PNGaseF From New England Biolabs

PNGaseF (peptide: N-glycosidase F [EC 3.5.15.2, N-linked-glycopeptide-(N-acetyl-beta-D-glucosaminyl)-L-asparagine amidohydrolase]) is an amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins, thus revealing the native polypeptide size of a protein. The enzyme provided by New England Biolabs (NEB) is purified from Flavobacterium meningosepticum. The enzyme is supplied in 20mM Tris-HCl (pH 7.5 @ 25^oC), 50mM NaCl, 5mM Na₂EDTA and 50% glycerol and must be kept at -20^oC at all times. For optimal performance in high-performance liquid chromatography (HPLC), the enzyme can be supplied without glycerol. The enzyme works well on denatured glycoproteins but longer incubation times and additional enzyme are needed to digest native glycoproteins. Incubation times and the amount of enzyme need to be determined for each particular protein digestion, but the enzyme is very easy to work with and the testing is not difficult at all.

I have adjusted the protocol to my experimental needs while working with proteins such as Tyrosinase, Tyrosinase-related protein 1, Dopachrome tautomerase. The main technique I use after the digestion is Western blotting. For my protocol, I have used a protein extraction buffer containing 1% Nonidet P-40 (Calbiochem, San Diego, CA, USA) in phosphate buffer saline (PBS – Gibco, Grand Island, NY) and a protease inhibitor cocktail (Boehringher Mannheim, Mannheim, Germany). This extraction buffer has proven to work well with the enzyme’s buffer. If possible, it is important to avoid the use of sodium dodecyl sulphate (SDS) as it will inactivate the PNGaseF. If SDS cannot be avoided, make sure that NP-40 is present to prevent enzyme inactivation. The protein concentration of the samples to be digested varies from 0.5 to 20ug. In my experiments, I obtained very good signal on my Western blots after using an average of 1-2ug protein in each sample. I normally use PBS to dilute samples if their concentration is too high for my experiments. Briefly, the protocol is as follows: cell lysates are denatured in the PNGaseF denaturing buffer (5% SDS and 10% 2-mercatptoethnaol) for 5 minutes at 100^oC, denatured lysates are then cooled and mixed with 1/10 volume each of concentrated PNGaseF reaction buffer, 0.5 M sodium phosphate, pH 7.5, and 10% NP-40. Samples are then digested with 500 units of PNGaseF for 3 hours at 37^oC. After this time, the reaction is stopped by the addition of the sample buffer (Bio-Rad, Hercules, CA) containing 2-mercaptoethanol. The reaction is then incubated at 100^oC for 5 minutes. For the glycoproteins that I worked with, digesting with this amount of enzyme gave the same results after 1, 3 or 24 hour incubation time. It is critical to keep in mind when calculating the volumes of the reaction that the denaturing buffer and PNGaseF reaction buffer are both 10X and NP-40 is 10%. Also, all the calculations have to be made taking into account the final volume of the sample that can be loaded in the gel. In my case, this was a maximum of 30ul. Also, after each step, especially those involving high temperatures or long incubation times, it is critical to spin down the tubes before going to the next step. Prior to starting the digestion, it is important to equilibrate all reagents (except the enzyme) to the room temperature. The enzyme must be stored at -20^oC and kept on ice when working with it. For long incubation times, it is important to cover the top of the eppendorf tubes with parafilm or to use screw cap tubes to avoid evaporation.

I have also been using the enzyme for a limited PNGaseF digestion using 0.4, 1, 10, 50, 100, 500 or 1,000 U and incubating over night at 37^oC in order to obtain information about the glycoproteins resistance to PNGaseF and about the occupancy of the N-glycosylation sites. There is room for improving the method but it works pretty well if the appropriate reaction conditions are chosen: enzyme amount, time, etc.

The enzyme can also be successfully used for in-gel deglycosylation of glycoproteins and release of the glycans. This method employs sodium dodecyl suflate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation followed by Coomassie visualization. The bands of interest are then excised from the gel, undergo a protein alkylation step and are finally completely dried in a SpeedVac (Savant Instruments, Farmingdale, NY). The dried pieces of gel are then incubated with PNGaseF and other enzyme combinations that will release the sugars. The sugars are then analyzed by normal phase high-performance liquid chromatography (NP-HPLC) or other methods.

In conclusion, the enzyme is very important for several lab applications involving N-glycosylated proteins, from the very “easy” ones like Western blotting to more complicated applications, such as normal phase HPLC.

Gertrude-Emilia Costin, PhD
Senior Research Scientist
Avon Products, Inc.
R&D, New Technology Department