Academic literature on the topic 'Lactosamine'

We have studied the elongation of oligosaccharides containing N-acetyl-lactosamine repeats using glycosylated human lysozyme mutants as a model. We reported previously that a combination of glycosylation sites at the 49th (site IV) and 68th (site II) amino acid residues of the protein particularly stimulates the synthesis of N-acetyl-lactosamine repeats [Melcher, Grosch, Grosse and Hasilik (1998) Glycoconjugate J. 15, 987-993]. In the present study we show that it is the carbohydrate attached to site IV that is selectively affected. It contains more N-acetyl-lactosamine repeats when site II is glycosylated in the same molecule. As a corollary of the glycosylation at site II, the synthesis of a third antenna at site IV is increased. The triantennary oligosaccharides at site IV contain more N-acetyl-lactosamine repeats than the biantennary ones. Thus placing a carbohydrate at site II stimulates the branching and the elongation of the carbohydrate at the other site.

beta 1,4-N-Acetylgalactosaminyltransferase (beta 1,4GalNAc-transferase) and alpha 2,3-sialyltransferase are both involved in the biosynthesis of the Sda blood group antigen, which is also present in cells of large intestine. The expression of these enzymes and of alpha 2,6-sialyltransferase activity towards N-acetyl-lactosamine was investigated in rat intestinal cells and correlated with both cell differentiation and extent of postnatal maturation. The beta 1,4GalNAc-transferase activity was exclusively found in epithelial cells of the large intestine, preferentially in the proximal segments suggesting a proximal-distal gradient of expression. The beta 1,4GalNAc-transferase and alpha 2,3-sialyltransferase activity towards N-acetyl-lactosamine were expressed in all cell fractions of the colonic crypt, with a maximum activity in the deeply located cells; therefore Sda antigen biosynthesis appears to occur preferentially at a specific stage of cell differentiation. By using N-acetyl-lactosamine as an acceptor, the predominant sialyltransferase in the colon cells was that capable of adding sialic acid in the alpha 2,3- linkage, whereas in the ileum cells the major enzyme was that forming the alpha 2,6-isomer. There were dramatic changes in the expression of colonic beta 1,4GalNac-transferase and of alpha 2,6-sialyltransferase activity towards N-acetyl-lactosamine during postnatal maturation. The former enzyme, practically absent at birth, increased slowly in the first days of life and then rapidly after weaning; by contrast, the latter enzyme was largely expressed only in newborn animals. As the colonic alpha 2,3-sialyltransferase activity towards N-acetyl-lactosamine did not change during the postnatal period, the ratio between the alpha 2,6- and alpha 2,3-sialyltransferase activities was reversed after weaning.

Given that galectin-3 (Gal-3) is a β-galactoside-binding lectin promoting tumor growth and metastatis, it could be a valuable target for the treatment of Gal-3-expressing neoplasms. An aromatic group introduced to the C-3′ position of lactosamine increased its affinity for Gal-3. Herein, we aimed at developing a radiopharmaceutical for the detection of Gal-3 positive malignancies. To enhance tumor specificity, a heterodimeric radiotracer capable of binding to both Gal-3 and αvβ3 integrin was also synthetized. Arginine-glycine-asparagine (RGD) peptide is the ligand of angiogenesis- and metastasis-associated αvβ3 integrin. Following the synthesis of the chelator-conjugated (2-naphthyl)methylated lactosamine, the obtained compound was applied as a precursor for radiolabeling and was conjugated to the RGD peptide by click reaction as well. Both synthetized precursors were radiolabeled with 68Ga, resulting in high labeling yield (>97). The biological studies were carried out using B16F10 melanoma tumor-bearing C57BL6 mice. High tumor accumulation of both labeled lactosamine derivatives—detected by in vivo PET and ex vivo biodistribution studies—indicated their potential for melanoma detection. However, the heterodimer radiotracer showed high hepatic uptake, while low liver accumulation characterized chelator-conjugated lactosamine, resulting in PET images with excellent contrast. Therefore, this novel carbohydrate-based radiotracer is suitable for the highly selective determination of Gal-3-expressing melanoma cells.

ABSTRACT Adenovirus isolate NADC-1, a strain of porcine adenovirus type 4, has a fiber containing an N-terminal virus attachment region, shaft and head domains, and a C-terminal galectin domain connected to the head by an RGD-containing sequence. The crystal structure of the head domain is similar to previously solved adenovirus fiber head domains, but specific residues for binding the coxsackievirus and adenovirus receptor (CAR), CD46, or sialic acid are not conserved. The structure of the galectin domain reveals an interaction interface between its two carbohydrate recognition domains, locating both sugar binding sites face to face. Sequence evidence suggests other tandem-repeat galectins have the same arrangement. We show that the galectin domain binds carbohydrates containing lactose and N-acetyl-lactosamine units, and we present structures of the galectin domain with lactose, N-acetyl-lactosamine, 3-aminopropyl-lacto-N-neotetraose, and 2-aminoethyl-tri(N-acetyl-lactosamine), confirming the domain as a bona fide galectin domain.

