IDENTIFICATION OF A NOVEL GM3 SYNTHASE mRNA ISOFORM FROM HUMAN PLACENTA AND FUNCTIONAL ANALYSIS OF THE 5’-FLANKING REGION OF THE TRANSCRIPT

GM3 synthase is a key enzyme in the biosynthesis of gangliosides that, as mediators of cell-cell interactions and modulators of signaling transduction, play fundamental roles in many both physiological and pathological cellular processes (i.e., proliferation, differentiation, oncogenesis) (1,2). GM3 synthase 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. Up to date GM3 synthase cDNA has been isolated and characterized from both TPA-differentiated HL-60 cells and human fetal and adult brain (3,4) and five mRNA variants have been identified. They differ in the 5’-untranslated region, but all of them predict a 41.7 kDa protein having substrate activity highly restricted to lactosylceramide. On the contrary, our study, directed to the isolation of the full length GM3 synthase cDNA from human placenta, led to the identification of an unusual GM3 synthase mRNA variant that strongly differs from all the other GM3 synthase transcripts until now identified. In fact, its 5’-terminal region introduces a new translation initiation site located upstream and in-frame with that usually considered as translation initiation codon in the GM3 synthase gene. Studies of in vitro translation and in vivo expression indicate that the human placental cDNA leads to the synthesis of two polypeptides with apparent molecular mass, in SDS-PAGE, of 42.1 and 38.3 kDa, respectively. Moreover, the stable transfection of the human placental cDNA into mammalian MG1361 cells resulted in a threefold increase of GM3 synthase activity, associated to a broader substrate specificity, and, more importantly, in significant alterations of cell ganglioside profile. Therefore, our data provide the first demonstration of the existence of two GM3 synthase isoforms and indicate that, at least in the human placenta, they apparently derive from a single transcript. In addition, to elucidate the mechanism that regulates the tissue-specific expression of the human GM3 synthase gene we isolated the genomic DNA region (~3 kb) flanking the placental GM3 synthase transcript. Sequence analysis with the MatInspector 2.2 program revealed that this region lacks canonical TATA e CAAT boxes, but contains several putative transcription factor binding sites including AP1, GATA1, NFAT, MZF1, IK2; unfortunately no placenta-specific transcription factor is reported in none of the available transcription factor databases. Up to date, functional analysis of the isolated genomic region has been assessed in human placental choriocarcinoma JEG3 cells by transient transfection, but preliminary data seem to indicate that it doesn’t promote transcription of the reporter SEAP gene. Future plans are to analyze the functional activity of DNA fragments containing varying lengths of the 5’-flanking genomic sequence in both JEG3 cells and other suitable cell lines so to evaluate the eventual presence of positive and negative regulatory elements and factors in both the DNA sequence and the cell line. 1) Hakomori S.I. (2000) Glycoconj. J. 17, 143. 2) Hakomori S.I. (2000) Glycoconj. J. 17, 627. 3) Ishii A. et al. (1998) J. Biol. Chem. 273, 31652. 4) Kapitonov D. et al.(1999) Glycoconj J. 16, 337.