Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol.

Inhibitors of glycosylation provide a tool for studying the biology of glycoconjugates. One class of inhibitors consists of glycosides that block glycoconjugate synthesis by acting as primers of free oligosaccharide chains. A typical primer contains one sugar linked to a hydrophobic aglycone. In this report, we describe a way to use disaccharides as primers. Chinese hamster ovary cells readily take up glycosides containing a pentose linked to naphthol, but they take up hexosides less efficiently and disaccharides not at all. Linking phenanthrol to a hexose improves its uptake dramatically but has no effect on disaccharides. To circumvent this problem, analogs of Xyl beta 1-->6Gal beta-O-2-naphthol were tested as primers of glycosaminoglycan chains. The unmodified disaccharide did not prime, but methylated derivatives had activity in the order Xyl beta 1-->6Gal(Me)3-beta-O-2-naphthol > Xyl beta 1-->6Gal (Me)2 beta-O-2-naphthol >> Xyl beta 1-->6Gal(Me)beta-O-2-naphthol. Acetylated Xyl beta 1-->6Gal beta-O-2-naphthol also primed glycosaminoglycans efficiently, suggesting that the terminal xylose residue was exposed by removing the acetyl groups. The general utility of using acetyl groups to create disaccharide primers was shown by the priming of oligosaccharides on peracetylated Gal beta 1-->4GlcNAc beta-O-naphthalenemethanol. This disaccharide inhibited sialyl Lewis X expression on HL-60 cells.

Approche de synthèse d'analogues du sialyl Lewis X

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Approche de synthèse d'analogues du sialyl Lewis X

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

GDP-L-fucose: synthesis and role in inflammation

GDP-L-fucose: synthesis and role in inflammation The migration of leukocytes from intravascular locations to extravascular sites is essential to the immune responses. The initial attachment of leukocytes to the endothelium and the rolling step of the leukocyte extravasation cascade are mediated by selectins, a family of cell adhesion molecules on cell surfaces. Selectins are able to recognize glycoproteins and glycolipids containing the tetrasaccharide sialyl Lewis x (sLex, Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc). Several glycosyltransferases are involved in the biosynthesis of sLex, fucosyltransferase VII (Fuc-TVII) being the last enzyme to modify the sLex structure. Fuc-TVII transfers L-fucose from GDP-L-fucose to sialylated N-acetyllactosamine. GDP-L-fucose is synthesized in the cytosol via two different metabolic pathways. The major, constitutively active de novo pathway involves conversion of GDP-α-D-mannose to GDP-β-L-fucose. In the alternative salvage pathway, L-fucokinase synthesizes from free fucose L-fucose-1-phosphate, which is further converted to GDP-L-fucose by GDP-L-fucose pyrophosphorylase. GDP-L-fucose is translocated from the cytosol to Golgi for fucosylation via the GDP-fucose transporter. This thesis involved the study of the synthesis of GDP-L-fucose via the salvage pathway: cloning and expression of murine L-fucokinase and GDP-L-fucose pyrophosphorylase. The gene expression levels of these enzymes were found to be relatively high in various tissues; the mRNA levels were highest in brain, ovary and testis. This study also describes molecular cloning of rat fucosyltransferase VII (FUT7) and its expression as a functional enzyme. Gene expression levels of GDP-L-fucose synthesizing enzymes, GDP-fucose transporter and FUT7 were determined in inflamed tissues as well as cancer cells. Our results revealed a clear upregulation of the enzymes involved in the synthesis of GDP-L-fucose via de novo pathway, GDP-fucose transporter and FUT7 in inflamed tissues and in cancer cells. On the contrary, the GDP-L-fucose salvage pathway was found to be irrelevant in inflammation and in tumorigenesis. Furthermore, our results indicated the transcriptional coregulation of Golgi transporters involved in the synthesis of sulfo sLex, i.e. CMP-sialic acid, GDP-fucose and 3 phosphoadenosine 5 -phosphosulfate transporters, in inflammation.

