Application of Metabolic 13C Labeling in Conjunction with High-Field Nuclear Magnetic Resonance Spectroscopy for Comparative Conformational Analysis of High Mannose-Type Oligosaccharides

Abstract

High mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, resulting in various processing intermediates with exposed glycotopes that are recognized by a series of lectins involved in glycoprotein fate determination in cells. Although recent crystallographic data have provided the structural basis for the carbohydrate recognition of intracellular lectins, atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of the energetics of carbohydrate–lectin interactions. Carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, but often hampered by poor spectral resolution and lack of recombinant techniques required to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for the preparation of a homogeneous high mannose-type oligosaccharide with 13C labeling using a genetically engineered yeast strain. We herein successfully extended this method to result in the overexpression of 13C-labeled Man9GlcNAc2 (M9) with a newly engineered yeast strain with the deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeled M9 in comparison with its processing product lacking the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches interact with the core in both glycoforms, and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be significantly associated with lectins and glycan-trimming enzymes.

Paramagnetic NMR probes for characterization of the dynamic conformations and interactions of oligosaccharides

“…4). These results are qualitatively consistent with previous reports based on chemical shift, J coupling, NOE, and PRE data [55,62,[64][65][66][67][68][69][70] and, furthermore, give quantitative insights into conformational fluctuations of this biologically important class of oligosaccharides [71,72].…”

“…A triantennary oligosaccharide containing nine mannose residues, M9, as well as its derivative M8B was overexpressed in genetically engineered yeast cells as fully 13 C-labeled forms [62,63] and attached to an EDTA-based lanthanide-chelating tag (Fig. 3a).…”

Exploration of Conformational Spaces of High‐Mannose‐Type Oligosaccharides by an NMR‐Validated Simulation

“…The former is enzymatically converted into the latter in the endoplasmic reticulum, which is considered to be a key step for triggering glycoprotein degradation. [15] The yeast cells were cultivated in a medium containing uniformly 13 Clabeled glucose as a carbon source, which gave rise to fully 13 C-labeled M9 and M8B. [15] The yeast cells were cultivated in a medium containing uniformly 13 Clabeled glucose as a carbon source, which gave rise to fully 13 C-labeled M9 and M8B.…”

New NMR Tools for Characterizing the Dynamic Conformations and Interactions of Oligosaccharides

“…15 In this study, the paramagnetic tag was covalently attached to the undecasaccharide that was overexpressed in genetically engineered yeast cells. 16 By this hybrid approach, combining cell engineering and chemistry, a source of long-distance atomic information was provided via PRE. This revealed that this triantennary oligosaccharide, which is involved in glycoprotein-fate-determining processes in cells, adopted foldback conformations.…”

“…31 This method was extended to the overexpression of 13 Clabeled Man9GlcNAc2 (M9) using an engineered yeast strain created by the deletion of four genes involved in N-glycan processing (Figure 9). 16 Using these techniques, comparative NMR analyses were performed to elucidate conformational differences between these two glycoforms (M8B and M9). Chemical shift differences were observed not only in the ManA residue, which is exposed as the nonreducing terminus in M8B, but also in the inner parts, indicating that removal of the ManD2 residue induces conformational rearrangements of the triantennary structure.…”