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Protocol 3

3. Deglycosylation of Glycoproteins Using Endoglycosidases


T.H. Plummer, Jr. and A.L. Tarentino, Department of Biochemistry, New York State Department of Health, Wadsworth Center for Laboratories and Research, Albany, New York 12201


Parameters of deglycosylation vary with every glycoprotein and must be determined empirically (Methods in Enzymology, 1994, 230: 44-57). Human transferrin is an example of a glycoprotein containing two asparagine-linked -fucosyl-biantennary chains, one of which is only released slowly by endoglycosidases even after denaturation in SDS. A typical test reaction on human transferrin is described in the protocol below.

 

Materials:

Transferrin

Sodium Dodecyl sulfate

Nonidet P-40

Gel electrophoresis apparatus

Enzymes, Substrates and Buffers:

PNGase F1

Didansyl-fetuin glycopeptide (L-A-N(CHO)-AeC-S, triantennary), 100 mM Hepes, pH 8.6

Endo F1

Dansyl-N-(GlcNAc)2(Man)6 Ovalbumin, high mannose, Sodium acetate, 100 mM, pH 5.8

Endo F2

Dansyl-L-M-G-E-N(CHO)-R Fibrinogen, biantennary Sodium acetate,100 mM, pH 4.5

Endo F3

Tridansyl-Y-K-N(CHO)-N-S-D-I-S-S-T-R IGM, biantennary with core 1-6 fucose, Sodium acetate 100 mM pH 4.5

 

 

Protocol:

1. Heat denature transferrin by incubation of 1 mg/ml of the glycoprotein in 0.5% SDS for 3 min. at 90C.

2. Add 2 g of denatured transferrin (1 mg/ml) into 4 l of buffer appropriate for the endoglycosidases used.

3. Add 3 l of 3.3% (v/v) nonidet P-40.

4. Add 1 l of appropriate enzyme stock. Enzyme addition must be preceded by addition of nonidet P-40.

5. Incubate digest at 37C 4 times ranging from 1 hr. to overnight.

6. Monitor release of oligosaccharides by observing altered protein band migration by SDS gel electrophoresis. If reaction is incomplete, more deglycosylating enzyme can be added.

Note: For human transferrin, PNGase F and Endo F2 release all oligosaccharides in 1 hr. at 37C, whereas Endo F3 requires 16 hr. for 50% release of the one oligosaccharide chain.

When large scale release of intact oligosaccharide chains is required, the glycoprotein is often partially degraded by proteases and then subjected to deglycosylation, thereby lowering the amount of enzyme required for deglycosylation, often by a factor of 10 or more.

References:

(1) Tarentino, A.L. and Plummer, T.H., Jr. (1994) Methods in Enzymology, 230: 44-47.

 


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