Protein N-glycosylation, which is ubiquitous in eukaryotes, is a complex pathway involving numerous gene families. Early stages of the glycosylation pathway show a high degree of conservation among eukaryotes, yet diversification of the number and size of gene families involved in the later stages of the pathway has led to the evolution of increasingly complex N-glycan structures and functions in various organisms. The overall purpose of this research project has been to characterize the diversity within the α-mannosidase gene family of filamentous fungi. The α-1,2-mannosidases are involved in mannose removal in the intermediate stages of the N-glycosylation pathways, and diversification of this gene family may have provided the first significant divergence in these pathways among major lineages. Four novel α-mannosidases were identified and characterized from the filamentous fungus Aspergillus nidulans. These genes were designated Class II α-mannosidase, Class I α-1,2-mannosidase IA, Class I α-1,2-mannosidase IB and Class I α-1,2-mannosidase IC, based on their similarity to other Class I and Class II α-mannosidase sequences. The Class II α-mannosidase was highly similar to the rat ER/cytosolic and yeast vacuolar Class II α-mannosidases, and these three proteins formed a phylogenetically distinct subgroup, Class IIC. The Class I enzymes were highly related to each other, and to other fungal Class I α-1,2-mannosidases. Phylogenetic analysis indicates these genes duplicated and diverged subsequent to the divergence of fungi from insects and mammals. In addition to this research on A. nidulans, a single Class I α-1,2-mannosidase was identified and characterized from the Dutch Elm pathogen, Ophiostoma novo-ulmi, which was highly related to the A. nidulans Class I α-1,2-mannosidase IA and IC enzymes, and less so to the A. nidulans Class I α-1,2-mannosidase IB. Analysis of the function and/or biochemical properties of these enzymes was examined using several methods. Disruption and overexpression of the A. nidulans Class IIC α-mannosidase did not have any noticeable effect on the growth or morphology of the organism, indicating that this gene was not essential for growth. Biochemical characterization of the A. nidulans Class I α-1,2-mannosidase IC was initiated by recombinant secretion of the enzyme into culture media. Successful expression of the enzyme showed that the α-1,2-mannosidase IC did not exert any cytotoxic effects when overexpressed, suggesting that high levels of expression and purification should be feasible. Finally, disruption of the Class I α-1,2-mannosidase from O. novo-ulmi slightly altered the morphology of the organism, but was not lethal. The possible presence of multiple Class I α-1,2-mannosidases in this organism could explain the non-lethality of this mutation. Elucidation of the N-glycosylation pathways of A. nidulans may be useful in host strain improvement for heterologous protein expression systems. Modulation of the N-glycosylation pathways to produce specific N-glycan structures would increase the utility of the host for the production of human therapeutic glycoproteins which require these N-glycans for efficacy. Additionally, investigation of the genetic components of the N-glycosylation pathways of the Dutch Elm pathogen may provide global antifungal targets with broad applicability in other fungi. / Graduate

Glycosylation is a fundamental post-translational modification of proteins that occurs in all eukaryotes, but the biological significance of specific glycosylation structures is still largely unknown. We have chosen to study this process by cloning and characterizing the genes involved in the N-linked glycosylation pathway in Drosophila melanogaster and analysing mutants of these genes. Since this biochemical pathway for the production of N-linked glycans is considerably conserved, studying it in Drosophila, a useful organism for genetic research, should yield results applicable to other eukaryotes. This thesis describes the attempt to identify a Drosophila homologue of rat ER mannosidase and how this investigation resulted in the identification of a Drosophila cDNA with considerable homology to calnexin, an enzyme involved in the folding of N-glycosylated proteins. It also describes the cloning and characterization of the genomic sequence for the putative Golgi mannosidase II (GmII)* in Drosophila. The expression and control of expression of GmII have been investigated further, and have suggested expression of this gene throughout Drosophila development. Analysis of the GmII promoter region has shown expression from a TATA-less promoter contained within 288bp of sequence 5' to the start of transcription.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 6 uppsatser.

