I want to check if a protein produced in Saccharomyces is always glycosilated. And if so, it could display an immunlogical reaction in humans?
Is your protein being secreted? if so, does it have N-glycosylation sites. If both are 'yes', does it appear as a smear on SDS-PAGE (or WB?), then it is likely N-glycosylated. Confirm by a PNGaseF digest to remove the N-glycans.
It could also be O-glycosylated, in case you still see a smear on gel after PNGaseF digestion, that will likely be the case.
Both N- and O-glycans of S. cerevisiae can elicit an immune response in humans. The alpha-1,3 mannose glycosidic linkages found in N-glycans are known to be immunogenic.In general, hyperglycosylated structures are easily recognized by the immune system as well, making them to be cleared rapidly from circulation after recognition by mannose receptors found on fro example macrophages.
Is your protein being secreted? if so, does it have N-glycosylation sites. If both are 'yes', does it appear as a smear on SDS-PAGE (or WB?), then it is likely N-glycosylated. Confirm by a PNGaseF digest to remove the N-glycans.
It could also be O-glycosylated, in case you still see a smear on gel after PNGaseF digestion, that will likely be the case.
Both N- and O-glycans of S. cerevisiae can elicit an immune response in humans. The alpha-1,3 mannose glycosidic linkages found in N-glycans are known to be immunogenic.In general, hyperglycosylated structures are easily recognized by the immune system as well, making them to be cleared rapidly from circulation after recognition by mannose receptors found on fro example macrophages.
Thanks a lot for the asnwer. Indeed the enzyme does have N-glycosylation sites and that's the reason i'm thinking to express in a bacterial system like E. coli. My doubt was if the presence of sugars always elicit immune reactions. Thanks a lot for the help
it depends on the protein, even having sites could be that it is not glycosylated since other features are important (culture conditions, for example, or residues around the sequon). Anyway being in Sacharomyces there is a high probability of N-glycosylation and O-manosylation and this yeast usually hyperglycosylate proteins and also add Man alpha-1,3 Man linkahes, as Bran Laukens told you, both features making the protein immunogenic. If you need a yeast to express the protein (for example trying to favor Cys-bonds formation or favoring higher expression levels than in Ecoli) you can try also Pichia instead of Saccharomyces, since the former does not hyperglycosylate and does not add Mana1,3Man linkages to the protein.
I am not sure about O-mannosylation, but you can try to prevent N-glycosylation by adding tunicamycin to your growth medium when you induce expression of your protein. It may be difficult to find the right balance, since you do not want your Saccharomyces to die, nor stop growth etc., but you also do not want low amounts of N-glycosylation on your protein.
Thanks a lot for the answers. If using the tunicamycin i'm sure that i'll be able to reduce/prevent N-glycosylation?
Artur, Tunicamycin blocks N-glycosylation. If your concentration is high enough the yeast cannot perform N-glycosylation. But it will definately hamper growth to a certain extent.
I see, if using tunicamycin i've to find a compromise between growth and absence of N-glycosylation. Thanks for the help
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