ABSTRACT Previously, we found that asialo-lactosamine sequences served as receptors for enteropathogenic Escherichia coli (EPEC) binding to Chinese hamster ovary (CHO) cells. In the present report, we have extended these earlier results by examining the ability of lactosamine- or fucosylated lactosamine-bovine serum albumin (BSA) glycoconjugates to inhibit EPEC, strain E2348/69, binding to HEp-2 cells. We found that, consistent with our previous findings with CHO cells, N-acetyllactosamine-BSA was the most effective inhibitor of EPEC localized adherence to HEp-2 cells, with Lewis X-BSA being the next best inhibitor. Further investigation revealed that coincubating EPEC E2348/69 with these BSA glycoconjugates alone caused a decrease in the expression of the bundle-forming pilus structural subunit (BfpA) and intimin by the bacteria. BfpA and intimin expression were reduced to the greatest extent byN-acetyllactosamine–BSA and Lewis X-BSA, respectively. These results suggest that the glycoconjugate inhibition of EPEC binding to HEp-2 cells might be achieved, wholly or in part, by an active mechanism that is distinct from simple competitive antagonism of receptor-adhesin interactions.

The envelope glycoprotein 130 (‘130’ referring to an Mr of 130,000) of simian immunodeficiency virus from sooty mangabey (Cercocebus atys) (SIVSM) was isolated from the cell-free supernatant of the SIVSM-infected human T-cell line H9, metabolically labelled with D-[6-3H]glucosamine. After digestion with Staphylococcus aureus V8 proteinase, radiolabelled N-glycans were liberated from resulting glycopeptides by sequential treatment with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F and fractionated by h.p.l.c. and gel filtration. Individual oligosaccharide species were characterized by enzymic microsequencing, chromatographic analyses and, in part, by acetolysis. The oligosaccharide structures thus established include oligomannosidic glycans with five to nine mannose residues as well as fucosylated and partially sialylated bi-, tri- and tetra-antennary N-acetyl-lactosaminic oligosaccharide species, the latter of which carry, in part, additional galactose residues or N-acetyl-lactosamine repeats. In comparison with the corresponding envelope glycoprotein 120 from human immunodeficiency virus type 1 (HIV-1), propagated in the same cell line [Geyer, Holschbach, Hunsmann and Schneider (1988) J. Biol. Chem. 263, 11760-11767], carbohydrates of the simian glycoprotein were found to consist of decreased amounts of oligomannosidic glycans and increased quantities of higher-branched N-acetyl-lactosaminic species.

We applied a horseradish peroxidase-Erythrina cristagalli agglutinin (HRP-ECA) conjugate for histochemical staining of tissue sections from various formalin-fixed, paraffin-embedded human tissue specimens. The HRP-ECA conjugate showed broad reactivity, but there was a distinct distribution of native (not masked by sialic acid) and sialic acid-masked ECA binding sites in the various organs. Free ECA binding sites could be detected on red blood cells, lymphocytes of thymus, tonsil, lymph node, and in mucous substances of different organs. Independent of blood group type, the vascular endothelium exhibited strong ECA reactivity. Free ECA binding sites occurred in the cytoplasm of Kupffer's cells in liver, in histiocytic cells of thymus, lymph node, tonsil, and in bone marrow. Podocytes of kidney glomerulus, syncytiotrophoblasts of placenta, megakaryocytes in bone marrow, myelin sheath of nerve, medullary thymocytes, and hepatocytes, as well as islet cells of pancreas, contained only sialic acid-capped ECA binding sites. Inhibiting studies with galactose, lactose, and N-acetyl-lactosamine, as well as other sugars, revealed that this lectin is specific for galactosyl residues. In comparison to galactose and lactose, N-acetyl-lactosamine exhibited the highest inhibitory activity on lectin binding, supporting the concept that this lectin is most reactive with N-acetyl-lactosamine-type (type 2 chain) glycoconjugates.

