CHARGE-DISTRIBUTION OF FLANKING AMINO-ACIDS INFLUENCES O-GLYCAN ACQUISITION IN-VIVO

The elements that regulate O-glycosylation are poorly understood, We h ave developed a novel in vivo system to analyze the role of flanking s equence on the modification of a single well characterized O-glycosyla tion site derived from human von Willebrand factor (PHMAQVTVGPGL), A s ecreted chimeric reporter protein, containing the human von Willebrand factor sequence, an antibody recognition epitope, and a heart muscle kinase site, was engineered and expressed in COS7 and MCF-7 cells, Gly cosylated and non-glycosylated forms of the immunoprecipitated reporte r were resolved electrophoretically and their relative amounts quantit ated, Using mutational analysis we find that the glycosylation apparat us of COS7 cells can accommodate a broad range of changes in the flank ing sequence without compromising glycosylation, but that the distribu tion of charged amino acids flanking the O-glycosylation site can have a profound influence on glycosylation with position -1 relative to th e glycosylation site being particularly sensitive, A combination of ac idic residues at positions -1 and +3 almost completely eliminates glyc osylation of the reporter in both COS7 and MCF-7 cells. The overall de nsity of charged amino acids is less important since substitution of a cidic residues at position -2, +1, and +2 had no effect in the level o f glycosylation observed.