Foi realizado o pirossequenciamento de duas bibliotecas de cDNA do intestino médio de Tenebrio molitor e as sequências foram submetidos à montagem através do programa Newbler. Visando sanar alguns questionamentos a respeito de muitos tipos de transportadores que pudessem estar envolvidos com funções presumíveis em tamponamento luminal, absorção de nutrientes, envolvimento em mecanismos de secreção de enzimas como a α-manosidases e secreção e absorção de água, foram analisadas sequências de interesse que pudessem esclarecer os fenômenos fisiológicos em questão. O pirossequenciamento revelou 19 sequencias de α-manosidases. Após alinhamentos múltiplos, desconfiou-se que o contig 12 era a continuação da sequência original da α-manosidase. Utilizando-se de iniciadores apropriados, a suspeita foi confirmada e uma sequência completa foi obtida e denominada de TmMan1. Através de cladogramas gerados com as sequências de todos os contigs obtidos, assim como de sequências representativas das famílias 38 e 47 das glicosídeos hidrolases, mostrou que todas a nossas sequências, exceto o contig 6 e 7, pertencem à família 38. Todas as sequências com mais de 100 reads (exceto o contig 9) tiveram a sua expressão tecidual avaliada por RT-PCR. Os resultados mostraram que só são expressos no intestino ou intestino e túbulo de Malpighi, implicando na possibilidade de serem digestivas. Dessas sequências, as únicas com peptídeo sinal são a TmMan1 (contig 12) e o contig 14 e, portanto, devem corresponder às atividades Man1 e Man2. Levando em conta o número de reads, TmMan1 deveria corresponder a Man2 e o contig 14 à Man1. É possível, embora necessite de confirmação, que os contigs 8 e 15 sejam de expressão lisossômica. Um peptídeo sintetizado que correspondia a sequencia única da TmMan1 foi usado para gerar anticorpos, que reconheceram a Man2, mas não a Man1, confirmando a identificação de TmMan1 com a Man2. Esse anticorpo foi também utilizado para imunolocalizar a TmMan1 nas células intestinais de T. molitor. Os resultados mostraram que a TmMan1 é secretada de forma apócrina pela região anterior de intestino de T. molitor. Esse trabalho é o primeiro que mostra a ocorrência de α-manosidases com especificidade similar àquelas lissossômicas, mas que são secretadas apócrinamente para fora da célula, devendo agir no lúmen intestinal, removendo resíduos de manoses de oligossacarídeos manosilados. Foram identificados 10 tipos diferentes de transportadores e na elaboração dos modelos fisiológicos só foram levados em conta aqueles expressos exclusivamente no intestino médio ou no intestino médio e túbulos de Malpighi. A V-ATPase em T.molitor parece ser uma bomba usada para energizar muitos dos transportes ao longo do intestino médio como, por exemplo, o de oligopeptídeos. Já as bombas de Na+ e K+ são responsáveis pelo equilíbrio de cargas e, portanto estão presentes na maioria dos tipos celulares. Duas sequências de cotransportadores de oligopeptídeos/H+ foram encontradas no pirossequenciamento e sua expressão é maior na região posterior, uma vez que ali é a última possibilidade de absorção dos oligopeptídeos que ainda estiverem no lúmen, remanescentes da digestão final de proteínas. Foi demonstrado que T.Molitor absorve aminoácidos e açúcares ao longo de todo o intestino médio, pois estes tipos de transportadores possuem uma expressão uniforme ao longo do intestino médio. Já a expressão dos transportadores de NH3/NH4+ em T.molitor, encontra-se confinada ás regiões onde o pH do intestino médio do inseto é mais ácida. Também há uma expressão de transportadores de cloreto que se manifesta mais intensamente na região anterior. Podemos visualizar que a distribuição dos contigs dos transportadores de bicarbonato encontra-se mais expressiva na região posterior do intestino médio. Os resultados sugerem que a acidificação na região anterior do intestino de T.molitor pode resultar da secreção de NH4+ acompanhado do íon cloreto e a alcalinização na região posterior do lançamento no lúmen de bicarbonato. A importância dos canais de cloreto é que o mesmo balanceia as cargas e desta forma pode ser útil juntamente com o transporte de NH4+, que gera uma carga no lado onde é transportado. Há absorvição de água (junto com glicose) ao longo de todo o intestino médio, enquanto que a secreção de água ocorreria apenas nos dois