Re: [ccp4bb] Glycoprotein expression question

2017-04-12 Thread Zhijie Li 

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

--

Bernhard Rupp

Crystallographiae Vindicis Militum Ordo

http://www.hofkristallamt.org/

b...@hofkristallamt.org 

+1 925 209 7429

+43 767 571 0536

--

:(){ :|: & };:

--

[ccp4bb] Fwd: Re: [ccp4bb] Glycoprotein expression question

2017-04-12 Thread radu 
Hi Savvas,

Thank you for kindly pointing to our review. Bernhard, in my experience your
case is a rare exception rather than the rule, so indeed lucky. Adrian
summarised very nicely the wide impact glycans may have on folding,
trafficking and/or function. To keep things simple, there is no need to mutate
the N-glycosilation sites for structural work (other than perhaps to probe
their impact, but people don't seem to do this normally). PMID: 17355862
describes how to deal with these if the aim is to improve crystal quality. For
cryo-EM, glycans should definitely stay on. Why would one risk solving
meaningless structures?

Best wishes,

Radu

-- 
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: +44-(0)1223-267049
fax: +44-(0)1223-268305


 Original message 
>Date: Wed, 12 Apr 2017 10:24:10 +0200
>From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Savvas
Savvides <savvas.savvi...@ugent.be>)
>Subject: Re: [ccp4bb] Glycoprotein expression question
>To: CCP4BB@JISCMAIL.AC.UK
>
>Dear Bernhard
>Our campaigns over the years aiming to produce mammalian cytokines and the
ectodomains of cytokine receptors via eukaryotic expression systems (mainly
in several HEK293 flavors) for structural biology, have taught us that the
N-linked glycosylation issue remains a very empirical exercise. Our protein
targets are typically 15-70 kDa and have 2-6 predicted N-linked glycosylation
sites. For instance, we have seen that elimination of even one such site by
mutagenesis can abrogate protein secretion. So for those cases one may even
make an argument for a glycosylation ‘hotspot'.  Also, eliminating all
possible N-linked glycosylation sites at once in the couple of cases tried
has been synonymous with zero protein secretion. Our consensus of the
‘magic combo’ in terms of expression levels and stability is a reduction
of N-linked glycosylation sites by up to 1/3. Such reduced levels of
glycosylation also appear to be amenable for enzymatic glycan trimming in
subsequent stages of sample preparation with good results.
>The article http://dx.doi.org/10.1016/j.sbi.2013.04.003 by Aricescu and Owens
may provide some additional perspectives.
>
>best wishes
>Savvas
>
>
>> On 11 Apr 2017, at 22:34, Bernhard Rupp <hofkristall...@gmail.com> 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
>>
>> --
>> Bernhard Rupp
>> Crystallographiae Vindicis Militum Ordo
>> http://www.hofkristallamt.org/
>> b...@hofkristallamt.org
>> +1 925 209 7429
>> +43 767 571 0536
>> --
>> :(){ :|: & };:
>> --

Re: [ccp4bb] Glycoprotein expression question

2017-04-12 Thread Savvas Savvides 
Dear Bernhard
Our campaigns over the years aiming to produce mammalian cytokines and the 
ectodomains of cytokine receptors via eukaryotic expression systems (mainly in 
several HEK293 flavors) for structural biology, have taught us that the 
N-linked glycosylation issue remains a very empirical exercise. Our protein 
targets are typically 15-70 kDa and have 2-6 predicted N-linked glycosylation 
sites. For instance, we have seen that elimination of even one such site by 
mutagenesis can abrogate protein secretion. So for those cases one may even 
make an argument for a glycosylation ‘hotspot'.  Also, eliminating all possible 
N-linked glycosylation sites at once in the couple of cases tried has been 
synonymous with zero protein secretion. Our consensus of the ‘magic combo’ in 
terms of expression levels and stability is a reduction of N-linked 
glycosylation sites by up to 1/3. Such reduced levels of glycosylation also 
appear to be amenable for enzymatic glycan trimming in subsequent stages of 
sample preparation with good results.  
The article http://dx.doi.org/10.1016/j.sbi.2013.04.003 by Aricescu and Owens 
may provide some additional perspectives.
 
