Hi Bernhard,
I guess you knew all these and is really asking for people's experience,
but please excuse me to start from the theory: N-glycans in eukaryotes
are known to be involved in glycoprotein folding in the ER. They allow
the nascent protein to get into the calnexin/calreticulin cycle in which
these lectins/chaperones can recruit the disulfide isomerase ERP57. The
N-glycans also serve as degradation signals in the ERAD pathways, where
certain structures signify the cells that all efforts had failed on this
molecule. Of course, for recombinant overproduction of proteins, these
factors can both be dispensible: the bad proteins, as long as they can
get secreted, might get preferentially lost during purification or
crystallization. In fact considering that N-glycans either tether the
not-so-folded protein to the Cxn/Crt cycle, or direct the terminally ill
proteins to degradation, and that the quality control of cells could be
leaky (which I think was exploited in some CFTR-related cases), one may
even expect that in certain cases not-so-badly folded proteins may get
to the surface easier if it does not contain N-glycans - pure
conjectures though. But indeed there are a lot of disulfide-containing
eukaryotic proteins that are completely not N-glycosylated in certain
species while fairly well N-glycosylated (to the common extent of ~1
site /80-100 residues) in many other species (although the surfaces of
the proteins will be significantly different too). So, it seems that the
requirement for N-glycan - chaperone - ERAD may not be very strong for
every protein.
On the other hand, the first GlcNAc in the N-glycan is sometimes found
to be very closely associated with the amino acid components of
proteins, even half-inserted into the folded domain (eg., 1HCN, A/NAG94
), which is a consequence of the co-translational addition of the
N-glycans. In such case I would expect that the removal of the
particular N-glycan by mutagenesis may be quite destructive to folding
in certain cases. I know that there are reports in which people removed
N-glycans one by one and observed very significant differences among
sites on secretion/solubility.
So, my view is that many of the N-glycan sites are removable by
mutagenesis, but certain sites are not. Therefore if one is faced with
highly N-glycosylated proteins, it is more of a matter of luck or
thoroughness if he/she starts to explore the combination of mutations.
To complicate this further, one may also consider trying different ways
of mutating the sites - besides changing the Asn to Asp, one can also
consider mutating the S/T at the third position, which is in fact often
how an N-glycosylation site gets lost among species. There is also the
space of the Asn mutants to explore. What I did in the past was mutating
the Asn to Gln, which does not introduce a charge difference.
Zhijie
On 11/04/2017 4:34 PM, Bernhard Rupp wrote:
Hi Fellows,
a humble question for our glyco-expressionists:
I have mutated out the Asns of the N-glycoslation consensus sites for Asp
(Asp simply because the PNGaseF treated protein stays stable so I
thought that might be a good guess)
and indeed the unglycosilated mutant expresses well and gets secreted
as planned.
But rumor has it that glycoproteins that are mutated to non-glyc often
are not processed correctly and
that we had just dumb luck.
May I poll the educated opinion of the erudite here?
Cheers, BR
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Bernhard Rupp
Crystallographiae Vindicis Militum Ordo
http://www.hofkristallamt.org/
[email protected] <mailto:[email protected]>
+1 925 209 7429
+43 767 571 0536
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