Novel Zebrafish Mono-α2,8-sialyltransferase (ST8Sia VIII): An Evolutionary Perspective of α2,8-Sialylation

Abstract

The mammalian mono-α2,8-sialyltransferase ST8Sia VI has been shown to catalyze the transfer of a unique sialic acid residues onto core 1 O-glycans leading to the formation of di-sialylated O-glycosylproteins and to a lesser extent to diSia motifs onto glycolipids like GD1a. Previous studies also reported the identification of an orthologue of the ST8SIA6 gene in the zebrafish genome. Trying to get insights into the biosynthesis and function of the oligo-sialylated glycoproteins during zebrafish development, we cloned and studied this fish α2,8-sialyltransferase homologue. In situ hybridization experiments demonstrate that expression of this gene is always detectable during zebrafish development both in the central nervous system and in non-neuronal tissues. Intriguingly, using biochemical approaches and the newly developed in vitro MicroPlate Sialyltransferase Assay (MPSA), we found that the zebrafish recombinant enzyme does not synthetize diSia motifs on glycoproteins or glycolipids as the human homologue does. Using comparative genomics and molecular phylogeny approaches, we show in this work that the human ST8Sia VI orthologue has disappeared in the ray-finned fish and that the homologue described in fish correspond to a new subfamily of α2,8-sialyltransferase named ST8Sia VIII that was not maintained in Chondrichtyes and Sarcopterygii.

Vertebrate Alpha2,8-Sialyltransferases (ST8Sia): A Teleost Perspective

“…The orthologues of ST8Sia I and ST8Sia V involved in gangliosides biosynthesis are identified in all the investigated genomes, suggesting a high conservation of the gangliosides biosynthetic pathways in vertebrates (Supplemental Table S1). Similarly, the ST8Sia III and the recently described fish ST8Sia VIII [17] could be found in all the Actinopterygii (ray-finned fishes) genomes ( Figure 1; Supplemental Table S1). Intriguingly, multiple copies of st8sia-related gene sequences were identified in Teleost genomes and their number varied considerably from one fish order or species to another.…”

“…Interestingly, our recent studies pointed to the fact that the st8sia gene family appears to be much larger in teleost fish genomes [14,17]. The emergence of several novel vertebrate mono-α2,8-sialyltransferases subfamilies like ST8Sia VII and ST8Sia VIII was described in this first group of ST8Sia and their enzymatic specificities remain to be determined.…”

“…The emergence of several novel vertebrate mono-α2,8-sialyltransferases subfamilies like ST8Sia VII and ST8Sia VIII was described in this first group of ST8Sia and their enzymatic specificities remain to be determined. These mono-α2,8-sialyltransferase genes have arisen as a consequence of whole genome duplications (WGDs, R1 and R2) at the base of vertebrates and were maintained in fish, whereas some others such as st8sia6, maintained in Tetrapods, have disappeared in fish [17,18]. In the second ST8Sia group, the enzymes responsible for the biosynthesis of sialic acid polymers, the poly-α2,8-sialyltransferases ST8Sia II and ST8Sia IV and the oligo-α2,8-sialyltransferase ST8Sia III, have been cloned and characterized from mammalian tissues, essentially the brain, where they act on α2,3-sialylated N-glycans of the neural cell adhesion molecule (NCAM), leading to an increased neuronal plasticity in embryos [19][20][21][22][23][24][25][26].…”

Direct Visualization of Live Zebrafish Glycan via Single-step Metabolic Labeling with Fluorophore-tagged Nucleotide Sugars

“…To first validate that the Cy5-tagged CMP-Sia analog can be incorporated by sialyltransferases in live cells, we examined the feasibility of transferring Sia-sulfo-Cy5 onto the cell-surface of sialylation-defect Chinese Hamster Ovary (CHO) mutant Lec2, using three recombinant human STs (hST3Gal1, hST3Gal4, hST6Gal1). 21 ST6Gal1/2 and ST3Gal 1/2/3/4/5 are evolutionarily conserved in human and zebrafish genomes and STs in both species share high homology, [22][23][24] therefore it is reasonable to use the human homologs to examine the donor substrate scope. After incubation with STs and probe A, we detected intense Cy5 fluorescence on Lec2 cells, but not in the cells treated without STs ( Fig.…”

“…For example, we observed that in the head region, fucosylatd glycans have a quicker turnover rate than sialylated glycans. 24,36,37 Studies by Stanley, Haltiwanger, and Taniguchi have shown that knockout of FUT8, POFUT1 or POFUT2 is lethal to mice. [38][39][40][41] In addition, congenital mutations of the Golgi localized GDP-fucose transporter SLC35C1 cause leukocyte adhesion deficiency type II, whose manifestation is severe developmental and immune deficiencies.…”