Evolutionary history of the alpha2,8-sialyltransferase (ST8Sia) gene family: Tandem duplications in early deuterostomes explain most of the diversity found in the vertebrate ST8Sia genes

<p>Abstract</p> <p>Background</p> <p>The animal sialyltransferases, which catalyze the transfer of sialic acid to the glycan moiety of glycoconjugates, are subdivided into four families: ST3Gal, ST6Gal, ST6GalNAc and ST8Sia, based on acceptor sugar specificity and glycosidic linkage formed. Despite low overall sequence identity between each sialyltransferase family, all sialyltransferases share four conserved peptide motifs (L, S, III and VS) that serve as hallmarks for the identification of the sialyltransferases. Currently, twenty subfamilies have been described in mammals and birds. Examples of the four sialyltransferase families have also been found in invertebrates. Focusing on the ST8Sia family, we investigated the origin of the three groups of α2,8-sialyltransferases demonstrated in vertebrates to carry out poly-, oligo- and mono-α2,8-sialylation.</p> <p>Results</p> <p>We identified in the genome of invertebrate deuterostomes, orthologs to the common ancestor for each of the three vertebrate ST8Sia groups and a set of novel genes named ST8Sia EX, not found in vertebrates. All these ST8Sia sequences share a new conserved family-motif, named "C-term" that is involved in protein folding, via an intramolecular disulfide bridge. Interestingly, sequences from <it>Branchiostoma floridae </it>orthologous to the common ancestor of polysialyltransferases possess a polysialyltransferase domain (PSTD) and those orthologous to the common ancestor of oligosialyltransferases possess a new ST8Sia III-specific motif similar to the PSTD. In osteichthyans, we have identified two new subfamilies. In addition, we describe the expression profile of ST8Sia genes in <it>Danio rerio</it>.</p> <p>Conclusion</p> <p>Polysialylation appeared early in the deuterostome lineage. The recent release of several deuterostome genome databases and paralogons combined with synteny analysis allowed us to obtain insight into events at the gene level that led to the diversification of the ST8Sia genes, with their corresponding enzymatic activities, in both invertebrates and vertebrates. The initial expansion and subsequent divergence of the ST8Sia genes resulted as a consequence of a series of ancient duplications and translocations in the invertebrate genome long before the emergence of vertebrates. A second subset of ST8sia genes in the vertebrate genome arose from whole genome duplication (WGD) R1 and R2. Subsequent selective ST8Sia gene loss is responsible for the characteristic ST8Sia gene expression pattern observed today in individual species.</p

Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

<p>Abstract</p> <p>Background</p> <p>The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of <it>fut8 </it>gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat <it>fut8 </it>gene.</p> <p>Results</p> <p>The cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat <it>fut8 </it>gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of <it>fut8 </it>in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 <it>fut8 </it>gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.</p> <p>Conclusion</p> <p>Altogether, these data contribute to a better knowledge of <it>fut8 </it>expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.</p

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

Glyco25, XXV International Symposium on Glycoconjugates

International audienc

Vertebrate Sialyltransferases

International audienc

Étude de la biosynthèse des motifs di-, oligo- et polysialylés chez les mammifères (identification et caractérisation d'une nouvelle sialyltransférase humaine (hST8Sia VI)responsable de la biosynthèse de motifs diSia sur des O-glycosylprotéines)

Chez les mammifères, les acides sialiques se trouvent aux extrémités terminales des glycoconjugués (glycoprotéines et glycolipides) ou des oligosaccharides libres. De part cette position terminale sur les glycannes des glycoconjugués et de leur charge négative, les acides sialiques conditionnent les propriétés physico-chimiques des sialoglycoconjugués et sont souvent impliqués dans des mécanismes d'interaction cellulaire. L'association de résidus d'acides sialiques par des liaisons en a2,8 forme des chaînes linéaires de longueur variable classées selon le degré de polymérisation en structures disialylées (diSia), oligosialylées (oligoSia) ou polysialylées (polySia, ou PSA pour Poly Sialic Acid). Les PSA portés par la N-CAM (Neural Cell Adhesion Molecule) ont été les premiers mis en évidence chez les mammifères. Ils sont impliqués dans de nombreux processus physiologiques tels que la migration des cellules neuronales, la croissance axonale ou la synaptogenèse. Depuis, d'autres structures di-, oligo- et polysialylées ont été décrites chez les mammifères et chez l'homme en particulier, mais très peu d'études concernant leurs implications biologiques et leur biosynthèse ont été menées. Grâce à une analyse in silico, nous avons reconstitué un gène putatif d'a2,8-sialyltransférase sur le chromosome 10 humain, que nous avons nommé hST8Sia VI. Les analyses de l' expression de ce gène dans différents tissus et cellules humains ont mis en évidence une expression faible et ubiquiste de ce gène. Nous avons cloné la totalité du cadre ouvert de lecture de ce gène à partir des cellules cancéreuses mammaires MCF-7 et avons produit une forme recombinante soluble de hST8Sia VI dans des cellules COS-7 et Sf-9 pour caractériser son activité enzymatique. Nos tests d'activité in vitro ont démontré que hST8Sia VI catalyse le transfert d'un seul résidu d'acide sialique sur les motifs glucidiques sialylés en a2-3 trouvés sur les O-glycannes des glycoprotéines pour former des motifs disialylés du type Neu5Aca2-8Neu5Aca23Galb 1-3GalNAc-. Afin de déterminer son activité enzymatique in vivo, nous avons créé un modèle de cellules cancéreuses mammaires (MDA) sur-exprimant stablement hST8Sia VI. Nos premiers résultats indiquent que hST8Sia VI utilise préférentiellement les motifs a2,3-sialylés, ce qui confirme les analyses réalisées in vitro.LILLE1-BU (590092102) / SudocSudocFranceF

Expression of glypican-1, syndecan-1 and syndecan-4 mRNAs protein kinase C-regulated in rat immature Sertoli cells by semi-quantitative RT-PCR analysis

International audienc

Catabolic pathway of oligosaccharide-diphospho-dolichol. Subcellular sites of the degradation of the oligomannoside moiety

International audienc

A study on the regulation of N-glycoloylneuraminic acid biosynthesis and utilization in rat and mouse liver

International audienc