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Profiling of N-linked glycans and O-GlcNAc modified proteins by incorporation of azido-sugar analogues in carbohydrates of melan-a, TM1 and TM5 cell lineages

Grant number: 09/02065-7
Support type:Regular Research Grants
Duration: June 01, 2009 - November 30, 2011
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:José César Rosa
Grantee:José César Rosa
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil


This proposal will investigate the profiling of N-glycans and O-GlcNAc modified proteins in melan-a, TM1 and TM5 cell lineages using a glycoproteomic approach. The cell lineages represent a murine model of melanoma progression: Melan-a, non tumorigenic lineage was obtained from normal melanocytes, while TM1 and TM5 are tumorigenic cells derived from melan-a that present phenotype of vertical growth phase (VGP). TM1 and TM5 were obtained after submitting melan-a to sequential cycles of forced anchorage impediment and represent cells that were resistant to anoikis. The following investigation will be done: 1) Studies of azido sugar metabolic incorporation in carbohydrates of those cell lineages using analogues of N-acetylglucosamine (GlcNAc) and N-acetylmanosamine (ManNAc) commercially available, as tetra-acetyl-N-azidoacetylglicosamine (Ac4GlcNAz) and tetra-acetyl-N-acetylmanosamine (Ac4ManNAz), 2) Characterization of N-glycans in glycoproteins and 3) glycoproteins modified by O-GlcNAc. The unnatural sugars Ac4GlcNAz and Ac4ManNaz incorporate into the carbohydrate molecules as N-acetylglucosamine and sialic acid in the cells using normal biosynthetic pathway. They contain a reactive azide group that reacts selectively with phosphine group (PH3) in a reaction denominated of Staudinger ligation which provides a wide variety of tags including fluorescent probes and affinity tags such as biotin and FLAG tags. The strategy for detection and isolation of post-translationally modified proteins based on tag attached to the modification substrate is referred to as tagging-via-substrate or the TAS technology. TAS technology will be used for detection and identification of O-GlcNAc modification and profiling of N-linked glycans by combination of FLAG/biotin/streptavidin complexes. The enriched glycoproteins will be analyzed by mass spectrometry after the separation of glycans and protein backbone by treatment of endoglycanase F and reverse phase chromatography for protein identification and glycan structural details. The focus of this investigation will be the global analysis of glycoproteins providing basic knowledge in glycobiology and producing candidate biomarkers for melanoma. (AU)