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.

Figure 1

(a) Schematic representation of Man₉GlcNAc₂ showing the linkage and branching patterns, together with the residue numbering scheme. (b) Scheme showing the processing pathway of N-linked oligosaccharide (adapted from reference [11] with modifications).

Figure 2

Elution profile of the pyridylamino (PA) derivative of N-linked oligosaccharides derived from the engineered S. cerevisiae cells on an Amide-80 column. The fraction indicated by the arrow corresponds to the PA derivative of Man₉GlcNAc₂ (M9), while those indicated by an asterisk contain no detectable oligosaccharides.

Figure 3

(a) ¹H-¹³C Heteronuclear Single Quantum Coherence (HSQC) spectra of the PA derivative of M9, metabolically ¹³C-labeled with D-[1-¹³C]glucose (black), D-[2-¹³C]glucose (blue), D-[3-¹³C]glucose (green), D-[4-¹³C]glucose (magenta), D-[5-¹³C]glucose (orange) or D-[6-¹³C]glucose (cyan). The six spectra were superposed and the ¹³C-labeled positions in the glucose isotopomers used as metabolic precursors are shown with circles in the same colors as the corresponding spectra. ¹H-¹³C HSQC experiments were performed at a proton observation frequency of 920.7 MHz with 256 (t₁) × 1024 (t₂) complex points and 16 scans per t₁ increment. The spectrum widths were 15.1 kHz for the ¹³C dimension and 5.8 kHz for the ¹H dimension. (b) ¹H-¹³C HSQC spectra of the PA derivative of M9 (black) and Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc glycoform (M8B) (red) uniformly labeled with ¹³C.

Figure 4

Part of the ¹³C-edited Nuclear Overhauser effect spectroscopy (NOESY) spectra of the PA derivatives of M8B and M9 uniformly labeled with ¹³C, showing connectivities from (a) GlcNAc2-Ac and (b) ManB-H₂. The spectrum was recorded at a proton observation frequency of 950.3 MHz with 64 (t₁) × 80 (t₂) × 1024 (t₃) complex points and two scans per t₁ increment with a mixing time of 200 ms. The number at the top of each slice is the chemical shift of ¹³C resonance.

Figure 5

Summary of the long-range Nuclear Overhauser effect (NOE) connectivities identified in the (a) M8B and (b) M9 oligosaccharides. Interresidue NOEs are shown except for those observed between neighboring residues and those unassigned due to peak overlap. They are classified based on normalized intensity as follows: > 0.2 (solid). 0.2–0.1 (dashed), 0.1–0.03 (dotted).