Glycopeptide specificity of the secretory protein folding sensor UDP–glucose glycoprotein:glucosyltransferase

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Abstract
Secretory and membrane N‐linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle. UDP–glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP–glucose to N‐linked Man9‐GlcNAc2 glycans. To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9‐GlcNAc2 glycopeptide substrates in vitro , and determined quantitatively the glucose incorporation into each glycan by mass spectrometry. A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT. The preference for amino‐acid residues close to N‐linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT.