> 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
---------------------------------------------------------------
Iain Wilson Institut fuer Chemie
Tel: 43-1-47654-6065 Universitaet fuer Bodenkultur
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E-mail: wilson at edv1.boku.ac.at A-1180, WIEN, Austria
http://www-bprc.mps.ohio-state.edu/cgi-bin/hpp/iain_wilson.html
