GM3 SYNTHASE FROM HUMAN PLACENTA : GENE CLONING AND PROTEIN CHARACTERIZATION

GM3 ganglioside is the first and the simplest ganglioside: it represents the common precursor for nearly all the naturally occurring ganglioside species, and it is ubiquitously distributed on the plasma membrane of all eukaryotic cells. Moreover, GM3 itself is known to have important functions in modulation of cellular growth, in cell differentiation and in cell adhesion/recognition processes through glycosphingolipid-enriched domains [50-64]. GM3 synthase (EC 2.4.99.9, ST3Gal V) catalyzes GM3 ganglioside biosynthesis via the transfer of a sialic acid moiety from CMP-sialic acid onto LacCer and, since this reaction represents the first committed step in the synthesis of nearly all ganglio-series gangliosides, it can be considered a key regulatory enzyme. GM3 synthase is, like all the glycosyltransferases, a type II transmembrane glycoprotein with a short NH2-terminal cytoplasmic tail which is not essential for its catalytic activity, followed by an hydrophobic segment that functions as signal anchor domain for its retention in the Golgi membrane, and by a large COOH-terminal catalytic domain that resides in the lumen [19]. In humans, GM3 synthase is mainly expressed in brain, muscle, testis and placenta: by now, GM3 synthase has been purified only by rat liver and brain, whereas human cDNAs has only been isolated by adult and fetal brain and by TPA-treated HL-60 cells. All the human cDNAs cloned up to now differ in their 5’ UTRs, indicating a gene expression transcriptionally regulated, but present an identically extended coding region: the encoded protein consists of 362 amino acid residues with a predicted molecular mass of 41.7 kDa, having an enzymatic activity highly restricted to LacCer [69-71]. On the contrary, rodent purified enzymes can use other glycolipids as acceptor substrates, including GalCer, GlcCer, aGM2, and aGM1, although with a lower extent of specificity respect to LacCer [64, 65]. In order to better understand the physiological and pathological roles of endogenous GM3, the aim of the research of my PhD was to investigate the regulation of the tissue-specific expression of the human GM3 synthase gene. The first step was to identify and isolate GM3 synthase cDNA from human placenta, where GM3 synthase is highly expressed [66, 70], and where GM3 expression levels vary in a dramatic way during gestation, suggesting a fine regulation of the activity and expression of this biosynthetic enzyme [78]. Interestingly, since the sequence analysis of this cDNA, highly homologous to all the other human GM3 synthase cDNAs, indicated the presence of a new putative translational start codon generating a protein with an NH2-extension of 33 amino acid residues, the corresponding gene product was biochemically characterized with both in vitro and in vivo approaches. Finally, to establish the mechanism that regulates the synthesis of this “unusual” GM3 synthase transcript in the human placenta, the genomic region flanking the 5’-terminus of this mRNA isoform was isolated and its possible functional activity as promoter analyzed.