exoglycosidase activity on native glycoproteins

Dr. Taj Mattu taj at glycob.ox.ac.uk
Tue Jun 24 07:05:06 EST 1997


Dr. David Hoover wrote 
>Does anyone have experiences with exoglycosidase reactions on native, >non-denatured glycoproteins?

Yes, we have used sialidase, galactosidase, and beta hexosaminidase on
the complex glycans of transferrin (Glycoconjugate J 1996, 13 pg
1031-1042) and the oligomannose glycans of RNAse B (Glycoconjugate J.
1992, 9, pg 86-91). The reactions were very slow for the mannosidase,
galactosidase, and hexosaminidase (>70hrs). However, the sialidase
(Arthobacter ureafaciens) was >90% completed within 5hrs. desialylation.  

> A discussion of the quality of available exoglycosidases (such
> as Sigma, Boehringer Mannheim, NEB, Glyko, etc.) would be very >helpful.

It depends upon your application. Sigma and Boehringer enzymes do have
some additional contaminating exoglycosidases, and I do not have
experience of Glyko and NEB exoglycosidases to comment. We only use
enzymes obtained from Oxford GlycoSystems or those prepared "in house",
since the specificity of the exoglycosidases is paramount. For your
particular application, I would suggest however

> Are there exoglycosidases available with high enough activity to allow this?

 Yes, the Sialidase, and hexosaminidase is supplied as freeze-dried
powders, so that they could be reconstituted with the galactosidase
giving rise to high activity for all three enzymes. Should further
increases in activity be required concentration by evaporation could be
considered.


In addition, 
kzhao at BIOVX1.BIOLOGY.UCLA.EDU wrote:

> 
> There is a problem to get native deglycosylated protein using PNGase F, as the
> enzyme requires denaturation of glycoproteins to be deglycosylated.  

This is not strictly true, deglycosylation of native glycoproteins can
be carried out. However, most proteins are not susceptible and those
which are may require extended incubation times with high enzyme
concentrations. So generally deglycosylation of native, non-
denatured glycoproteins can only be assessed on a case by case
situation. Our lab has found that in cases where PNGase F has failed and
Endo H and Endo D are not suitable, COMPLEX glycans (sialylated and core
fucosylated) can be removed using Endo F3 (obtained from Oxford
GlycoSystems). However again, each protein should be tested.

Hope this helps, and does confuse the issues already answered...

________________________________________________________________________
Dr. Taj S. Mattu
Glycobiology Institute
Dept. Biochemistry
Oxford University
South Parks Road, 
Oxford U.K.



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