Omniprésentes dans le règne animal et végétal, les galectines occupent de nombreuses fonctions biologiques au sein des organismes vivants. Cette famille de lectines, capable de se lier aux B-galactosides grâce à un domaine de reconnaissance des sucres conservé, compte quinze membres chez les mammifères. Ces protéines ont pu être identifiées comme des acteurs importants de plusieurs processus biologiques tels que le cycle cellulaire, la réponse immunitaire ou encore la migration cellulaire. Plus récemment les galectines ont été identifiés comme des cibles thérapeutiques de choix car la dérégulation de l'expression de ces protéines a pu être corrélée à plus d'une centaine de pathologies (cancer, diabète, maladie inflammatoire,…). L’un des enjeux majeurs concernant la recherche sur ces protéines, est de parvenir à synthétiser des outils permettant une meilleure compréhension de leurs rôles au sein de l’organisme. Portant une attention plus particulière à la galectine-3, nous avons mis au point des voies de synthèses permettant l’obtention d’inhibiteurs affins et sélectifs de cette dernière, et ce dans le but d'être utilisés comme outils dans l'étude du rôle des galectines dans le phénomène de migration cellulaire des cellules épithéliales de la peau. Appliquant une réaction d’azido-phenylsélénation au lactal ou par une approche chimio-enzymatique, nous avons pu synthétiser deux familles de nouveaux composés dérivés de la lactosamine. L’affinité et la sélectivité de ces composés vis-à-vis des galectines ont été déterminées par des techniques de bio-puces, de polarisation de fluorescence ou encore de microcalorimétrie
Galectins are ubiquitous in the animal and plant kingdom. They are involved in many biological processes in living organisms. This family of lectins, which recognize B-galactosides through a highly conserved carbohydrate recognition domain, is composed of fifteen members in mammals. These proteins have been identified as key players in many biological processes such as cell cycle, immune response or cell migration. More recently galectins have been identified as therapeutic targets since the deregulation of the expression of these proteins has been correlated to more than one hundred diseases (cancer, diabetes, inflammatory diseases ...). One of the major issues concerning research on these proteins is to achieve synthesis of tools to delineate their roles in cellulo and in vivo. Focusing on galectin-3, we have developed synthetic routes for obtaining potent and selective inhibitors of this galectin to be used as tools in the study of galectins in the process of cell migration of epithelial cells. Applying an azido-phenylselenation reaction on lactal or by a chemo-enzymatic approach, we have been able to access to novel potential families of inhibitors based on lactosamine. The affinity and selectivity of these compounds on galectins was determined by bio-microarrays, fluorescence polarization and microcalorimetry

La synthese d'analogues carbones de disaccharide, en particulier de c-disaccharides, molecules ou l'oxygene interglycosidique a ete remplace par un groupement methylene, suscite un interet croissant. Dans cette optique, la strategie de l'agrafe moleculaire a ete etendue a la synthese de c-disaccharides complexes. Elle met en jeu l'addition d'un radical anomere sur une double liaison presente dans la molecule. Une etude basee sur les derives d'acides 3-desoxy-ulosoniques, nous a permis de mettre en evidence le caractere inerte des radicaux tertiaires anomeres obtenus. Malgre l'elaboration de l'agrafe a la fonction ester, nous n'avons pas pu obtenir de c-disaccharides de cette famille. La n-acetyl-c-lactosamine a ete synthetisee suivant deux strategies. La voie directe met en jeu une unite accepteur de radical possedant deja la fonction acetamido. Dans la voie indirecte, le produit de cyclisation en serie neutre est fonctionnalise par une double inversion permettant d'introduire le groupement acetamido. La n-acetyl-c-lactosamine obtenue est glycosylee par voie enzymatique afin d'obtenir un lewis x mixte. Dans une troisieme partie, nous avons effectues la synthese du trisaccharide c,o-lewis a a partir d'un c-gal-beta-(1-3)glcnac, glycosyle par la suite. Apres introduction de la fonction acetamido sur le c-galactoside par la strategie mise au point dans le chapitre precedent, la fucosylation du c-disaccharide obtenu nous a conduit au lewis a mixte desire. Parallelement, nous avons synthetise un c-fuc-alpha-(1-4)glcnac, motif qui constitue le lewis a.

