Data di Pubblicazione:
2004
Citazione:
ISOLATION AND CHARACTERIZATION OF A NOVEL ST3Gal V mRNA ISOFORM
FROM HUMAN PLACENTA / P.V. Berselli, S. Zava, E. Sottocornola, I. Colombo. ((Intervento presentato al 49. convegno SIB tenutosi a Riccione nel 2004.
Abstract:
INTRODUCTION: ST3Gal V (EC 2.4.99.9, GM3 synthase) is a key enzyme in the biosynthesis of gangliosides, a large and heterogeneous family of sialic acid-containing glycosphingolipids that, as mediator of cell-cell interactions and modulators of signalling transduction, play fundamental roles in many both physiological and pathological cellular processes (i.e., proliferation, differentiation, oncogenesis) (1). It catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto LacCer producing GM3, a ganglioside ubiquitously distributed on the plasma membrane of all the eukaryotic cells, representing the common precursor of almost all ganglio-series gangliosides (2). However, the homologous rat protein purified from brain and liver can use other glycolipids as acceptor substrates, including GalCer, GlcCer, aGM2, and aGM1, although with a lower extent of specificity respect to LacCer (3). Contrasting results are also reported about the intracellular localization of the protein: GM3 synthase activity has been detected in both proximal and distal compartments of Golgi apparatus (4).
Human ST3Gal V results expressed in a tissue-specific manner, especially in brain, muscle, testis, and placenta, and only one transcript, of about 2.4 kb, has been detected in all the tissues (5). Human ST3Gal V cDNAs have been already cloned from both TPA-differentiated HL60 cells and fetal and adult brain, and several mRNA variants have been identified (5). They differ in the 5’-UTR sequences, but all of them seem to contain an identical coding region; the substrate activity of the encoded protein (362 aminoacids with a predicted molecular mass of 41.7 kDa) is highly restricted to LacCer. Studies on the structural organization and transcriptional regulation of the gene (6) also provided contrasting, but stimulating, results.
MATERIALS AND METHODS: The complete ST3Gal V cDNA from human placenta was obtained by the 5’- and 3’-RACE technology (SMART RACE cDNA Amplification Kit, Clontech) using total RNA as template. The sequence of the PCR product was determined by M-Medical’s DNA sequencing service. The transcription initiation site was evaluated by primer extension analysis. The translation initiation site was identified by in vitro experiments, using the TNT® T7 Quick coupled transcription/translation system (Promega), and it was confirmed by in vivo analyses. The isolated cDNA protein product, expressed as fusion protein with EGFP in CV1 cells using pEGFP-N3 as expression vector, was detected by western blot with specific anti-GFP antibodies. The enzymatic activity of the isolated cDNA protein product was assayed in rat mammary adenocarcinoma cells stably transfected with the isolated cDNA using the pRC/CMV (Promega) as expression vector; the enzymatic activity was determined by an in vitro radioactive assay using the microsomal enriched protein fraction as enzyme source, CMP-[14C]sialic acid as donor substrate, and different glycolipids as acceptor substrates. The genomic structure of the human ST3Gal V gene has been determined through a human genome BLAST homology search of the public database (GenBank) using the isolated cDNA as query sequence.
RESULTS: A cDNA, consisting of 2149 bp (the poli-A tail is lacking) and showing high sequence identity with all the human ST3Gal V cDNAs until now registered in GeneBank, has been successfully isolated and cloned from human placenta. Primer extension experiments confirmed the transcription initiation site. Not surprisingly, it differs from all the other human cDNAs in the 5’-end sequence, but, with respect to the other ones, it contains a new, possibly translation initiation, ATG codon; it is located upstream and in frame with the ATG indicated as translation initiation site in all the
Tipologia IRIS:
14 - Intervento a convegno non pubblicato
Elenco autori:
P.V. Berselli, S. Zava, E. Sottocornola, I. Colombo
Link alla scheda completa: