METHOD FOR PRODUCING REDUCED GLYCANS AND GLYCAN-ARRAYS
Production and usage of reduced glycans and glycan-arrays.
Glycosylation is the most abundant post-translational protein modification, playing an important role in protein folding, stability and function.
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Current methods for glycosylation analysis do not allow for a simultaneous assessment of the protein and the glycan (sugar) portion of a glycosylated protein. This is a major drawback for the investigation of glycosylation and for engi-neering and quality control of therapeutic proteins.
The invention presented here provides an improved method for the preparation of multiple types of glycans and their usage for scientific and medical applications. This new method of reductive alkaline hydrolysis provides simultaneous access to reduced glycitols, glycosylamines, reducing N-glycans and reduced 1-amino-glycitols. The core-process is the reaction of the glycoprotein starting material with borohydride in the presence of a base at 50 °C or higher. By use ofthis process glycoproteins resp. glycopeptides can be completely deglycosylated. If used in apreparative manner, the glycans provided by the process can be used for manufacturing glycan-arrays or glycan affinity matrices for scientific or medical use.
The product ratio between unreduced sugars (either regular sugar-moietiesor glycosylamines) and reduced sugars (1-amino-glycits resp. glycits, i.e. reduced glycans) can be easily varied by variation of temperature and/ or concentration of alkali resp. borohydride. Further major advantages are the avoidance of using hydrazine (dangerous poison) orenzymes (expensive) for the isolation of glycans.
The pool of glycans accessible herewith can well be used for manufacturing glycan arrays which may then be used for scientific or medical purposes, e.g. for testing the suitability of donor and recipient for organ transplantation. They may also be used as diagnostic tools for numerous other diseases and disorders, even cancer. Glycan arrays are also a powerful tool for the high-throughput elucidation of interactions of carbohydrate structures with biological targets including antibodies, proteins, viruses and cells. This technique is especially suitable for glycomics studies, for these arrays present carbohydrate ligands in a manner that mimics interactions at cell-cell interfaces. Glycan affinity matrices may be used for the isolation of specific carbohydrate proteins or the fractionation of cells depending on their reportoire of glycan binding surface proteins. Besides for the production of glycan arrays, the method can also be directly used for structure determination, for it provides stable sub-structuresof the protein starting material which can then easily be separated and analyzed, e.g. via HPLC, MALDI-TOF etc.
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StichworteN-glycans, reduced glycitols, glycosyl-amines, 1-amino-glycitols, glycomics, glycans, carbohydrate binding proteins, glycan-arrays