membranes leading to embryonic lethality while the absence of lysyl hydroxylase activity led to ultrastructural alterations in muscle and basement membranes and disorganization of collagen fibrils. The disruption of basement membrane was due to an intracellular accumulation of unglycosylated type IV collagen, whereas the ultrastructural alterations were related to the abnormal aggregation and distribution of underglycosylated type VI collagen. The results demonstrate that hydroxylysine-linked glycosylations are critical for the secretion of type IV collagen and its assembly into basement membranes, and for the assembly and distribution of type VI collagen.

No description available.

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>