terços finais do intestino médio com o auxílio de aquaporinas complementado por transportadores de íons, teria como consequência a abosorção líquida de água na região anterior e uma secreção líquida no final do intestino médio. Isso esclarece qual a base molecular para a ocorrência do contrafluxo intestinal evidenciado por experimentos fisiológicos. / Pyrosequencing was performed with two cDNA libraries in the midgut of Tenebrio molitor and the sequences were subjected to assembly with the Newbler program. In order to tackle questions concerning proteins which may be involved in midgut buffering, nutrient absorption, in the secretion of enzymes such as α-mannosidases , and water absorption and secretion, sequences of interest were analyzed in order to clarify those physiological phenomena. The pyrosequencing revealed 19 sequences of α- mannosidases . After multiple alignments, it was suspected that the contig 12 was the continuation of the original sequence of the α-mannosidase. Using appropriate primers, the hypothesis was confirmed and a complete sequence was obtained and named TmMan . Through cladograms generated from the sequences of the contigs obtained, as well as of sequences representing families 38 and 47 of glycoside hydrolases, it was showed that all sequences except the contig 6 and 7 belong to family 38. All sequences with over 100 reads (except contig 9) had their tissue expression assessed by RT-PCR. The results showed that they are expressed only in the midgut or midgut and Malpighian tubules, implying the possibility of having a digestive function. Among these sequences, the only ones with a signal peptide are TmMan1 (contig 12) and contig 14 and therefore they should correspond to the activities Man1 and Man2. Taking into account the number of reads, TmMan1 should correspond to Man2 and contig 14 to Man1. It is possible, though it requires confirmation, that the contigs 8 and 15 are lysosomal. A peptide corresponding to the unique sequence TmMan1 was synthesized and used to generate antibodies that recognized Man2 , but not Man1, confirming the identification of TmMan1 with Man2. This antibody was also used to immunolocalyze TmMan1 in the midgut cells of T. molitor. The results showed that TmMan1 is secreted in an apocrine way by the anterior region of T. molitor midgut. This is the first study that shows the occurrence of α-mannosidases with similar specificity to those lysosomal, but that are secreted in an apocrine way, acting in the midgut lumen, removing mannoses from mannosylated oligosaccharides. We identified 10 different types of carriers and in the development of physiological models it was only taken into account those expressed exclusively in the midgut or midgut and Malpighian tubules . The V- ATPase in T.molitor appears to be a pump used for powering many transports along the midgut, as of oligopeptides. The Na+ and K+ pumps are responsible for charge load balancing and therefore are present in most cell types. Two sequences of oligopeptide / H+ cotransporters were found in the transcriptome and their expression is higher in the posterior region. This agrees with the fact that there is the last possibility of oligopeptides remaining in the lumen to be absorbed. It is highly probable that T.molitor absorbs amino acids and sugars throughout the midgut, once these types of carriers have a uniform expression throughout the midgut . The expression of NH3/NH4+ transporters in T.molitor is confined to the regions where the pH of the insect midgut is more acidic. There is also chloride transporter expression there. The expression of bicarbonate transporters is more significant in the posterior midgut. The results suggest that acidification in the anterior T.molitor midgut may result from the secretion of NH4+ and chloride ions together, whereas the alkalization in the posterior midgut results from bicarbonate release. There is water absortion (along with glucose) throughout the midgut , while the water secretion occurs only in the final two-thirds of the midgut with the aid of aquaporins, complemented by ion transporters. It would result in the net absorption and net secretion of water in the anterior and postertior midgut, respectively. This clarifies the molecular basis of the midgut countercurrent fluxes evidenced by physiological experiments.

