The evolution of galactose α2,3-sialyltransferase: Cionaintestinalis ST3GAL I/II and Takifugu rubripes ST3GAL II sialylate Galβ1,3GalNAc structures on glycoproteins but not glycolipids

Abstract

Sialyltransferases are a family of enzymes catalyzing the transfer of sialic acid residues to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. Although expression of sialic acid is well documented in animals of the deuterostomian lineage, sialyltransferases have been predominantly described for relatively recent vertebrate lineages such as birds and mammals. This study outlines the characterization of the only sialyltransferase gene found in the tunicate Ciona intestinalis, the first such report of a non-vertebrate deuterostomian sialyltransferase, which has been discussed as a possible orthologue of the common ancestor of galactose alpha2,3-sialyltransferases. We also report for the first time the characterization of a ST3Gal II gene from the bony fish Takifugu rubripes. We demonstrate that both genes encode functional alpha2,3-sialyltransferases that are structurally and functionally related to the ST3Gal family of mammalian sialyltransferases. However, characterization of the recombinant, purified forms of both enzymes reveal novel acceptor substrate specificities, with sialylation of the disaccharide Galbeta1-3GalNAc and asialofetuin, but not GM1 or GD1b observed. This is in contrast to the mammalian ST3Gal II that predominantly sialylates gangliosides. Taken together the ceramide binding/recognition site previously proposed for the mouse ST3Gal II might represent a unique feature of mammalian ST3Gal II that is missing in the evolutionary more distant fish and tunicate species reported here. This suggests that during the evolution of the ST3Gal II, probably following the separation of the teleosts, a significant shift in substrate specificity enabling the sialylation of gangliosides took place.

Integrative View of α2,3-Sialyltransferases (ST3Gal) Molecular and Functional Evolution in Deuterostomes: Significance of Lineage-Specific Losses

“…However, information on invertebrate st3gal -related sequences is scarce. A unique cDNA homolog called ST3Gal I/II ( Harduin-Lepers et al 2005 ) was cloned from the tunicate Ciona intestinalis ( Lehmann et al 2008 ) and from the amphioxus Branchiostoma belcheri ( Guérardel et al 2012 ) suggesting that the ST3Gal family is present in a few copy number in tunicates and in cephalochordates.…”

Phylogenetic-Derived Insights into the Evolution of Sialylation in Eukaryotes: Comprehensive Analysis of Vertebrate β-Galactoside α2,3/6-Sialyltransferases (ST3Gal and ST6Gal)

“…The invertebrate ST genes are orthologous to the common ancestor of the vertebrate subfamilies and are named accordingly. For instance, the D. melanogaster st6gal1/2 gene (also known as DSiaT) described in [48] and the C. intestinalis st3gal1/2 gene [5]. …”

“…Sialic acids (SA) represent a broad family of nine-carbon electro-negatively charged monosaccharides commonly described in the deuterostomes and some microorganisms [1,2,3,4,5]. Interestingly, SA show a discontinuous distribution across evolutionary metazoan lineages.…”

“…Each family catalyzes the formation of different glycosidic linkages, α2–3, or α2–6 to the terminal Gal residue in N - or O -glycans, α2–6 to the terminal GalNAc residue in O -glycans and glycolipids and α2–8 to terminal SA residues in N - or O -glycans or glycolipids). The ST enzymatic activities have been documented mainly in mouse and human tissues and more recently in chicken [46,47], and to a lesser extent in the invertebrates like the fly D. melanogaster [48], the silkworm Bombyx mori [49], the amphioxus Branchiostoma floridae [1] and the tunicate C. intestinalis [5]. Each member of the mammalian ST3Gal and ST6Gal families shows exquisite acceptor specificities (for reviews, see [41,50,51].…”

Moraxella catarrhalis Lgt2, a galactosyltransferase with broad acceptor substrate specificity

“…Galactosyltransferase assays were performed using a modification of the method described in Lehmann et al 25 Briefly, purified Lgt2 B/C (50 lg) was combined in a final reaction volume of 100 lL with an appropriate acceptor substrate in 50 mM Tris (pH 6.8.) containing 10 mM Mg 2+ , and 0.5 mM UDP-Gal with 0.18 lCi UDP-[ 3 H]-Gal (GE Healthcare, specific activity 5 Ci mmol À1 ), at 30°C for 18 h. The temperature optimum was determined between 20 and 42°C, and the pH optimum was determined over a pH range from 5.6 to 8.8, using 50 mM citrate-phosphate buffer (pH 5.6), and 50 mM phosphate buffer (pH 6-8.8).…”