To elucidate the structure of the sugar moiety of O-glycoproteins, exoglycosidase digestion is useful. For example, the terminal sugar, α/β-anomer and linkage of the terminal sugar can be estimated using exoglycosidase treatment. On the other hand, a specific type of O-glycans can be removed from the peptides and/or proteins by endoglycosidase (O-glycanase or O-glycosidase). |
| Category | O-Glycans |
| Protocol Name | Glycosidase digestion of O-glycoproteins and related O-glycans |
Authors
 |
Hatsumi M. Goda
*
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
Makoto Ito
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University
*To whom correspondence should be addressed.
|
| Keywords | O-glycan
exoglycosidase
endoglycosidase |
Reagents
 |
| ● |
Neuraminidase (Arthrobacter ureafaciens) (Sigma-aldorich Co.) |
| ● |
O-glycosidase (endo-α-N-acetylgalactosaminidase) (Streptococcus pneumoniae) (Sigma-aldorich Co.) |
|
Instruments
 |
| ● |
Speed Vac Concentrator (SAVANT) |
|
| Methods |
|
1. |
Removal of sialic acids from fetuin O-glycans by Arthorobacter sialidase. |
| 1) |
Dissolve 30 μg of O-glycans (eg. Sialyl-T antigen) in 100 μl of 50 mM sodium acetate buffer, pH 5.0, with 50 mU of Arthrobacter sialidase. |
Comment 0
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| 2) |
Incubate the mixture at 37°C for 16 h. |
Comment 0
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| 3) |
Terminate the reaction to keep the sample in boiling water for 3 min. |
Comment 0
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| 5) |
Centrifuge at 15,000 rpm for 10 min. |
Comment 0
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| 6) |
Dry the supernatant with a Speed Vac Concentrator. |
Comment 0
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|
2. |
Removal of a core I disaccharide (Galβ1-3GalNAc) from an O-glycoprotein by endo-α-N-acetylgalactosaminidase. |
| 1) |
Dissolve 300 μg of O-glycoprotein (eg. asialofetuin) in 100 μl of 50 mM sodium phosphate buffer, pH 6.0, with 50 mU of O-glycosidase. |
Comment 1
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| 2) |
Incubate the mixture at 37°C for 16 h. |
Comment 0
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| 3) |
Terminate the reaction to keep the sample in boiling water for 3 min. |
Comment 0
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| 5) |
Centrifuge at 15,000 rpm for 10 min. |
Comment 0
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| 6) |
Dry the supernatant with a Speed Vac Concentrator. |
Comment 0
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| Figure & Legends |
Figure & Legends 
Fig. 1. Action points of glycosidases on O-glycoproteins and related O-glycans |
| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
|
| Date of registration：2012-11-09 16:15:56 |
Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ, Dwek RA, Rudd PM. (2002) An analytical and structural database provides a strategy for sequencung O-glycan from microgram quantities of glycoproteins. Anal Biochem 304:70-90. [PMID : 11969191]
Edge AS, Spiro RG. (1987) Presence of an O-glycosidically linked hexasaccharide in fetuin. J Biol Chem. 262:16135-16141. [PMID : 2445744] |
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Goda,
Ito,
" Glycosidase digestion of O-glycoproteins and related O-glycans " 2012. http://jcggdb.jp/GlycoPOD/ © Ritsumeikan University, AIST & JCGGDB.
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Goda,
Ito,
" Glycosidase digestion of O-glycoproteins and related O-glycans " 2012. http://jcggdb.jp/GlycoPOD/ © Ritsumeikan University, AIST & JCGGDB.
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