Carbohydrates are among the most abundant molecules found on the cell surfaces of bacteria, parasites, and viruses. Apart from the conventional roles of carbohydrates as energy sources and structural polymers, carbohydrates are also associated with cancer metastasis, protein stabilization, pathogen infection and the immune response. Cells of our body have sensors made out of carbohydrates on outer surface of plasma membrane and acts as sensors and can detect many kinds of stimuli, and can signal the immune system to respond. Carbohydrate-protein molecular recognition processes have pivotal roles in infections and in immune response to pathogens. To date, several vaccines based on isolated capsular polysaccharides (CPSs) are marketed against infectious diseases. However, the use of isolated capsular polysaccharide poses several limitations, as natural sources are generally limited and the isolation is very challenging. Additionally, the isolated polysaccharides are heterogeneous and often contains impurities. Furthermore, limited protection of certain CPS antigens impairs the efficiency of vaccines. To overcome limitations associated with isolated polysaccharides, synthetic oligosaccharides present an effective alternative with great potential to understand glycan immunology and rationally design effective antigens. Consequently, characterization and reconstruction of carbohydrate epitopes with authentic composition has become one of the major target in glycoscience. To this end, strategies are needed to facilitate the streamlined design and generation of these antigens. This thesis concerns the development of an effective synthetic strategy to obtain Group B Streptococcus (GBS) type II oligosaccharide for vaccine development. GBS, a Gram-positive bacterium, inhabits the intestinal and genitourinary tract of 10‐30% of humans. GBS is one of the primary causes of bacterial infections among neonates and pregnant women, resulting in many severe diseases such as sepsis, meningitis, abortion, and so on. Type II GBS is one of the predominant GBS serotypes and is associated with about 15% of the invasive infections in adults and infants; therefore, represents an important human pathogen. The development of effective preventive vaccine against GBS is much needed to help pregnant women protect their newborns. This thesis describes the effective synthetic strategy to synthesize GBS type II oligosaccharide to be applied for vaccine development. Herein, we present a new and convenient synthesis of the repeating unit of GBS type II capsular polysaccharide. The structure of GBS type II was elucidated in 1983 and the repeating unit of GBS type II is a heptasaccharide composed of α-Neu5Ac (2-3)-ß-D-Gal-(1-4)- ß-D-GlcNAc-(1-3)-[-ß-D-Gal-(1-6)]-ß-D-Gal-(1-4)-ß-D-Gal-(1-3)-ß-D-Glc. The presented synthetic strategy is based on the five subcomponents derived from the retro synthetic analysis. Suitably protected lactosamine and lactose derivatives are pivotal building blocks in our synthesis and both disaccharide fragments have been achieved from the cheap and readily available lactose. Having started from two disaccharides saves the efforts of glycosylation and reduces the number of synthetic steps. The building blocks have been obtained in good overall yield following the optimized synthetic approach. The synthesis of backbone linear chain trisaccharide [ß-D-Gal-(1-4)-ß-D-Gal-(1-3)-ß-D-Glc] and pentasaccharide [ß-D-Gal-(1-4)-ß-D-GlcNAc-(1-3)-ß-D-Gal-(1-4)-ß-D-Gal-(1-3)-ß-D-Glc] has been achieved in excellent yield (~80% yield). The final steps of the synthesis comprise- the incorporation of ß-D-Gal unit into the linear chain pentasaccharide (currently ongoing) followed by the enzymatic introduction of sialic acid (NeuNAc unit) and subsequent deprotection to yield the repeating unit of GBS type II capsular polysaccharide. To conclude, in this thesis we present an efficient and easy handling synthetic approach to the heptasaccharide repeating unit of GBS type II. Readily available and cheap dairy side-product lactose has been used as a key structure in the presented scheme, allowing the efficient synthesis of the pentasaccharide backbone of the target compound. The synthetic GBS II fragments will be used for glycan array and structural studies and immunochemical characterization with specific monoclonal antibodies. This thesis comprises of four main chapters and the experimental section containing the methods and synthetic procedures for the discussed schemes. Chapter one is a general introduction and deals with the necessity and the social importance of the described project. Chapter two of the thesis outlines the scientific background and pathogenesis of GBS, carbohydrates and their biological importance, and general introduction of vaccines and how the carbohydrates can be used as a suitable vaccine candidate. Chapter two establishes the importance of synthetic carbohydrates and how the synthetic carbohydrates can be used to develop suitable effective vaccines against GBS diseases. Chapter three of the thesis contains the general introduction and structural features of GBS II CPS and the retrosynthetic analysis of GBS II CPS to identify the building blocks for the synthesis of GBS CPS II. Chapter four of the thesis summarizes the synthetic strategies and results to achieve the building blocks described in chapter three and the recombination of fragments to achieve the final molecule GBS II CPS repeating unit. The last part of the thesis will consists of the experimental methods and synthetic procedures to achieve the proposed molecule along with the characterization data.