Foi realizado o pirossequenciamento de duas bibliotecas de cDNA do intestino médio de Tenebrio molitor e as sequências foram submetidos à montagem através do programa Newbler. Visando sanar alguns questionamentos a respeito de muitos tipos de transportadores que pudessem estar envolvidos com funções presumíveis em tamponamento luminal, absorção de nutrientes, envolvimento em mecanismos de secreção de enzimas como a α-manosidases e secreção e absorção de água, foram analisadas sequências de interesse que pudessem esclarecer os fenômenos fisiológicos em questão. O pirossequenciamento revelou 19 sequencias de α-manosidases. Após alinhamentos múltiplos, desconfiou-se que o contig 12 era a continuação da sequência original da α-manosidase. Utilizando-se de iniciadores apropriados, a suspeita foi confirmada e uma sequência completa foi obtida e denominada de TmMan1. Através de cladogramas gerados com as sequências de todos os contigs obtidos, assim como de sequências representativas das famílias 38 e 47 das glicosídeos hidrolases, mostrou que todas a nossas sequências, exceto o contig 6 e 7, pertencem à família 38. Todas as sequências com mais de 100 reads (exceto o contig 9) tiveram a sua expressão tecidual avaliada por RT-PCR. Os resultados mostraram que só são expressos no intestino ou intestino e túbulo de Malpighi, implicando na possibilidade de serem digestivas. Dessas sequências, as únicas com peptídeo sinal são a TmMan1 (contig 12) e o contig 14 e, portanto, devem corresponder às atividades Man1 e Man2. Levando em conta o número de reads, TmMan1 deveria corresponder a Man2 e o contig 14 à Man1. É possível, embora necessite de confirmação, que os contigs 8 e 15 sejam de expressão lisossômica. Um peptídeo sintetizado que correspondia a sequencia única da TmMan1 foi usado para gerar anticorpos, que reconheceram a Man2, mas não a Man1, confirmando a identificação de TmMan1 com a Man2. Esse anticorpo foi também utilizado para imunolocalizar a TmMan1 nas células intestinais de T. molitor. Os resultados mostraram que a TmMan1 é secretada de forma apócrina pela região anterior de intestino de T. molitor. Esse trabalho é o primeiro que mostra a ocorrência de α-manosidases com especificidade similar àquelas lissossômicas, mas que são secretadas apócrinamente para fora da célula, devendo agir no lúmen intestinal, removendo resíduos de manoses de oligossacarídeos manosilados. Foram identificados 10 tipos diferentes de transportadores e na elaboração dos modelos fisiológicos só foram levados em conta aqueles expressos exclusivamente no intestino médio ou no intestino médio e túbulos de Malpighi. A V-ATPase em T.molitor parece ser uma bomba usada para energizar muitos dos transportes ao longo do intestino médio como, por exemplo, o de oligopeptídeos. Já as bombas de Na+ e K+ são responsáveis pelo equilíbrio de cargas e, portanto estão presentes na maioria dos tipos celulares. Duas sequências de cotransportadores de oligopeptídeos/H+ foram encontradas no pirossequenciamento e sua expressão é maior na região posterior, uma vez que ali é a última possibilidade de absorção dos oligopeptídeos que ainda estiverem no lúmen, remanescentes da digestão final de proteínas. Foi demonstrado que T.Molitor absorve aminoácidos e açúcares ao longo de todo o intestino médio, pois estes tipos de transportadores possuem uma expressão uniforme ao longo do intestino médio. Já a expressão dos transportadores de NH3/NH4+ em T.molitor, encontra-se confinada ás regiões onde o pH do intestino médio do inseto é mais ácida. Também há uma expressão de transportadores de cloreto que se manifesta mais intensamente na região anterior. Podemos visualizar que a distribuição dos contigs dos transportadores de bicarbonato encontra-se mais expressiva na região posterior do intestino médio. Os resultados sugerem que a acidificação na região anterior do intestino de T.molitor pode resultar da secreção de NH4+ acompanhado do íon cloreto e a alcalinização na região posterior do lançamento no lúmen de bicarbonato. A importância dos canais de cloreto é que o mesmo balanceia as cargas e desta forma pode ser útil juntamente com o transporte de NH4+, que gera uma carga no lado onde é transportado. Há absorvição de água (junto com glicose) ao longo de todo o intestino médio, enquanto que a secreção de água ocorreria apenas nos dois terços finais do intestino médio com o auxílio de aquaporinas complementado por transportadores de íons, teria como consequência a abosorção líquida de água na região anterior e uma secreção líquida no final do intestino médio. Isso esclarece qual a base molecular para a ocorrência do contrafluxo intestinal evidenciado por experimentos fisiológicos. / Pyrosequencing was performed with two cDNA libraries in the midgut of Tenebrio molitor and the sequences were subjected to assembly with the Newbler program. In order to tackle questions concerning proteins which may be involved in midgut buffering, nutrient absorption, in the secretion of enzymes such as α-mannosidases , and water absorption and secretion, sequences of interest were analyzed in order to clarify those physiological phenomena. The pyrosequencing revealed 19 sequences of α- mannosidases . After multiple alignments, it was suspected that the contig 12 was the continuation of the original sequence of the α-mannosidase. Using