Analysis of the proximal promoter of the human colon-specific B4GALNT2 (Sda synthase) gene: B4GALNT2 is transcriptionally regulated by ETS1

13siopenBackground: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. Methods: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. Results: A short cis-regulatory region restricted to the −72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. Conclusions: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.openWavelet-Vermuse C.; Groux-Degroote S.; Vicogne D.; Cogez V.; Venturi G.; Trinchera M.; Brysbaert G.; Krzewinski-Recchi M.-A.; Bachir E.H.; Schulz C.; Vincent A.; Van Seuningen I.; Harduin-Lepers A.Wavelet-Vermuse, C.; Groux-Degroote, S.; Vicogne, D.; Cogez, V.; Venturi, G.; Trinchera, M.; Brysbaert, G.; Krzewinski-Recchi, M. -A.; Bachir, E. H.; Schulz, C.; Vincent, A.; Van Seuningen, I.; Harduin-Lepers, A

Étude de l'expression et des mécanismes de régulation transcriptionnelle tissu-spécifique du gène ST6GAL2

La sialylation est l une des dernières étapes de la biosynthèse des chaînes glycaniques des glycoprotéines et des glycolipides. La sialylation en a2,6 des structures N-acétyllactosaminiques (Galß1-4GlcNAc) est souvent retrouvée en périphérie des glycannes et est impliquée dans de nombreux mécanismes d adhésion et de reconnaissance cellule / cellule ou hôte / pathogène. Chez l Homme, deux sialyltransférases synthétisent ce type d épitope glycanique : hST6Gal I et hST6Gal II. Elles se distinguent par leur spécificité de substrat accepteur et par leur profil d expression tissulaire. Alors que le gène ST6GAL1 codant hST6Gal I est exprimé dans la plupart des tissus, ST6GAL2 présente une expression tissulaire plus restreinte, se limitant essentiellement au cerveau embryonnaire et adulte. Par ailleurs, hST6Gal II présente des similitudes en termes de spécificité de substrat et d expression tissulaire avec la sialyltransférase identifiée chez D. melanogaster et semble avoir conservé certaines propriétés ancestrales essentielles pour le développement du tissu nerveux. Plusieurs études ont montré que l expression des sialyltransférases est contrôlée au niveau transcriptionnel par l utilisation de promoteurs tissulaires régulant l expression de manière tissu-spécifique. Si les données concernant ST6GAL2 sont encore limitées, il apparaît cependant que l expression de ce gène est finement contrôlée par des mécanismes apparemment conservés au cours de l évolution. Le projet de thèse que nous avons mené a eu pour but d identifier les régions 5 -non traduites de ST6GAL2 et de caractériser les régions promotrices associées. A partir d un modèle cellulaire de neuroblastome en culture, nous avons identifié par 5 RACE trois types de transcrits qui diffèrent par leur premier exon non traduit. Ces exons, appelés EX, EY et EZ, sont situés à plus de 42 kpb du premier exon commun codant et ne sont séparés que de 124 et 87 pb, respectivement. Par Q-PCR en duplex avec le gène normalisateur HPRT, nous avons montré que les transcrits initiés par l exon EX et EY étaient prépondérants par rapport aux transcrits contenant EZ, à la fois dans plusieurs lignées cellulaires à caractère neuronal et dans des échantillons de tissu cérébral humain. Nous avons également montré que la protéine hST6Gal II est exprimée dans les différents lobes du cortex cérébral, dans le cervelet et dans l hippocampe. Nous avons isolé différentes régions génomiques situées en amont et à l intérieur de la région EX/EY/EZ que nous avons sous cloné en amont du gène de la luciférase pour des tests d activité. Nous avons défini deux régions promotrices, en amont des exons EX et EY. Des expériences de mutagenèse dirigée couplées à des analyses bioinformatiques nous ont révélé que les facteurs de transcription NF-?B et NRSF sont probablement des répresseurs de la transcription, alors que les facteurs Sox5, SP1, Pura et Olf1 agirait comme des éléments activateurs de la transcription de ST6GAL2. Les facteurs NRSF, Sox5, Pura et Olf1 régulent notamment la transcription de gènes impliqués dans le fonctionnement et le développement neuronal, suggérant un rôle de ST6GAL2 dans les fonctions neuronales. Enfin, nous avons mis en