O-linked glycosylation

Iain Wilson WILSON at edv1.boku.ac.at
Fri Mar 17 11:37:38 EST 1995


>Date: 17 Mar 1995 15:19:14 -0000
>Sender: lpddist at mserv1.dl.ac.uk
>Message-ID: <3kc99i$nvt at mserv1.dl.ac.uk>
>Original-To: glycosci at dl.ac.uk
>
>I have produced a recombinant protein in the yeast Pichia pastoris that is
>partially glycosylated with O-liked sugar (there are no N-linked sites in the
>protein). I wish to seperate the glycosylated form the unglycosylated protein.
>I have tried using ConA-Sepharose (Pharmacia) and Glycine-Max (Sigma) without
>success (neither seemed to bind the glycoprotein). The ConA came with
>instructions, but I have not been able to dig out any protocol for using the
>Glucine-Max and Sigma were not forthcoming.

Going by the 'rules', Con A would not bind O-linked mannose. (See 
Kobata and Yamashita in Glycobiology A Practical Approach, p. 110 - 
they refer to a previous Kobata paper and state that ConA 'interacts 
with sugar chains which have two non-substituted or C-2 substituted 
alpha-mannopyranosyl residues in one molecule') That said, they 
probably did not study O-linked mannose. According to another article 
in the same book (Berlin and Hanover, p. 343). Soybean agglutinin 
(Glycine max) would bind D-GalNAc (but there's no more detail than 
that). I don't know whether Clinton Ballou or Widmar Tanner (who have 
looked at yeast O-mannosylation) have examined any lectin binding to 
O-mannose.

The question is do you really want this protein glycosylated or not? 
The only problem with yeast mutants of this pathway is that (if I 
remember the work of Peter Orlean on Dol-P-Man synthetase mutants) 
the O-mannosylation is required for viability. Also Tanner's work 
appears to indicate that there are two transferases for the first 
step in O-mannosylation.

A novel way round your problem may be to investigate the specificity 
of mannose binding protein (animal origin) and see if a recombinant 
form of MBP could bind your enzyme!

---------------------------------------------------------------
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

http://www-bprc.mps.ohio-state.edu/cgi-bin/hpp/iain_wilson.html



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