appropriate primers, the hypothesis was confirmed and a complete sequence was obtained and named TmMan . Through cladograms generated from the sequences of the contigs obtained, as well as of sequences representing families 38 and 47 of glycoside hydrolases, it was showed that all sequences except the contig 6 and 7 belong to family 38. All sequences with over 100 reads (except contig 9) had their tissue expression assessed by RT-PCR. The results showed that they are expressed only in the midgut or midgut and Malpighian tubules, implying the possibility of having a digestive function. Among these sequences, the only ones with a signal peptide are TmMan1 (contig 12) and contig 14 and therefore they should correspond to the activities Man1 and Man2. Taking into account the number of reads, TmMan1 should correspond to Man2 and contig 14 to Man1. It is possible, though it requires confirmation, that the contigs 8 and 15 are lysosomal. A peptide corresponding to the unique sequence TmMan1 was synthesized and used to generate antibodies that recognized Man2 , but not Man1, confirming the identification of TmMan1 with Man2. This antibody was also used to immunolocalyze TmMan1 in the midgut cells of T. molitor. The results showed that TmMan1 is secreted in an apocrine way by the anterior region of T. molitor midgut. This is the first study that shows the occurrence of α-mannosidases with similar specificity to those lysosomal, but that are secreted in an apocrine way, acting in the midgut lumen, removing mannoses from mannosylated oligosaccharides. We identified 10 different types of carriers and in the development of physiological models it was only taken into account those expressed exclusively in the midgut or midgut and Malpighian tubules . The V- ATPase in T.molitor appears to be a pump used for powering many transports along the midgut, as of oligopeptides. The Na+ and K+ pumps are responsible for charge load balancing and therefore are present in most cell types. Two sequences of oligopeptide / H+ cotransporters were found in the transcriptome and their expression is higher in the posterior region. This agrees with the fact that there is the last possibility of oligopeptides remaining in the lumen to be absorbed. It is highly probable that T.molitor absorbs amino acids and sugars throughout the midgut, once these types of carriers have a uniform expression throughout the midgut . The expression of NH3/NH4+ transporters in T.molitor is confined to the regions where the pH of the insect midgut is more acidic. There is also chloride transporter expression there. The expression of bicarbonate transporters is more significant in the posterior midgut. The results suggest that acidification in the anterior T.molitor midgut may result from the secretion of NH4+ and chloride ions together, whereas the alkalization in the posterior midgut results from bicarbonate release. There is water absortion (along with glucose) throughout the midgut , while the water secretion occurs only in the final two-thirds of the midgut with the aid of aquaporins, complemented by ion transporters. It would result in the net absorption and net secretion of water in the anterior and postertior midgut, respectively. This clarifies the molecular basis of the midgut countercurrent fluxes evidenced by physiological experiments.

The thesis is divided in to five chapters. Chapter I: Synthesis of New thiolevomannosan derivatives In this chapter, a general and efficient method for the synthesis of conformationally locked thiosugars has been discussed. An unprecedented synthesis of a novel thioorthoester and its synthetic utility in glycosylation has been demonstrated. Chapter II: Studies on -Mannosidase Inhibitors The synthesis and evaluation of novel, conformationally locked glycomimic, thiolevomannosan and its analogs sulfoxide and sulfone starting from readily available D-mannose is discussed in this chapter. This is the first report of thiosugar derivatives with enhanced potency compared to kifunensine. Docking and biochemical studies have been discussed. Chapter III: Studies on Novel Cyclic Tetraselenides of Mannose In this chapter, the syntheses and structural properties of novel cyclic tetraselenides starting from mannose have been discussed. These tetraselenides are the first of their kind where all four selenium atoms are arranged in a cyclic manner as the backbone of mannose. X-ray structures have been reported for the tetraselenides. Chapter IV: Novel Chalcogenides of Uridine and Thymidine: Synthesis and Applications An efficient and simple method to synthesise disulfides and diselenides of thymidine and uridine derivatives has been demonstrated in this chapter. The utility of these disulfides in various ring opening reactions as well in Michael addition reactions has been demonstrated. Chapter V: Studies on New, Potent Urease Inhibitors In this chapter, a facile one-pot synthesis of thio and selenourea derivatives under mild conditions by the reaction of amines and commercially available Viehe’s iminium salt in the presence of benzyltriethylammonium tetrathiomolybdate as sulfur transfer reagent and tetraethylammonium tetraselenotungstate as selenium transfer reagent has been discussed. A few of the urea derivatives have shown potent inhibitor activity in the nanomolar range for jackbean urease.