I would like to remove glycosylation from the C-terminal part of the GPCR receptor CCR5 to do further study on ligand binding. Does anybody know if it exist an enzyme that can be added to the cell medium so that all receptor on cell surface are de-glycosylated?
Many thanks
I achieved complete deglycosylation on a purified protein (with N-glycosylations) in its native form using EndoH from NEB. EndoH cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins hence it just leaves the N-acetylglucosamine core to the Asn in the protein backbone.
I know there are cocktails designed to provide a complete deglycosylation on proteins (E-DEGLY).
if you are planning to test different solutions I would skip PNGase; in my experience it doesn't work on native proteins but only after denaturation. Look for enzymes that do not need to access the protein backbone; a trimming of the glycosylation might be enough to see an effect of the specific aspect you are studying.
I always worked with purified protein, therefore I don't know how the presence of medium would influence the reaction. Maybe you might try to place the cell in PBS (instead of medium) and test the efficiency of the system.
last suggestion. if you are interested in studying the effect of a specific glycosylation on a specific protein have you ever considered to perform a site specific mutation to knock out the glycosylation site?
Hi Ilaria if you really want to go nuclear on glysosylated proteins, I would recommend Pierce Thermo EDC and all the destructive powers that they have.
https://www.thermofisher.com/search/results?query=edc&resultPage=1&resultsPerPage=15&autocomplete=
Can you please provide specific position of glycosylation sites in the c-terminus?? As per information from uniprot, CCR5 has 4 o-linked glycosylation sites out of which 2 are important for ligand binding. Therefore, you'll have to be careful in designing your assays. For cell-based studies, you can mutate these four residues or work with O-glycosidase (neb) if planning to use lysate.
If residue mutation is an option for you, the GPCRdb Construct design tool (http://gpcrdb.org/construct/design) lists those in the N-terminus. For any segment, you can find N- and O-linked glycosylations with these two servers http://www.cbs.dtu.dk/services/NetNGlyc/ and http://www.cbs.dtu.dk/services/NetOGlyc/.
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