UDP-GlcNac transporter SLC35A3 participation in Xenopus laevis development

HAEL A; AYBAR M; BREDESTON LM

Tucuman

Congreso; XLI Reunion Anual de la Sociedad Argentina de Biofisica; 2012

Sociedad Argentina de Biofisica

Nucleotide-sugar transporters (NST, SLC35 family) control the flux of activated sugar to the lumen of Golgi apparatus where glyconjugate biosynthesis take place by specific transferases. SLC35A3 is a member of the NST family with 326 aminoacids and a prediction of 8-10 transmembrane helices (TM). Missense mutations in bovine SLC35A3 cause vertebral complex malformation, characterized by misshapen and fused vertebrae around the cervico-thoracic junction, by an unknown molecular mechanism (Thomsen B et al., 2006). Here we propose the use of Xenopus laevis as a model to study the role of XlSLC35A3 on embryonic development. Results shown: 1) Xenopus tropicalis SLC35A3 complement the yeast mutant Kl3 , lacking a UDP-GlcNac transporter, confirming its specificity for UDP-GlcNac. 2) Fusion of GFP to the C-terminal of XtSLC35A3 does not affect transport activity and can be used as an expression indicator. 3) A mutant of XtSLC35A3 lacking the only two cysteines is active indicating that these residues are not essential for transport activity. Mutation of residues E47C in TM2 impairs activity but not expression. 4) RT-PCR studies of SLC35A3 using X. laevis embryos show expression between stages 28 and 35. 5) Localization of SLC35A3 transcripts in X. laevis embryos from stages 18 to 33, by whole mount in situ hybridization, showed specific labelling of the notochord from stage 28 to 33. 6) Overexpression of wild type SLC35A3 affected mesoderm, the embryonic sheet from which notochord is derived, suggesting that precise levels of transporter expression are necessary for the normal development of the mesoderm. 1. Thomsen B et al. (2006) Genome Res. 16, 97-105 Acknowledgment: supported by CIUNT, CONICET and ANPCYT-FONCYT.