o-linked deglycosylation of HLA-DR

Iain Wilson WILSON at edv1.boku.ac.at
Sat Apr 1 08:34:59 EST 1995

> Date:          31 Mar 1995 23:01:32 GMT
> From:          srps at galactose.mc.duke.edu (Steven Pirie-Shepherd)
> Reply-to:      srps at galactose.mc.duke.edu (Steven Pirie-Shepherd)
> To:            "bionet.glycosci mail newsgroup" <bionet-news at dl.ac.uk>
> Subject:       Re: o-linked deglycosylation of HLA-DR

> George Weiner (george-weiner at uiowa.edu) wrote:
> : We have produced an antibody that we think reacts with an o-linked
> : glycosylation variant of HLA-DR.  Is there an enzyme or culture
> : techniques we can use to remove the o-linked glycosylation of surface
> : proteins from intact cells or or lysates?
> O-glycosidases are liminted in scope as they only play with Gal->GalNAc as
> a substrate, further substitutions such as SA, fucose etc MUST be triommed
> with the appropriate exoglycosidases prior to the O-glycosidase (Glyko in
> novata CA have a comprehensive list of decent glycosidases). Otherwise it 
> takes hydrazine to remove all the O-linked sugar on a protein.. I am not 
> sure what effect tunicamycin has on O-linked glycosylation? Mabe someone 
> lse has that info?

Tunicamycin should have no effect on O-linked glycosylation since it 
affects GlcNAc-1-phosphotransferase - the first enzyme of the 
dolichol-linked pathway for N-links. However, there was some paper in 
Biochemistry, on erythropoietin and one of the colony stimulating 
factors, last year which used some technique for preventing addition 
of O-links (an ionophore perhaps?). I don't have the reference since 
I left most of mine at my last lab in the UK before I came here. 
Perhaps someone else remembers the paper I refer to.

Naturally, hydrazine would be useless for deglycosylating whilst 
retaining an intact protein, since it attacks amide bonds: hence 
requirement for re-N-acetylation after removal of N-links. (I do not 
know the chemical mechanism for hydrazinolysis with respect to 
O-links - neither do I have personal experience as to the efficiency 
of hydrazinolysis with O-links.) TFMS is another possible treatment 
but I do not know whether it is truly effective for removal of sugar, 
while not modifying the protein.

I would think that any study attempting to determine whether the 
glycosylation is involved should examine the carbohydrate structure. 
Techniques would then have to be used to verify removal of that 
structure before making any claims that a certain treatment affects 
binding of an antibody. There is a lot of very vague data on binding 
of antibodies or other molecules to carbohydrate - I find it in some 
papers dealing with allergy that I read in connection to my own 
project. Absence of binding after TFMS or periodate treatment doesn't 
tell you an awful lot! Similarly papers which deal with mutagenesis 
of glycosylation sites can't rule out the effects 'due to 
glycosylation' are not artefacts of altered protein structure. 
Perhaps a general discussion on how to design experiments to prove 
that glycosylation has a role in a certain instance would be 
generally interesting and educational.


Iain Wilson                        Institut fuer Chemie           
Tel: 43-1-47654-6065               Universitaet fuer Bodenkultur   
Fax: 43-1-310-5176                 Gregor-Mendel-Strasse 33
E-mail: wilson at edv1.boku.ac.at     A-1180, WIEN, Austria


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