Forced Dissociation of Selectin-ligand Complexes Using Steered Molecular Dynamics Simulation

Selectin-ligand interactions are crucial to such biological processes as inflammatory cascade or tumor metastasis. How transient formation and dissociation of selectin-ligand bonds in blood flow are coupled to molecular conformation at atomic level, however, has not been well understood. In this study, steered molecular dynamics (SMD) simulations were used to elucidate the intramolecular and intermolecular conformational evolutions involved in forced dissociation of three selectin-ligand systems: the construct consisting of P-selectin lectin (Lec) and epidermal growth factor (EGF)-like domains (P-LE) interacting with synthesized sulfoglycopeptide or SGP-3, P-LE with sialyl Lewis X (sLeX), and E-LE with sLeX. SMD simulations were based on newly built-up force field parameters including carbohydrate units and sulfated tyrosine(s) using an analogy approach. The simulations demonstrated that the complex dissociation was coupled to the molecular extension. While the intramolecular unraveling in P-LESGP-3 system mainly resulted from the destroy of the two anti-parallel sheets of EGF domain and the breakage of hydrogen-bond cluster at the Lec-EGF interface, the intermolecular dissociation was mainly determined by separation of fucose (FUC) from Ca2+ ion in all three systems. Conformational changes during forced dissociations depended on pulling velocities and forces, as well as on how the force was applied. This work provides an insight into better understanding of conformational changes and adhesive functionality of selectin-ligand interactions under external forces.

PSGL-1酪氨酸变异体的动力学分析

<正>在炎症反应过程中,选择素/配体相互作用介导了白细胞在血管内皮表面的滚动和结合。选择素/配体相互作用的化学反应速率、反应亲和性和力学强度对白细胞的滚动起决定性作用。选择素家族的所有成员(P-、E-、和L-选择素)都有一个共同的配体PSGL-1(P-selectinglycoproteinligand-1)——一种白细胞表面的同源双体粘液素蛋白。PSGL-1与P-或L-选择素发生粘附需要同时含有两个必要结构:46、48和51位酪氨酸的硫酸化;57位苏氨酸的0型糖苷,并经过sLe~x(sialyl

Papel da fucana sulfatada FucSulf I na interação entre células tumorais e o endotélio in vitro

Para formar metástases, as células tumorais devem se desprender do tumor primário e migrar através do endotélio num processo denominado intravasamento. Uma vez na circulação, elas devem aderir ao endotélio do tecido alvo e extravasar para o novo sítio de colonização, onde irão proliferar. A interação das células tumorais com o endotélio é mediada por selectinas, seguida pela interação com integrinas. As células tumorais apresentam um padrão anormal de glicosilação, expressando ligantes de selectinas, formados por polissacarídeos fucosilados, como sialyl Lewis a/x. Durante o processo metastático, células tumorais secretam diversos fatores de crescimento. Além de modular diferentes tipos celulares que constituem o microambiente tumoral, estes fatores de crescimento também atuam nas células tumorais de forma autócrina, ativando vias de sinalização envolvidas na proliferação e migração celular. Polissacarídeos sulfatados como a heparina, podem atuar como inibidores de P e L-selectinas, além de se ligar a fatores de crescimento, impedindo a ativação de seus receptores. Neste trabalho, avaliamos o papel de fucanas sulfatadas extraídas de diferentes espécies de invertebrados marinhos (L. variegatus, S. franciscanus, S. pallidus, A. lixula e S. droebachiensis) na modulação da interação entre células tumorais com o endotélio in vitro e comparamos seu efeito com o da heparina. Também avaliamos o papel destas moléculas na proliferação de células tumorais. Para isso, utilizamos duas linhagens tumorais de próstata (DU-145 e PC-3) e culturas primárias de células endoteliais de veia umbilical humana (HUVECs). Ao avaliar o efeito das fucanas na adesão das células tumorais às HUVECs, observamos que todas as fucanas testadas inibiram a adesão da linhagem DU-145 à monocamada endotelial, enquanto apenas a fucana extraída da espécie L. variegatus (FucSulf I) e da espécie S. franciscanus inibiram a adesão da linhagem PC-3. A FucSulf I foi uma das fucanas que apresentou maior potencial inibitório nas duas linhagens e foi a única que inibiu a adesão da linhagem DU-145 à matriz subendotelial, não interferindo na adesão da linhagem PC-3. A FucSulf I mostrou-se capaz de diminuir também a migração transendotelial das linhagens tumorais DU-145 e PC-3. A heparina mostrou efeito significativo apenas nos ensaios de transmigração, inibindo este evento de forma similar a FucSuf I. Sabe-se que o VEGF aumenta a permeabilidade endotelial, facilitando a passagem de células tumorais através do vaso. Observamos que as duas linhagens secretam VEGF e que a FucSulf I se liga a este fator. Estes dados sugerem que a interação da FucSuf I com o VEGF pode impedir a ação deste fator nas células endoteliais, diminuindo a migração transendotelial das células tumorais testadas. Também verificamos que a FucSulf I inibiu a proliferação das linhagens celulares na ausência de fatores exógenos ou na presença de soro fetal bovino ou VEGF. Por fim, avaliamos que a FucSulf I interfere na ativação de proteínas específicas de vias de sinalização disparadas por fatores de crescimento. A FucSulf I inibe a ativação da AKT na linhagem PC-3, enquanto nas células DU-145 observamos uma inibição da ativação da ERK. Esses dados indicam que a FucSulf I modula diversas etapas da progressão tumoral e pode ser um potencial candidato para o uso em terapias antitumorais

