Site-to-site cross-talk in OST-B glycosylation of hCEACAM1-IgV

Abstract

N-glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus sequence, N-X-S/T. While this sequence is required for glycosylation, the initial transfer of a high-mannose glycan by oligosaccharyl transferases A or B (OST-A or OST-B) can lead to incomplete occupancy at a given site. Factors that determine the extent of transfer are not well understood, and understanding them may provide insight into the function of these important enzymes. Here, we use mass spectrometry (MS) to simultaneously measure relative occupancies for three N-glycosylation sites on the N-terminal IgV domain of the recombinant glycoprotein, hCEACAM1. We demonstrate that addition is primarily by the OST-B enzyme and propose a kinetic model of OST-B N-glycosylation. Fitting the kinetic model to the MS data yields distinct rates for glycan addition at most sites and suggests a largely stochastic initial order of glycan addition. The model also suggests that glycosylation at one site influences the efficiency of subsequent modifications at the other sites, and glycosylation at the central or N-terminal site leads to dead-end products that seldom lead to full glycosylation of all three sites. Only one path of progressive glycosylation, one initiated by glycosylation at the C-terminal site, can efficiently lead to full occupancy for all three sites. Thus, the hCEACAM1 domain provides an effective model system to study site-specific recognition of glycosylation sequons by OST-B and suggests that the order and efficiency of posttranslational glycosylation is influenced by steric cross-talk between adjoining acceptor sites.

Keywords: glycobiology; mass spectrometry; oligosaccharyltransferase.

Fig. 1.

(A) Diagram of OST-A and OST-B function. (Left) A nascent polypeptide is shown emerging from the ribosome (light blue, PDB 6FTI), passing into the ER lumen via the sec61 channel (green) and being scanned by OST-A (red, PDB 6S7O). (Right) OST-B (blue, PDB 6S7T) is shown interacting with a partially folded peptide substrate. A dolichol pyrophosphate-linked oligosaccharide donor is shown in the membrane to the left of OST-A. (B) Model of glycosylation pathways for hCEACAM1-IgV via OST-B. After protein translation, glycans can be added in any order to each of the three N-glycan sequons (indicated by an “N” in the linear polypeptide representation), and each addition is given its own rate constant. Glycans are shown with the Symbol Nomenclature for Glycans symbol for GlcNAc, for simplicity.

Fig. 2.

(A) Mass spectrum of hCEACAM1-IgV-WT showing a region containing the 12+ charge distribution. Three peaks are observed corresponding to the protein with one, two, or three GlcNAc residues (blue squares). In the case of one or two GlcNAc residues, the location of the modification is ambiguous and indicated with brackets. (B) Comparison of mass spectra for hCEACAM1 glycosylation site mutants. The number within the blue square indicates the number of GlcNAc residues. (C) Comparison of mass spectra for hCEACAM1-WT samples expressed in cells with and without the presence of OST-B inhibitor. (D) Example fragmentation map from top-down ECD MS/MS spectra of monoglycosylated hCEACAM1-IgV. Lines drawn between residues indicate an assigned fragment ion. The notch at the top of a line indicates a c-type fragment ion, while a notch at the bottom indicates an assigned z ion. Residue N111 is highlighted, indicating the presence of a GlcNAc PTM.

Fig. 3.

Flowchart describing the kinetic model of OST-B glycosylation. Glycosylation state is shown as a three-bit binary representation, for simplicity. The order of the three bits (e.g., [010]) corresponds to sites N104, N111, and N115, respectively, where a “0” represents an unglycosylated residue and a “1” represents the presence of a GlcNAc at the respective site. Each arrow indicates a separate reaction. In the case of the processes removing protein from the ER, only one arrow is drawn, for clarity, but the model includes a separate process for each species to exit the ER. The numbers above each arrow indicate the value of the corresponding optimized rate constant from the kinetic model, while the numbers below each arrow indicate the SD. The thickness of each line has been scaled in proportion to the value of the rate constant. The orientation of the graph mirrors the diagram in Fig. 1B.

Fig. 4.

Model of unfolded hCEACAM1-IgV peptide (blue ribbon) docked into the catalytic site of STT3B (blue surface). Site 1 (N104) is docked in the catalytic site, and a Glc₃Man₉GlcNAc₂ N-glycan (transparent surface) is attached at site 2 (N111). The structure of OST-B was taken from PDB 67ST.