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Considerado um problema de saúde pública, o câncer tem tido um grande impacto em toda a comunidade, seja em países desenvolvidos ou em países em desenvolvimento. Em mulheres, destacam-se os cânceres de mama e útero, sendo o primeiro o de maior incidência entre as mulheres. Uma detecção precoce desses dois tipos de câncer pode significar um aumento da sobrevida do paciente e de sua cura. Uma das áreas de pesquisa voltada para o estudo do câncer é a busca por biomarcadores que possam ser úteis em sua detecção e/ou tratamento. É de notório conhecimento que uma expressão alterada de glicoconjugados de superfície celular podem contribuir para o fenótipo maligno em eventos oncogênicos. Entre os glicanos desses glicoconjugados, a polilactosamina (poliLacNAc) tem sido alvo de diversos estudos devido ao seu potencial papel em diversas eventos oncogênicos, em especial um envolvimento com o potencial metastático. Assim, a expressão de poliLacNAc e outros carboidratos (GlcNAc) foram avaliados, através de histoquímica com lectinas, em amostras normais e transformadas de colo uterino e mama em diferentes estágios de transformação. Também foram verificados a expressão, utilizando imunohistoquímica, de glicosiltransferases (MGAT5, β3GnT2 e β3GnT3) que podem alterar a expressão das poliLacNAc e galectina-3 (Gal-3), uma proteína que tem afinidade por polilactosaminas que também tem sido associada a eventos oncogênicos, incluído a metástase. Nas amostras de tumores de colo uterino foi observado a presença de cadeias de poliLacNAc foi observada mais expressas nas amostras com maior grau de transformação (carcinomas) quando comparado com as outras amostras normais e pré-malignas, além de que uma expressão nuclear também foi observada significantemente nesse tipo de amostra. Nas amostras de tecidos de mama as poliLacNAc foram pouco expressas em todos as amostras. No entanto, uma expressão de MGAT5, uma enzima que pode regular a expressão de poliLacNAc e que também associada à metástase foi mais observada nas amostras com grau de transformação mais elevado. Essas observações reforçam o potencial envolvimento das cadeias de poliLacNAc e de enzimas chaves que regulam sua expressão, como a MGAT5, durante os eventos de transformação oncogênica.
A public health problem, cancer has a major impact n the entire community, in developed countries or in developing countries. In women, breast and uterine cancers are the most prevalent, being breast cancer the first. An early detection of both cancer can allow an increasing of patient survival and rate of health. One of the goals of cancer science is the biomarker research to use them at detection and/or treatment. It is known that an altered expression of glycoconjugates of cell surface can contribute to the malignant phenotype at oncogenic events. Among these glycans, polylactosamine (polyLacNAc) has been target of many studies for its potential role in many oncogenic events, in especial at the metastatic potential. Thus, the expression of polyLacNAc and others carbohydrates (GlcNAc) was evaluated, using lectin histochemistry, in normal and transformed samples of uterine and breast tissue at different stages. It was also evaluated the expression, by immunohistochemistry, of glycosyltrasnferases (MGAT5, β3GnT2 e β3GnT3) which could regulate polyLacNAc chains and of galectin-3 (Gal-3), a binding protein for polyLacNAc which has been correlated to oncogenic events, including metastasis. At uterine samples, was observed the presence of polyLacNAc chains at high levels in samples which higher degree of transformation (carcinoma) when compared to the normal and pre-malignant samples, and was observed a significant nuclear expression at these samples. In breast samples, polyLacNAc was observed at lower levels at all samples. Besides, MGAT5 expression was observed at higher levels in samples which higher transformation degree. These findings reinforces the potential involvement of polyLacNAc chains and the enzymes involved in its expression, like MGAT5, at oncogenic events.

Carbohydrate-binding proteins of non-immunological origin -lectins- have been recognized over the last decades as decisive players in numerous biological processes, ranging from cellcell communication, fertilization, pathogen-cell adhesion to metastasis. Consequently, there is an increasing interest in finding powerful and nanosized tools to screen for these molecules and to study their carbohydrate interactions in detail. Here, two complementary approaches are described to characterize lectin-carbohydrate interactions with high sensitivity, low sample consumption, and without the need for sample labelling: SPR and CREDEX-MS. In SPR, we have developed an approach where the sugar is immobilized onto a sensor surface through a tailor-made peptide module that allows (1) to capture the lectin, (2) to characterize the interaction through kinetic and thermodynamic parameters, and (3) to identify the interacted protein by mass spectrometry. In CREDEX-MS, based on proteolytic excision of proteincarbohydrate complexes and mass spectrometric analysis, the peptides comforming the carbohydrate binding domain are identified.
