Cloning and expression of two key enzymes involved in glycosphingolipid biosynthesis during Xenopus early development

MELCHOR E. LUQUE; MARÍA E. MÓNACO; MANUEL J. AYBAR; SARA S. SÁNCHEZ

Guarujá, Brasil

Congreso; Second International Meeting of the Latinamerican Society of Developmental Biology; 2005

Glycosphingolipids (GSL) are believed to be integral for the dynamics of many cell membrane events, including cellular interactions, signaling and trafficking. In previous studies we investigated the ganglioside profile, cellular expression, and biosynthesis during the early development of the Bufo arenarum amphibian embryo. Since molecular information about the participation of gangliosides in the early developmental processes is lacking, we decided to clone key enzymes involved in the biosynthesis metabolism of such molecules. In this work we report the molecular cloning of UDP-glucose:ceramide glucosyl transferase (CGT), that catalizes the first committed step of glycosphingolipids synthesis, and the cloning of a2,8-sialyltrasnsferase (GD3 synthase, GD3s), a key enzyme that catalizes the first step of b-series gangliosides synthesis. We have isolated a 1.62 Kb length transcript for CGT and 2.88 Kb length transcript for GD3s, encoding proteins of 394 and 338 aminoacid residues, respectively. Expression of both enzymes was analyzed during early development of Xenopus embryos by RT-PCR and in situ hybridization. We detected transcripts of CGT from oogenesis and first segmentation stages, while GD3s mRNA showed zygotic expression detectable from stage 10 that increases at later stages of development. The spatio-temporal comparative analysis of expression patterns of both enzymes and specific markers by in situ hybridization reveals that they are developmentally regulated. CGT and GD3s were expressed at ectodermal and mesodermal tissues at early stages. GD3s mRNA was found in dorsal blastopore lip at gastrula stages. At early neurula stage, the expression of GD3s appeared in dorsal ectodermal regions and in the paraxial mesoderm. Our results support a role for gangliosides in the development of mesoderm and dorsal structures of Xenopus laevis embryos.