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.
Highlights
The carbohydrate chains that modify proteins play key roles in a variety of physiological and pathological functions, such as intercellular communications, viral infections and immune reactions [1].These biological processes involve molecular recognition mediated by carbohydrate–protein and carbohydrate–carbohydrate interactions, which can be potential therapeutic targets [2,3,4,5]
We have recently developed a methodology for the overexpression of a homogeneous high mannose-type oligosaccharide with 13C labeling with a genetically engineered yeast strain [11]
From the glycoprotein mixture harvested from the cells, N-linked oligosaccharides were released by hydrazinolysis, re-N-acetylated with 13C-labeled acetic anhydride and subsequently fluorescencelabeled with 2-aminopyridine
Summary
The carbohydrate chains that modify proteins play key roles in a variety of physiological and pathological functions, such as intercellular communications, viral infections and immune reactions [1] These biological processes involve molecular recognition mediated by carbohydrate–protein and carbohydrate–carbohydrate interactions, which can be potential therapeutic targets [2,3,4,5]. In the endoplasmic reticulum (ER), a nascent polypeptide chain is covalently linked to the triantennary oligosaccharide, Glc3Man9GlcNAc2 (G3M9), which is subsequently trimmed by glucosidases and mannosidases to result in a variety of high mannose-type oligosaccharides These processing intermediates exhibit specific glycotopes that are recognized by intracellular lectins functioning as molecular chaperones and cargo receptors and by those involved in ER-associated protein degradation. The glycotopes as fate determinants of glycoproteins are embedded in the triantennary structure of G3M9 and exposed with trimming by the ER enzymes
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