Las lectinas (proteínas de origen no inmune capaces de reconocer azúcares) se han revelado en las últimas décadas como participantes cruciales en multitud de procesos biológicos, tales como la comunicación célula-célula, la fertilización, la adhesión del patógeno a la célula y la metástasis, entre muchos otros. Por lo tanto, existe un gran interés en el desarrollo de técnicas analíticas potentes para el estudio de las interacciones lectina-carbohidrato. En este trabajo, se describen dos aproximaciones complementarias mediante las cuales se pueden caracterizar las interacciones lectinas-azúcar con gran sensibilidad, poca utilización de muestra y sin la necesitad de ningún marcaje. En la técnica basada en resonancia de plasmón superficial (SPR), el azúcar es inmovilizado sobre una superficie a través de un módulo peptídico, lo cual permite (1) capturar la lectina, (2) caracterizar su interacción mediante parámetros cinéticos y termodinámicos y (3) identificar posteriormente la proteína mediante espectrometría de masas. Complementariamente, la técnica CREDEX-MS, basada en la excisión proteolítica del complejo proteína-azúcar y posterior análisis por espectrometría de masas, nos permite identificar los péptidos que forman parte del dominio de unión al azúcar.

[EN] Human milk contains a large number of oligosaccharides, either free or bound to proteins and lipids, and their physiological role is mostly unknown. These oligosaccharides are resistant to host gastrointestinal digestion and therefore, a significant proportion reaches the infant colon, where they can be substrates for the resident microbiota. Lacto-N-biose (LNB) and galacto-N-biose (GNB) are the core type-1 sugar structures in HMO and mucin glycoproteins, respectively. They are fermented by species of the genus Bifidobacterium, but, there is no data about their utilization by the genus Lactobacillus. There is also no information for this genus about the metabolism of N-acetyllactosamine (LacNAc), which constitutes the type-2 sugar core in HMO, and lacto-N-triose, which forms part of the structure of lacto-N-tetraose, one of the most abundant HMOs. Lactobacillus casei is a lactic acid bacteria isolated from several environmental niches such as milk, meat, and reproductive and gastrointestinal tracts of animals and humans. In addition, some strains are used as starter cultures in the dairy industry and also as probiotics. The capability of L. casei species to survive in the gastrointestinal tract would depend in part of its ability to metabolize the available carbohydrates. In this Thesis we have shown that this strain is able to grow using LNB, GBN, LacNAc, and lacto-N-triose as carbon sources, and we have characterized the corresponding metabolic pathways. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR. Upstream of the gnb operon, there are two genes, bnaG and manA, encoding a b-N-acetylglucosaminidase precursor and a mannose-6P isomerase. It has been shown that BnaG is an extracellular wall-attached enzyme and that it is involved on lacto-N-triose metabolism. ManA enzyme is involved in the utilization of the mannose moiety of 3'-N-acetylglucosaminyl-mannose, which is a carbon source for L. casei BL23. Finally, in this strain, LacNAc is transported and phosphorylated by the lactose PTS and it is intracellularly hydrolyzed by the phospho-b-galactosidase LacG into galactose-6P and GlcNAc. Transcriptional analysis showed that the lac operon, in addition to lactose, is also induced by LacNAc. In an effort to better understand the metabolism and bioactive potential of HMO, sufficient quantities are required. In order to have enough amounts of LNB and GNB to test their biological activities, both disaccharides have been synthesized in vitro using the transglycosylation activity of the GnbG glycosyl hydrolase isolated from L. casei. Transglycosylation reactions were scaled and the resulting products were purified, and the yields obtained were 10.7 ± 0.2 g/L of LNB and 10.8 ± 0.3 g/l of GNB. Both disaccharides were used in vitro to determine their potential prebiotic properties using 33 Lactobacillus strains corresponding to 13 different species. It was determined that 21 strains, corresponding to the species L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri and Lactobacillus johnsonii were able to metabolize both GNB as LNB. Recently, some scientific evidences suggest an immunomodulatory function for HMO. In vitro analyses to assay this function have been performed for LNB and GNB, and for two fucosyloligosaccharides (Fuc-a-1,3-GlcNAc and Fuc-a-1,6-GlcNAc) previously synthesized in our laboratory. The four disaccharides were able to significantly increase IFN-g production in Peripheral Blood Mononuclear Cells (PBMC). Fuc-a-1,6-GlcNAc was also able to significantly reduce the production of IL13. These results suggest a stimulatory effect on the immune system, and in the particular case of Fuc-a-1,6-GlcNAc, a polarizing effect of immune response Th1 / Th2 towards Th1 populations.
