Glycosylation of closely spaced acceptor sites in human glycoproteins

Abstract

Asparagine linked glycosylation of proteins by the oligosaccharyltransferse (OST) occurs when acceptor sites or sequons (N-X≠P-T/S) on nascent polypeptides enter the lumen of the rough endoplasmic reticulum. Metazoan organisms assemble two isoforms of the oligosaccharyltransferase that have different catalytic subunits (STT3A or STT3B) and partially non-overlapping cellular roles. Potential glycosylation sites move past the translocation channel associated STT3A complex at the protein synthesis elongation rate. Here, we have investigated whether close spacing between acceptor sites in a nascent protein promotes site skipping by the STT3A complex. Biosynthetic analysis of four human glycoproteins revealed that closely spaced sites are efficiently glycosylated by an STT3B-independent process unless the sequons contain non-optimal sequence features including extreme close spacing between sequons (e.g., NXTNXT) or the presence of paired NXS sequons (e.g., NXSANXS). Many, but not all, glycosylation sites that are skipped by the STT3A complex can be glycosylated by the STT3B complex. Analysis of a murine glycoprotein database revealed that closely spaced sequons are surprisingly common, and are enriched for paired NXT sites when the gap between sequons is less than three residues.