Galactosyltransferase 4 is a major control point for glycan branching in N-linked glycosylation

Abstract

Protein N-glycosylation is a common post-translational modification that produces a complex array of branched glycan structures. The levels of branching, or antennarity, give rise to differential biological activities for single glycoproteins. However, the precise mechanism controlling the glycan branching and glycosylation network is unknown. Here, we constructed quantitative mathematical models of N-linked glycosylation that predicted new control points for glycan branching. Galactosyltransferase, which acts on N-acetylglucosamine residues, was unexpectedly found to control metabolic flux through the glycosylation pathway and the level of final antennarity of nascent protein produced in the Golgi network. To further investigate the biological consequences of glycan branching in nascent proteins, we glycoengineered a series of mammalian cells overexpressing human chorionic gonadotropin (hCG). We identified a mechanism in which galactosyltransferase 4 isoform regulated N-glycan branching on the nascent protein, subsequently controlling biological activity in an in vivo model of hCG activity. We found that galactosyltransferase 4 is a major control point for glycan branching decisions taken in the Golgi of the cell, which might ultimately control the biological activity of nascent glycoprotein.

Transfection of glycoprotein encoding mRNA for swift evaluation of N‐glycan engineering strategies

“…23 At most processing steps in the Golgi, N-glycans are potential substrates for multiple enzymes at a time and changes in glycosyltransferase expression levels have previously been described to shift the equilibrium of possible further modifications and therefore impact the resulting N-glycan profiles. 19,28,29 Competition between ST3GAL and B3GNT has been observed previously for interferon γ N-glycan processing. 9 LacNAc synthesis is rather poorly characterized for CHO cells, but overall this N-glycan modification is regarded as undesirable as it increases N-glycan heterogeneity.…”

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

“…Although the degree to which individual enzymes have been studied may vary, evidence of differential localization of glycosylation enzymes has been unambiguously demonstrated. Thus, in contrast to previous studies that attempted a non-compartmentalized view on Golgi reaction topology (McDonald et al, 2014), we find it imperative to carefully consider differential Golgi enzyme localization for modeling purposes. We are aware, though, that exclusive assignment of enzymes to distinct compartments likely represents an oversimplification since enzyme activity actually only peaks in a certain compartment but spans multiple compartments (Nilsson et al, 1993b; Rabouille et al, 1995).…”

Controlling the time evolution of mAb N‐linked glycosylation ‐ Part II: Model‐based predictions

“…This modeling approach was further expanded by incorporating fucosylation, galactosylation, and sialylation resulting in a large network with several thousands of substances and reactions . Furthermore, the microheterogeneity model has been used for glycosylation controllability analysis toward real‐time glycosylation online control and for the identification of key enzymes that control glycan branching . Del Val et al have recently developed a dynamic model of monoclonal antibody glycosylation that links cellular metabolism via nucleotide sugar donors.…”