HUMAN ST3Gal V ISOFORMS, BOTH TARGETED TO GOLGI, DERIVE FROM USE OF TWO ALTERNATE TRANSLATION START SITES

ST3Gal V (or GM3 synthase) is the sialyltransferase that catalyses the initial step in the biosynthesis of most complex gangliosides from lactosylceramide. ST3Gal V plays a key regulatory role in determining the cell surface ganglioside profile and, consequently, in modulating a large variety of ganglioside-dependent cellular events, such as cell proliferation and differentiation, adhesion, apoptosis, oncogenesis. In addition, a homozygous loss-of-function mutation in the hST3Gal V gene is cause of an autosomal recessive infantile–onset symptomatic epilepsy syndrome. Recently, we have identified a hST3Gal V mRNA variant containing an additional translation start codon, located upstream and in-frame with that considered unique translation initiation site in the human GM3 synthase gene, providing the first evidence of the existence of two differentially N-terminal extended isoforms of the protein [1]. The functional relevance of the longer isoform has to be defined yet, and a study to define its sub-cellular localization could provide useful findings to approach this topic. Here, we have recombinantly expressed the c-myc C-terminal tagged longer isoform in COS-7 cells and verified its N-glycosylation state by treatment of transfected-cell lysates with PNGase F and endo-H; the protein expression and the glycosidase-digested products have been analyzed by western blot with anti-c-myc antibodies. Surprisingly, the transient transfection of COS-7 cells with the hST3Gal V cDNA corresponding to mRNA variant isolated by us resulted in the expression of both hST3Gal V isoforms suggesting the possibility of an alternate initiation of translation by context-dependent leaky scanning. The hypothesis has been confirmed by site-specific mutagenesis experiments. Results of PNGase F and endo-H treatments indicated that the longer isoform carries N-glycans of complex type as the shorter form, strongly supporting the conclusion that both isoforms are targeted to the Golgi. Analyses to determine eventual differences in the Golgi compartment (cis, medial, or trans) localization of the two hST3Gal V isoforms are in progress. 1. Berselli P et al, 2006, Human GM3 synthase: a new mRNA variant encodes an NH2-terminal extended form of the protein, Biochim Biophys Acta 1759, 348-358