Molecular basis for glycan recognition and reaction priming of eukaryotic oligosaccharyltransferase

Abstract

Oligosaccharyltransferase (OST) is the central enzyme of N-linked protein glycosylation. It catalyzes the transfer of a pre-assembled glycan, GlcNAc2Man9Glc3, from a dolichyl-pyrophosphate donor to acceptor sites in secretory proteins in the lumen of the endoplasmic reticulum. Precise recognition of the fully assembled glycan by OST is essential for the subsequent quality control steps of glycoprotein biosynthesis. However, the molecular basis of the OST-donor glycan interaction is unknown. Here we present cryo-EM structures of S. cerevisiae OST in distinct functional states. Our findings reveal that the terminal glucoses (Glc3) of a chemo-enzymatically generated donor glycan analog bind to a pocket formed by the non-catalytic subunits WBP1 and OST2. We further find that binding either donor or acceptor substrate leads to distinct primed states of OST, where subsequent binding of the other substrate triggers conformational changes required for catalysis. This alternate priming allows OST to efficiently process closely spaced N-glycosylation sites.

Investigation of the Catalytic Mechanism of a Soluble N-glycosyltransferase Allows Synthesis of N-glycans at Noncanonical Sequons

“…OST enzymes have highly conserved WWD and MXXI/DK motifs (the MXXI motif in bacteria, the DK motif in archaea and eukaryotes) responsible for recognizing the N-X-S/T sequon (Figures S6E–G). ,− In NGT, we wonder if the highly conserved 215 DVYM 218 motif fulfills a similar role (Figure S6H).…”

Optimizing Phosphate Couplings for Dolichyl Diphosphochitobiose to Enable Protein N‐Glycosylation Studies

“…of the lipid phosphate was used. A 8 : 2 : 1 AcOEt/ i PrOH/H 2 O solvent system was used to elute imidazole, and subsequent elution with 4 : 2 : 1 ratio delivered the pure product (8).…”

“…This process involves the stepwise assembly of the lipid‐linked oligosaccharide (LLO) 1a from the corresponding polyprenyl diphosphochitobiose precursor 2a , followed by transfer of its tetradecasaccharide unit to the asparagine side‐chain of nascent proteins by a series of membrane‐bound glycosyl transferases ( Figure 1). [1,2] While the natural substrates 1a and 2a cannot be handled properly due to their long polyprenol chain which renders them insoluble in water, the synthetic analog 2b , dolichyl diphosphochitobiose (GlcNAc 2 ‐PP‐Dol 25 ), carrying a simplified C 25 ‐dolichyl lipid, is accepted as substrate and processed by the entire set of glycosyl transferases all the way up to the tetradecasaccharide ( 1b ) and its transfer to acceptor proteins, enabling detailed structural and biochemical studies of several of these enzymes [3–9] . Herein, we report our optimization of the chemical synthesis of LLO precursor 2b , whose continuous supply is critical to enable biochemical and structural studies of protein N ‐glycosylation and its further engineering to produce glycosylated biologics.…”

“…[1,2] While the natural substrates 1a and 2a cannot be handled properly due to their long polyprenol chain which renders them insoluble in water, the synthetic analog 2b, dolichyl diphosphochitobiose (GlcNAc 2 -PP-Dol 25 ), carrying a simplified C 25dolichyl lipid, is accepted as substrate and processed by the entire set of glycosyl transferases all the way up to the tetradecasaccharide (1b) and its transfer to acceptor proteins, enabling detailed structural and biochemical studies of several of these enzymes. [3][4][5][6][7][8][9] Herein, we report our optimization of the chemical synthesis of LLO precursor 2b, whose continuous supply is critical to enable biochemical and structural studies of protein N-glycosylation and its further engineering to produce glycosylated biologics. This optimization focused on three low-yielding phosphate coupling steps and the final product purification.…”

The role of N-glycosylation modification in the pathogenesis of liver cancer

“…Subsequently, the N-glycan is recognized by the oligosaccharyltransferase (OST) complex, and the OST complex mediates its transfer to the N site on the nascent polypeptide chain that harbors an NXS/T(X ≠ P) sequence combination [ 7 ]. Finally, the N-glycan of the bound polypeptide chain will enter the Golgi for further processing and modification to form three different N-glycan classes: oligomannose, hybrid and complex [ 9 ]. The abovementioned is the main process of N-glycosylation.…”