Charge Distribution of Flanking Amino Acids Influences O-Glycan Acquisition in Vivo

Keith Nehrke,Fred K Hagen,Lawrence A Tabak

doi:10.1074/jbc.271.12.7061

Keith Nehrke, Fred K Hagen + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.12.7061

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Abstract

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.

Highlights

Must exist to specify which Ser and Thr residues acquire Oglycans
In the present study we have examined a small chimeric reporter protein to determine the role played by flanking amino acids on in vivo O-glycosylation
Glycosylation of a Chimeric Reporter Protein in COS7 Cells— Wild-type rHVF was O-glycosylated when expressed from COS7 cells acquiring a common O-glycan structure consisting of sialic acid residues attached to a Gal␤1,3GalNAc core. rHVF migrated as a single species with a relative molecular mass of approximately 6,000 Da (Fig. 2, lane 2)

Summary

Introduction

Must exist to specify which Ser and Thr residues acquire Oglycans. From surveys of known O-glycosylation sites, it is apparent that charged residues are rarely found at positions flanking glycosylated Thr or Ser, while Pro and clusters of Ser and Thr often flank glycosylated residues (5–7). Construction of Vector Encoding Chimeric Reporter—The reporter protein (rHVF) contains the amino acid sequence PHMAQVTVGPGL, which is a known glycosylation site of human von Willebrand factor and which is an in vitro substrate for ppGaNTase (8).

Results

Conclusion