best wishes
Savvas


> On 11 Apr 2017, at 22:34, 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
>  
> --
> Bernhard Rupp
> Crystallographiae Vindicis Militum Ordo
> http://www.hofkristallamt.org/
> b...@hofkristallamt.org
> +1 925 209 7429
> +43 767 571 0536
> --
> :(){ :|: & };:
> --

Re: [ccp4bb] Glycoprotein expression question

2017-04-12 Thread Adriana Erica Miele 
Good morning, Bernhard,

I do not think that you had pure luck (though serendipity helps a lot). You 
said that the PNGaseF treated protein was indeed stable, that was already a 
good hint.

In my little experience with N- and O-glycoproteins, with not a high percentage 
of sugar content, I saw a difference.
N-glycosylated proteins with Asn mutated to Asp tend to be more stable, because 
you are putting negative charges on the surface.
O-glycosylated proteins expressed unglycosylated or mutated do not gain in 
stability, and are prone to precipitation (whenever they are expressed). This 
is possibly due to the fact that  -OH on the surface have not the same effect 
as the negative charges.
Moreover, in the few entries in the PDB with O-linked sugars, these seems to be 
like un umbrella, covering/masking a larger portion of the protein's surface. 
Hence trimming the sugars off will expose potentially "sticky" parts.

That's my 2 cents.

Best regards,
Adriana

Re: [ccp4bb] Glycoprotein expression question

2017-04-12 Thread Goldman, Adrian 
I think it completely depends on the protein: in some proteins, they are 
required for folding; in some (eg Fcs), they are not required for folding but 
for function); in some, some of them are required and others not; in some they 
are required _during_ folding, but afterwards they can be removed without 
affecting apparent stability.  We had that experience with some UDP glycosyl 
transferases.

Adrian


> On 12 Apr 2017, at 07:52, Jan Dohnalek  wrote:
> 
> We had experience with a relatively small glycoprotein - when glycosylation 
> sites were deleted, solubility went drastically down - we could not express 
> soluble any more. Back to eukaryotic expression system which worked.
> 
> So may be you were really lucky.
> 
> Jan
> 
> 
> On Tue, Apr 11, 2017 at 10:34 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
> 
> 
> 
> --
> 
> Bernhard Rupp
> 
> Crystallographiae Vindicis Militum Ordo
> 
> http://www.hofkristallamt.org/ 
> b...@hofkristallamt.org 
> +1 925 209 7429 
> +43 767 571 0536 
> --
> 
> :(){ :|: & };:
> 
> --
> 
> 
> 
> 
> 
> 
> --
> Jan Dohnalek, Ph.D
> Institute of Biotechnology
> Academy of Sciences of the Czech Republic
> Biocev
> Prumyslova 595
> 252 50 Vestec near Prague
> Czech Republic
> 
> Tel. +420 325 873 758



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Re: [ccp4bb] Glycoprotein expression question

2017-04-12 Thread Jan Dohnalek 
We had experience with a relatively small glycoprotein - when glycosylation
sites were deleted, solubility went drastically down - we could not express
soluble any more. Back to eukaryotic expression system which worked.

So may be you were really lucky.

Jan


On Tue, Apr 11, 2017 at 10:34 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
>
>
>
> --
>
> Bernhard Rupp
>
> Crystallographiae Vindicis Militum Ordo
>
> http://www.hofkristallamt.org/
>
> b...@hofkristallamt.org
>
> +1 925 209 7429 <(925)%20209-7429>
>
> +43 767 571 0536 <+43%207675%20710536>
>
> --
>
> :(){ :|: & };:
>
> --
>
>
>



-- 
Jan Dohnalek, Ph.D
Institute of Biotechnology
Academy of Sciences of the Czech Republic
Biocev
Prumyslova 595
252 50 Vestec near Prague
Czech Republic

Tel. +420 325 873 758

[ccp4bb] Glycoprotein expression question

2017-04-11 Thread Bernhard Rupp 
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

 

--

Bernhard Rupp

Crystallographiae Vindicis Militum Ordo

  http://www.hofkristallamt.org/

  b...@hofkristallamt.org

+1 925 209 7429

+43 767 571 0536

--

:(){ :|: & };:

--