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Native N-Linked Deglycosylatation

Native N-Linked Deglycosylation Strategies

For some glycoproteins, no cleavage by PNGase F occurs unless the protein is denatured. For others, some or all of the oligosaccharides can be removed from the native protein after extensive incubation of three days or longer. PNGase F will remain active under reaction conditions for at least three days allowing extended incubations of native glycoproteins. In general, it appears that particular residues, due to their location in the native protein structure, are resistant to PNGase F and can not be removed unless the protein is denatured.

Endoglycosidases F1, F2, and F3 are less sensitive to protein conformation than PNGase F and are more suitable for deglycosylation of native proteins. Sigma’s N-DEGLY kit supplies all three of these enzymes with reaction buffers and detailed instructions. The linkage specificities of Endoglycosidases F1, F2, and F3 suggest a general strategy for deglycosylation of proteins that may remove all classes of N-linked oligosaccharides without denaturing the protein. Initially complex oligosaccharides can be reduced to the trimannosyldiacetylchitobiose using neuraminidase, b-galactosidase, and N-acetylglucosaminidase. kit. Fucosidases may be required in some situations. The remaining trimannosyldiacetylchitobiose core structures can be removed with Endoglycosidase F3. Bi- and triantennary structures can be immediately removed by Endoglycosidases F2 and F3, respectively.

High mannose (oligomannose) and hybrid structures can be removed by Endoglycosidase F1, but not complex, oligosaccharides.

Endo F2 and Endo F3 have the ability to cleave complex structures. Endo F2 cleaves biantennary complex and to lesser extent high mannose oligosaccharides. Fucosylation has little effect on Endo F2 cleavage of biantennary structures. Endo F2 will not cleave hybrid structures. Endo F3 cleaves biantennary and triantennary complex oligosaccharides. However, non-fucosylated biantennary and triantennary structures are hydrolyzed at a slow rate by Endo F3 Core fucosylated biantennary structures are efficient substrates for Endo F3 oligosaccharides. Core fucosylation of biantennary structures increases activity up to 400-fold. Endo F3 has no activity on oligomannose and hybrid molecules.

Endo F3 will cleave fucosylated and non-fucosylated trimannosyl core structures on free and protein-linked glycans. Native deglycosylation of complex tetrantennary glycans requires sequential hydrolysis down to the trimannosyldiacetylchitobiose core.

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