Charge distribution of flanking amino acids influences O-glycan acquisition in vivo.
The elements that regulate O-glycosylation are poorly understood. We have developed a novel in vivo system to analyze the role of flanking sequence on the modification of a single well characterized O-glycosylation site derived from human von Willebrand factor (PHMAQVTVGPGL). A secreted 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. Glycosylated and non-glycosylated forms of the immunoprecipitated reporter were resolved electrophoretically and their relative amounts quantitated. Using mutational analysis we find that the glycosylation apparatus of COS7 cells can accommodate a broad range of changes in the flanking sequence without compromising glycosylation, but that the distribution of charged amino acids flanking the O-glycosylation site can have a profound influence on glycosylation with position -1 relative to the glycosylation site being particularly sensitive. A combination of acidic residues at positions -1 and +3 almost completely eliminates glycosylation of the reporter in both COS7 and MCF-7 cells. The overall density of charged amino acids is less important since substitution of acidic residues at position -2, +1, and +2 had no effect in the level of glycosylation observed.
Nehrke
K
K
Department of Dental Research, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA.
Hagen
F K
FK
Tabak
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LA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011134:Polysaccharides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013696:Temperature; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D009838:Oligodeoxyribonucleotides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor