A hierarchical structure in the N-glycosylation process governs the N-glycosylation output: prolonged cultivation induces glycoenzymes expression variations that are reflected in the cellular N-glycome but not in the protein and site-specific glycoprofile of CHO cells.

N-glycosylation is a central component in the modification of secretory proteins. One characteristic of this process is a heterogeneous output. The heterogeneity is the result of both structural constraints of the glycoprotein as well as the composition of the cellular glycosylation machinery. Empirical data addressing correlations between glycosylation output and glycosylation machinery composition are seldom due to the low abundance of glycoenzymes. We assessed how differences in the glycoenzyme expression affected the N-glycosylation output at a cellular as well as at a protein-specific level. Our results showed that cellular N-glycome changes could be correlated with the variation of glycoenzyme expression, whereas at the protein level differential responses to glycoenzymes alterations were observed. We therefore identified a hierarchical structure in the N-glycosylation process: the enzyme levels in this complex pathway determine its capacity (reflected in the N-glycome), while protein-specific parameters determine the glycosite-specificity. What emerges is a highly variable and adaptable protein modification system that represents a hallmark of eukaryotic cells.