Population-based study reveals new risk-stratification biomarker panel for Barrett's esophagus

BACKGROUND & AIMS: The risk of progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) is low and difficult to calculate. Accurate tools to determine risk are needed to optimize surveillance and intervention. We assessed the ability of candidate biomarkers to predict which cases of BE will progress to EAC or high-grade dysplasia and identified those that can be measured in formalin-fixed tissues. METHODS: We analyzed data from a nested case-control study performed using the population-based Northern Ireland BE Register (1993-2005). Cases who progressed to EAC (n = 89) or high-grade dysplasia =6 months after diagnosis with BE were matched to controls (nonprogressors, n = 291), for age, sex, and year of BE diagnosis. Established biomarkers (abnormal DNA content, p53, and cyclin A expression) and new biomarkers (levels of sialyl Lewis(a), Lewis(x), and Aspergillus oryzae lectin [AOL] and binding of wheat germ agglutinin) were assessed in paraffin-embedded tissue samples from patients with a first diagnosis of BE. Conditional logistic regression analysis was applied to assess odds of progression for patients with dysplastic and nondysplastic BE, based on biomarker status. RESULTS: Low-grade dysplasia and all biomarkers tested, other than Lewis(x), were associated with risk of EAC or high-grade dysplasia. In backward selection, a panel comprising low-grade dysplasia, abnormal DNA ploidy, and AOL most accurately identified progressors and nonprogressors. The adjusted odds ratio for progression of patients with BE with low-grade dysplasia was 3.74 (95% confidence interval, 2.43-5.79) for each additional biomarker and the risk increased by 2.99 for each additional factor (95% confidence interval, 1.72-5.20) in patients without dysplasia. CONCLUSIONS: Low-grade dysplasia, abnormal DNA ploidy, and AOL can be used to identify patients with BE most likely to develop EAC or high-grade dysplasia.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

Study of glycosidic changes in lung cancer: potential effectors in hematogenous metastasis