[ES] La leche humana contiene una gran cantidad de oligosacáridos, libres o unidos a lípidos y proteínas, y su función fisiológica es mayoritariamente desconocida. Estos oligosacáridos son resistentes a la digestión y una gran proporción llega al intestino del lactante, donde pueden ser substratos para la microbiota. La lacto-N-biosa (LNB) y la galacto-N-biosa (GNB) son las estructuras tipo-1 que forman el núcleo de los oligosacáridos de la leche humana (OLH) y de las glicoproteínas de la mucina, respectivamente. Los dos azúcares son fermentados por especies del género Bifidobacterium, pero no hay datos acerca de su utilización por el género Lactobacillus. Tampoco hay información para este género acerca del metabolismo de la N-acetil-lactosamina (LacNAc), que constituye la cadena de azúcar tipo-2 en los OLH, y de la lacto-N-triosa, que forma parte de la estructura de la lacto-N-tetraosa, uno de los OLH más abundantes. La capacidad de Lactobacillus casei, de prevalecer en el sistema gastrointestinal dependerá en parte de su versatilidad metabólica para utilizar los carbohidratos disponibles. En la presente Tesis Doctoral se ha demostrado que la cepa L. casei BL23 se puede cultivar en presencia de LNB, GBN, LacNAc, y lacto-N-triosa como fuentes de carbono. En el metabolismo de la LNB, GNB y también N-acetilgalactosamina (GalNAc) está implicado el operon gnbREFGBCDA. Análisis transcripcionales demostraron que el operon gnb está regulado por inducción del substrato mediada por el represor transcripcional GnbR. Por encima del operon gnb, hay dos genes bnaG y manA, que codifican para el precursor de una b-N-acetilglucosaminidasa y una manosa-6P isomerasa. Se ha demostrado que BnaG es un enzima extracelular unido a la pared celular y que está implicado en el metabolismo de la lacto-N-triosa. El enzima ManA está implicada en el metabolismo de la manosa presente en el disacárido 3'-N-acetilglucosaminil-manosa, el cual es también una fuente de carbono para L. casei BL23. Por último, en esta cepa se ha demostrado que la LacNAc es transportada y fosforilada por el PTS de la lactosa e hidrolizada intracelularmente por la fosfo-b-galactosidasa LacG en galactosa-6P y GlcNAc. Análisis transcripcionales demostraron que el operon lac, además de por lactosa, está también inducido por LacNAc. Para intentar comprender mejor el metabolismo y potencial bioactivo de los OLH se necesitan cantidades adecuadas de éstos. Con el objeto de disponer de LNB y GNB en cantidad suficiente para ensayar su actividad biológica, se han sintetizado in vitro ambos disacáridos utilizando para ello la capacidad de transglicosidación de la glicosil hidrolasa GnbG aislada de L. casei. Las reacciones de transglicosidación se escalaron, los productos resultantes se purificaron, y se obtuvieron rendimientos de 10.7 ± 0.2 g/l de LNB y 10.8 ± 0.3 g/l de GNB. Ambos disacáridos fueron utilizados in vitro para determinar sus propiedades prebióticas potenciales con 33 cepas de Lactobacillus correspondientes a 13 especies diferentes. Se determinó que 21 de las cepas, correspondientes a las especies L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri y Lactobacillus johnsonii fueron capaces de metabolizar tanto GNB como LNB. Existen evidencias científicas recientes que les atribuyen a los OLH propiedades inmunomoduladoras, así que se han determinado éstas in vitro para LNB y GNB, y para dos fucosiloligosacáridos (Fuc-a-1,3-GlcNAc y Fuc-a-1,6-GlcNAc) sintetizados anteriormente en nuestro laboratorio. Se ha demostrado que los cuatro disacáridos son capaces de incrementar significativamente la producción de IFN-g en Células Mononucleares de Sangre Periférica. El Fuc-a-1,6-GlcNAc además fue capaz de reducir significativamente la producción de IL13. Estos resultados sugieren un efecto estimulante del sistema inmune y en el caso particular del Fuc-a-1,6-GlcNAc un efecto polarizador de la respuesta inmune Th1/Th2