évidence une forte augmentation de l expression ST6GAL2 au cours de la différentiation en neurones des cellules NT2/D1 sous l action de l acide rétinoïque, suggérant un rôle potentiel de cette enzyme au cours de la différentiation neuronale.Sialylation is one of the last step of the biosynthesis of glycan chains carried by glycoproteins and glycolipids. The a2,6-sialylation of N-acetyllactosaminyl (Galß1-4GlcNAc) structures is commonly found at the end of glycan chains and is involved in numerous cell / cell or host / pathogen adhesion and recognition events. In Human, two sialyltransferases synthesise this glycan epitope, namely hST6Gal I and hST6Gal II. They differ from each other in substrate specificity an in tissue-specific pattern of expression. Whereas the gene encoding hST6Gal I, ST6GAL1, is expressed in almost all tissues, ST6GAL2 shows a narrower pattern of tissue expression essentially limited to fetal and adult brain. In addition, hST6Gal II exhibits similarities in terms of substrate specificity and gene expression pattern with the sialyltransferase identified in D. melanogaster and therefore, seems to have conserved ancestral properties required for brain function and growing nervous tissue. Several studies have shown that the expression of sialyltransferases is controlled at the transcriptional level by the use of specific promoters that regulate their expression in a tissue-specific fashion. Data about ST6GAL2 are rather limited; however, it appears the expression of this gene is finely regulated by mechanisms likely conserved through evolution. The aim of this thesis was to identify the 5 non translated regions of the ST6GAL2 gene and to characterize the associated promoter regions. From a neuroblastoma cultured cell model, we identified by 5 RACE three types of transcripts which are different only in their first non-coding exon. Those exons, named EX, EY and EZ, are located more then 42 kbp upstream of the first common coding exon and are only separated by 124 and 87 bp, respectively. Using Taqman duplex Q-PCR technology we have shown that the transcripts initiated by EX and EY are predominantly expressed compared to EZ both in several cell lines and in human brain tissue samples. We also demonstrated that the hST6Gal II protein is expressed in the different lobes of the human cerebral cortex, the cerebellum and the hippocampus. We isolated different genomic sequences upstream EX and within EX/EY/EZ region and inserted them in a reporter vector for luciferase assays. We could define two promoter sequences upstream EX and ZY. PCR site-directed mutagenesis experiments along with bioinformatics analysis revealed that transcription factors NF-?B and NRSF are likely to act as transcription inhibitors, whereas the Sox5, SP1, Pura and Olf1 factors would be involved in the transcriptional activation of ST6GAL2. The NRSF, Sox5, Pura and Olf1 transcription factors are notably involved in the transcriptional regulation of genes related to neuronal functions and the neuronal development. Eventually, we have shown evidence of a strong increased ST6GAL2 expression during neuronal differentiation of the NT2/D1 cell line under acid retinoic treatment, suggesting of putative role this enzyme in neuronal differentiation.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain.

The nucleotide sequence of the short and long transcripts of beta1,4- N -acetylgalactosaminyltransferase have been submitted to the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under accession nos AJ517770 and AJ517771 respectively. The human Sd(a) antigen is formed through the addition of an N -acetylgalactosamine residue via a beta1,4-linkage to a sub-terminal galactose residue substituted with an alpha2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4- N -acetylgalactosaminyltransferase (Sd(a) beta1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sd(a) beta1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1(S) and E1(L), leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to alpha2,3-sialylated acceptor substrates, to form the GalNAcbeta1-4[Neu5Acalpha2-3]Galbeta1-R trisaccharide common to both Sd(a) and Cad antigens