RESUMO: O cancro do pulmão (LC), uma das principais causas de mortalidade relacionada com cancro em Portugal, pode levar à formação de metástases hematogénicas. A adesão das células tumorais ao endotélio é considerada um dos passos fundamentais envolvidos na metástase. Em células sanguíneas, esta adesão é mediada por ligandos de E-selectina (E-SL), glicoproteínas ou glicolípidos decorados principalmente com sialyl-Lewis x (sLex) e sialyl-Lewis a (sLea). Tem sido descrito a expressão destes antigénios em LC, contudo o seu papel funcional em permitir a adesão das células de LC ao endotélio é ainda pouco compreendido. Foram analisadas amostras emparelhadas normais e tumorais de pacientes com cancro de pulmão de não-pequenas células (NSCLC) e três linhas celulares de LC. Immunoblotting assays com anti-sLex/sLea e molécula quimérica de E-selectina demonstraram que tecidos tumorais de LC sobreexpressam significativamente E-SL e resultados de citometria de fluxo demonstraram uma expressão elevada de E-SL nas linhas celulares. Para compreender o mecanismo da sobreexpressão de E-SL em tecidos tumorais e linhas celulares de LC, foi analisada a expressão de genes envolvidos na biossíntese de E-SL, nomeadamente FUT3, FUT4, FUT6, FUT7, ST3GAL3, ST3GAL4, ST3GAL6, β4GALT1, GCNT1 e GALNT3. Observou-se a sobreexpressão das fucosiltransferases FUT3, FUT6 e FUT7 em tecidos tumorais de LC e FUT3 em linhas celulares de LC, sendo que neste último, esta expressão é correlacionada com um aumento da adesão das células de LC às selectinas endoteliais. Foi observado que uma baixa expressão de FUT4 em tecidos tumorais está associada com estadios menos avançados de NSCLC. Foram analisadas ainda proteínas decoradas com sLex/sLea, tendo-se identificado como E-SL o antigénio carcinoembrionário em NSCLC. Em resumo, esta tese contribuiu para uma melhor compreensão das alterações glicosídicas e moléculas que podem influenciar a progressão tumoral do LC, podendo permitir identificar futuramente novos biomarcadores de diagnóstico/prognóstico e potenciais alvos terapêuticos para o NSCLC.--------------------------ABSTRACT: Lung cancer (LC), one of the major causes of mortality related to cancer in Portugal, may lead to hematogenous metastasis. Adhesion of cancer cells to endothelium is considered one of the crucial steps involved in metastasis. In blood cells, this adhesion is initiated by endothelial selectin ligands (E-SL) that are glycoproteins or glycolipids decorated mostly with sialyl-Lewis x (sLex) and sialyl-Lewis a (sLea). While LC has been described as expressing these sialyl Lewis antigens, its functional role in allowing LC adhesion to endothelium is still poorly understood. We analyzed paired tumor and normal tissues samples from non-small cell lung cancer (NSCLC) patients and three LC cell lines. Immunoblotting assays with anti-sLex/sLea and E-selectin chimera demonstrated that LC tumor tissues significantly overexpress E-SL and flow cytometry results indicated that E-SL are also abundantly expressed in LC cell lines. To understand the mechanism behind the overexpression of E-SL in LC tissues and cell lines, we analyzed the expression of genes involved in its biosynthesis, namely FUT3, FUT4, FUT6, FUT7, ST3GAL3, ST3GAL4, ST3GAL6, β4GALT1, GCNT1 and GALNT3. It was observed the overexpression of fucosyltransferases FUT3, FUT6 and FUT7 in LC tumor tissues and FUT3 in LC cell lines, being this last one correlated with an increased reactivity of the LC cells to endothelial selectins. It was described that low expression of FUT4 in tumor tissues is correlated with early stages of NSCLC. We also analyzed scaffolds proteins of sLex/sLea and it was identified the carcinoembryonic antigen as an E-SL in NSCLC. In summary, this thesis contributed to a better understanding of the glycosidic changes and molecules that can influence tumor progression of LC, allowing identifying in the future new diagnosis/prognosis biomarkers and potential therapeutic targets for NSCLC.

Synthèse d'une librairie d'analogues monomériques et dimériques du sLe X

Dans cet ouvrage sera décrite la synthèse de nouveaux analogues du sialyl Lewis X (sLex). A cet effet, nous avons préparé une librairie d’analogues synthétisée à partir d’une approche mettant en jeu un «espaceur» acyclique permettant d’avoir un biais conformationnel que nous avons défini comme la stratégie ATC-B. Nous avions déjà démontré que certains analogues portant un groupe benzoate en C-2 et en C-4 du galactose présentent une activité 50 fois supérieure à celle du sLex. Nous avions par ailleurs démontré qu’en l’absence du benzoate en C-2, l’activité devient alors trois fois plus faible. A présent, il paraissait interessant de synthétiser des analogues ayant seulement un groupe benzoate en C-4 pour evaluer l’impact de ce groupement sur la puissance de nos analogues. Par le passé, nous avions également mis en évidence le rôle des esters sur l’activité des analogues portant un «espaceur» acyclique dans le cadre de la stratégie ATC-B. Nous effectuerons donc des variations à ce niveau pour en évaluer l’impact. Enfin, nous avons préparé une nouvelle famille d’analogues de type dimère. Ceux-ci seront constitués de 2 unités des composés monomériques synthétisés précédemment. La synthèse de ces dimères fera l’emploi de la «Click Chemistry». Cette étude nous mènera a vous présenter la synthèse de ces composés et la méthodologie employée.

Glycan alterations of serum proteins as tumour markers. Prostate-specific antigen in prostate cancer and acute-phase proteins in pancreatic cancer

Els pacients amb càncer presenten una taxa de supervivència superior si es diagnostiquen a estadis inicials, per la qual cosa és indispensable disposar de marcadors tumorals adequats. Glicoformes de proteïnes específiques es podrian utilizar com marcadors tumorals. S’han investigat les subformes i glicosilació de l’Antígen Prostàtic Específic (PSA) per millorar la seva capacitat de diagnosis de pacients amb càncer de pròstata vs aquells amb hiperplàsia benigna prostàtica. També s’han avaluat glicoproteïnes sèriques amb alteracions glucídiques en pacients de càncer de pàncrees, comparat amb pacients amb pancreatitis crònica i controls. S’ha observat una disminució de la fucosilació core i sialilació del PSA en càncer de pròstata i un augment de la fucosilació core i Sialyl-Lewis X en algunes Proteïnes de fase Aguda en càncer de pàncrees. Aquest canvis s’haurien d’avaluar en un cohort de pacients més gran per determinar el seu paper en el cribratge, diagnòstic o monitorització dels cancers estudiats.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.

Simple mucin-type carbohydrate antigens (T, sialosyl T, Tn and sialosyl-Tn) in breast carcinogenesis

Immunohistochemical analysis of the expression of simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) was performed in a series of 43 cases of intraductal hyperplasia without atypia, 9 cases of intraductal hyperplasia with atypia, 54 cases of ductal carcinoma in situ (DCIS) and 26 cases of invasive breast carcinoma. We also studied 36 cases of isolated breast normal epithelium, 20 cases of 'normal' breast epithelium adjacent to neoplasms and 14 cases of apocrine metaplasia. All antigens were detected in different frequencies in normal, hyperplastic, metaplastic and neoplastic breast epithelium. Tn and sialyl-Tn are expressed more frequently in malignant than in benign breast epithelium; while Tn expression increases from normal to invasive carcinomas, sialyl-Tn increases until DCIS and drops in invasive carcinomas, suggesting that either there is a failure of a proportion of DCIS to progress to invasive carcinoma or loss of expression of sialyl-Tn when some carcinomas become invasive. The high frequency of Tn and sialyl-Tn expression in breast intraductal proliferations probably reflects incomplete glycosylation in these lesions, which is a well-known tumour-associated phenomenon and supports the assumption that such lesions are putative precursors of breast cancer. T antigen was expressed in all groups studied, but its prevalence differed significantly between normal and neoplastic epithelium. The expression of these antigens in epithelium adjacent to carcinomas is similar to that found in isolated normal breast epithelium, whereas apocrine metaplasia has a pattern of simple mucin-type glycosylation that is specific and distinct from that of the normal breast epithelium, with a high frequency of marked expression of Tn and sialyl-Tn. The similarity of the pattern of expression of simple mucin-type antigens in metaplasia and malignant neoplasia reduces the usefulness of these markers from a diagnostic standpoint.

Design, synthesis and the effect of 1,2,3-triazole sialylmimetic neoglycoconjugates on Trypanosoma cruzi and its cell surface trans-sialidase

This work describes the synthesis of a series of sialylmimetic neoglycoconjugates represented by 1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing galactose modified at either C-1 or C-6 positions, glucose or gulose at C-3 position, and by the amino acid derivative 1,2,3-triazole fused threonine-3-O-galactose as potential TcTS inhibitors and anti-trypanosomal agents. This series was obtained by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-functionalized sugars 1-N(3)-Gal (commercial), 6-N(3)-Gal, 3-N(3)-Glc and 3-N(3)-Gul with the corresponding alkyne-based 2-propynyl-sialic acid, as well as by click chemistry reaction between the amino acid N(3)-ThrOBn with 3-O-propynyl-GalOMe. The 1,2,3-triazole linked sialic acid-6-O-galactose and the sialic acid-galactopyranoside showed high Trypanosoma cruzi trans-sialidase (TcTS) inhibitory activity at 1.0 mM (approx. 90%), whilst only the former displayed relevant trypanocidal activity (IC(50) 260 mu M). These results highlight the 1,2,3-triazole linked sialic acid-6-O-galactose as a prototype for further design of new neoglycoconjugates against Chagas' disease. (C) 2011 Elsevier Ltd. All rights reserved.