Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.

Multiple sugar molecules that may or may not include mannose can be added to the precursor in the Golgi to generate complex oligosaccharides. N-linked sugars could be classified as high-mannose oligosaccharides based on whether sugars other than mannose and N-acetyl glucosamine are present. As soon as an N-linked oligosaccharide precursor generated in the ER enters the Golgi, it undergoes sugar trimming. Misfolded protein intermediates created during trimming are detected by a conformation sensing ER glucosyltransferase that adds a glucose residue, tagging the protein for another chance at folding. Proteins that fail to fold are transported to proteasomes for degradation.

In contrast, trimmed high-mannose oligosaccharides do not undergo sugar addition once they enter the Golgi apparatus. While the glycan core of the complex oligosaccharide is more accessible to adding sugars and tagging, the high mannose sugar cores remain tightly bound and inaccessible to the enzymes. Thus, resistance to specific glycosidases like Endo H is characteristic of maturing glycans. Because sugar addition is unidirectional, Endo H sensitivity is widely used to trace the forward transport of newly synthesized glycoproteins in the secretory pathway.