SPCA1 governs the stability of TMEM165 in Hailey-Hailey disease

International audienceTMEM165 is a Golgi protein whose deficiency causes a Congenital Disorder of Glycosylation (CDG). We have demonstrated that Mn2+ supplementation could suppress the glycosylation defects observed in TMEM165-deficient cells and that TMEM165 was a Mn2+-sensitive protein. In the Golgi, the other transmembrane protein capable to regulate Mn2+/Ca2+ homeostasis is SPCA1, encoded by the ATP2C1 gene. A loss of one copy of the ATP2C1 gene leads to Hailey-Hailey Disease (HHD), an acantholytic skin disorder in Humans. Our latest results suggest an unexpected functional link between SPCA1 and TMEM165. In order to clarify this link in case of partial SPCA1 deficiency, HHD fibroblasts were used to assess TMEM165 expression, subcellular localization and Mn2+-induced degradation. No differences were observed regarding TMEM165 expression and localization in HHD patients’ fibroblasts compared to control fibroblasts. Nevertheless, we demonstrated both for fibroblasts and keratinocytes that TMEM165 expression is more sensitive to MnCl2 exposure in HHD cells than in control cells. We linked, using ICP-MS and GPP130 as a Golgi Mn2+ sensor, this higher Mn2+-induced sensitivity to a cytosolic Mn accumulation in MnCl2 supplemented HHD fibroblasts. Altogether, these results link the function of SPCA1 to the stability of TMEM165 in a pathological context of Hailey-Hailey disease

Consequences of the expression of sialylated antigens in breast cancer

International audienceChanges in cell surface glycosylation are common modifications that occur during oncogenesis, leading to the over-expression of tumour-associated carbohydrate antigens (TACA). Most of these antigens are sialylated and the increase of sialylation is a well-known feature of transformed cells. In breast cancer, expression of TACA such as sialyl-Lewis(x) or sialyl-Tn is usually associated with a poor prognosis and a decreased overall survival of patients. However, the specific role of these sialylated antigens in breast tumour development and aggressiveness is not clearly understood. These glycosylation changes result from the modification of the expression of genes encoding specific glycosyltransferases involved in glycan biosynthesis and the level of expression of sialyltransferase genes has been proposed to be a prognostic marker for the follow-up of breast cancer patients. Several human cellular models have been developed in order to explain the mechanisms by which carbohydrate antigens can reinforce breast cancer progression and aggressiveness. TACA expression is associated with changes in cell adhesion, migration, proliferation and tumour growth. In addition, recent data on glycolipid biosynthesis indicate an important role of G(D3) synthase expression in breast cancer progression. The aim of this review is to summarize our current knowledge of sialylation changes that occur in breast cancer and to describe the cellular models developed to analyze the consequences of these changes on disease progression and aggressiveness

Sialyltransferases functions in cancers.

International audienceAbnormally elevated levels of sialylated tumor associated carbohydrate antigens are frequently described at the surface of cancer cells and/or secreted in biological fluids. It is now well established that this over-expression may result from deregulation in sialyltransferases enzymatic activity involved in their biosynthesis, but the precise molecular mechanisms remain unknown. Twenty different human sialyltransferases preside to the sialylation of glycoconjugates, either glycolipids or glycoproteins. This review summarizes the current knowledge on human sialyltransferases implicated in the altered expression of sialylated tumor associated antigens, the molecular basis of their regulated expression in cancer cells and the various tools developed by researchers and clinicians for their study in pathological samples

Carbohydrate-to-carbohydrate interactions between α2,3-linked sialic acids on α2 integrin subunits and asialo-GM1 underlie the bone metastatic behaviour of LNCAP-derivative C4-2B prostate cancer cells

Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2β1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2β1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2β1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2β1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes

The human sialyltransferase family

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Expression of Sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc α2,6-sialyltransferase (ST6GalNAc I) cDNA

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