Molecular and cellular regulation of beta1,4-GalNAcT-II in physiological and pathological states (gastrointestinal cancer) – GALFISH

This project aims at understanding the molecular and cellular mechanisms underlying the supramolecular organization and localization of Golgi-GTs implicated in the biosynthesis of Sda et sLex antigens. These carbohydrate determinants are found in the normal and cancerous gastrointestinal (GI) tract respectively: Sda antigen is highly expressed in normal GI whereas sLex is almost not present. In cancer GI, the opposite is observed: the sLex antigen appears in multiple tumor types at the expense of the Sda antigen, which disappears. Although intensively studied, the mechanisms of regulation leading to the biosynthesis of one or another of these antigens, which involves several alpha2,3-sialyltransferases, alpha1,3-fucosyltransferases and one beta1,4-N-acetylgalactosaminyltransferase (beta4GalNAcT-II) are not yet elucidated. Very recent advances made in the field have shown that the regulation of the beta4GalNAcT-II involved the biosynthesis of the Sda antigen is a key regulation point for this balance and that deregulation of its expression leads to the biosynthesis of the sLex antigen observed in cancer cells. Transcription of the unique B4GALNT2 gene leads to the biosynthesis of two transcripts differing in their 5’-untranslated region. It seems that epigenetic regulation through DNA methylation of the promoter region of the B4GALNT2 gene is not sufficient to explain the down regulated expression of this GT in cancer GI tissues, which up to date has never been studied. Using a classical molecular biology approach, we will determine if the expression of the B4GALNT2 gene relies on one or two promoter regions and we will identify the transcription factors implicated in this regulation, in particular in the diminished expression of the beta4GalNAcT-II observed in cancer GI tract. Interestingly, translation of the two transcripts leads to the potential synthesis of two protein isoforms differing only in their cytoplasmic tail. One shows a regular 6 AA cytoplasmic tail, whereas this other shows an unusually long 66 AA cytoplasmic tail. These differences in the length of the cytoplasmic tails could lead to different molecular interactions between the two beta4GalNAcT-II protein isoforms with cytosolic proteins implicated in the subcellular localization and traffic of GTs in the Golgi apparatus. In particular, recent studies of CDG (Congenital Disorders of Glycosylation) patients has shown that deficiencies in the proteins implicated in vesicular Golgi trafficking could affect subcellular localization of GTs and lead to the biosynthesis of abnormal glycans. Our second objective is thus to study the subcellular localization of the two beta4GalNAcT-II protein isoforms and their potential differential implication in GTs complexes by virtue of their different cytoplasmic tail. Finally, we will assess the implication of vesicular Golgi trafficking proteins, in particular those of the COG complex, in their subcellular localization. To achieve this, we will develop a cell biology approach and observation by fluorescent microscopy (confocal and biophotonique) with FRET techniques to characterize molecular interactions between GT and potentially other proteins. Our third objective is to determine whether these regulations only take place in the human GI tissue, using an integrative biology and a phylogenomic approach to trace back the evolutionary history of the B4GALNT2 gene in vertebrates and to set up a whole organism model for the study of these regulations in vivo, the zebrafish D. rerio.