[CAT] La llet humana conté una gran quantitat d'oligosacàrids, lliures o conjugats amb lípids o proteïnes i la seua funció fisiològica és majoritàriament desconeguda. Aquests oligosacàrids són resistents a la digestió i una gran proporció arriba a l'intestí dels lactants, on poden ser substrat per a la microbiota. La lacto-N-biosa (LNB) i la galacto-N-biosa (GNB) són les estructures tipus-1 que conformen el nucli dels oligosacàrids de la llet humana (OLH) i de les glicoproteïnes de la mucina, respectivament. Els dos sucres són fermentats per espècies del gènere Bifidobacterium, però no existeixen dades sobre l'ús pel gènere Lactobacillus. Tampoc no hi ha informació per a aquest gènere sobre el metabolisme de la N-acetil-lactosamina (LacNAc), que cosntitueix la cadena de sucre tipus-2 als OLH i de la lacto-N-triosa, que forma part de l'estructura de als OLH tipus-1 i 2. La capacitat de L. casei, una bactèria làctica aïllada de múltiples nínxols ambientals, de prevaler al sistema gastrointestinal dependrà en part de la seua versatilitat metabòlica per a utilitzar els carbohidrats disponibles. A la present tesi doctoral s'ha demostrat que la soca L. casei BL23 es pot cultivar en presència de LNB, GBN, LacNAc i lacto-N-triosa com a fonts de carboni. Al metabolisme de la LNB, GNB i també de la N-acetilgalactosamina (GalNAc) està implicat l'operó gnbREFGBCDA. Anàlisi transcripcionals demostraren que l'operó gnb està regulat per inducció del substrat mitjançant el repressor transcripcional GnbR. A sobre de l'operó gnb, hi ha dos gens bnaG i manA, que codifiquen per al precursor d'una b-N-acetilglucosaminidasa i una manosa-6P isomerasa. S'ha demostrat que BnaG és un enzim extracel·lular unit a la paret cel·lular i que està implicat en el metabolisme de la lacto-N-triosa. Aquesta és hidrolitzada per BnaG en lactosa i N-acetilglucosamina (GlcNAc). L'enzim ManA està implicada en el metabolisme de la manosa present al disacàrid 3-N-acetilglucosaminil-manosa, el qual també representa una font de carboni per a L. casei BL23. Per concloure, en aquesta soca s'ha demostrat que la LacNAc és transportada i fosforilada pel PTS de la lactosa i hidrolitzada intracel·lularment per la fosfo-b-galactosidasa LacG en galactosa-6P i GlcNAc. Anàlisi transcripcionals demostraren que l'operó lac, a més de per lactosa està induït per LacNAc. Per intentar comprendre millor el metabolisme i potencial bioactiu dels OLH es necessiten quantitats adequades d'aquests. A fi de disposar de LNB i GNB en quantitats suficients per a assajar la seua activitat biològica, s'han sintetitzat in vitro ambdós disacàrids utilitzant per a tal la capacitat de trasglicosidació de la glicosil hidrolasa GnbG aïllada de L. casei. Les reaccions de transglicosidació s'escalaren, els productes resultants es purificaren i s'obtingueren rendiments de 10.7 ± 0.2 g/l de LNB y 10.8 ± 0.3 g/l de GNB. Ambdós disacàrids foren utilitzats in vitro per a determinar les seues propietats prebiòtiques potencials amb 33 soques de Lactobacillus corresponents a 13 espècies diferents. Es determinà que 21 de les soques, corresponents a les espècies L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri i Lactobacillus johnsonii foren capaces de metabolitzar tant GNB com LNB. Existeixen evidències científiques recents que atribueixen als OLH propietats immunomoduladores, així que s'han determinat aquestes in vitro per a LNB i GNB i per als dos fucosiloligosacàrids (Fuc-a-1,3-GlcNAc y Fuc-a-1,6-GlcNAc) sintetitzats anteriorment al nostre laboratori. S'ha demostrat que els quatre disacàrids són capaços d'incrementar significativament la producció de IFN-g en Cèllules Mononuclears de Sang Perifèrica. El Fuc-a-1,6-GlcNAc a més, fou capaç de reduir significativament la producció de IL13. Aquests resultats suggereixen un efecte estimulant del sistema immune i en el cas particular del Fuc-a-1,6-GlcNAc un efecte pol
Bidart Costoya, G. (2016). Metabolismo y síntesis de oligosacáridos de la leche humana mediante la utilización de enzimas glicosil hidrolasas de Lactobacillus casei [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/70898
TESIS

碩士
國立臺灣師範大學
化學系
98
N-Acetyllactosamine (Gal-b-1,4-GlcNAc, LacNAc) and its 1,3-analogue often exist as repeating disaccharides resided at the non-reducing terminus of N-glycans. These carbohydrates are associated with numerous biological activities. For example, they are known to interact with various types of galectins for the immune-activity of B-cells. Recently the incorporation of sulfate groups to the hydroxyl group(s) was observed to greatly enhance the binding interaction with a variety of proteins. This thesis research aims at the synthesis of these sulfated Gal-1,3-GlcNAc, LacNAc and analogues in order for the purpose of systematic investigation. These target disaccharides were synthesized by the reactions steps of protecting group manipulations, glycosylation, selective deprotection and sulfation. We followed the previously established method to prepare the building blocks of Gal donor and GlcNAc-acceptor. The pre-activation method by using diphenyl sulfoxide-Tf2O was found to be suitable for formation of the desired b-1,3-linkage with satisfying high yields.