Studies involving N-glycosylated proteins always include several enzymatic digestions of the glycan part of the molecule. This digestion is necessary in order to obtain data about the composition of the glycan. New England Biolabs (NEB) provides several Endo- and Exo-glycosidases which cover a wide range of preliminary investigational needs. Endoglycosidase H (EndoH) is one of these enzymes. It is used in many labs interested in glycoproteins and its use it just one of the first (and many) steps that are needed to clarify the glycan composition of a glycoprotein. A well designed experiment should for sure include a digestion with other enzymes like (Peptide:N-glycosidase F) PNGaseF and more to give a better picture of the glycosylation of the proteins of interest. Several companies on the market provide de-glycosylation kits which include combination of enzymes to be used in these types of studies.
Endoglycosidase H [EC 3.2.1.96, glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-b-glucosaminohydrolase] is a recombinant glycosidase which cleaves the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins, a conversion that occurs in the medial Golgi region. When proteins are correctly processed through the endoplasmic reticulum (ER) and Golgi, they become resistant to EndoH, and sensitivity to EndoH indicates the presence of proteins that have not yet been processed beyond the ER. The enzyme provided by NEB was cloned from Streptomyces plicatus and then overexpressed in E. coli. The enzyme is supplied in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl and 5 mM Na2EDTA and is critical to be stored at -20oC for all the time.
I have been using this enzyme for many years now and I have adjusted the protocol to my experimental needs. The main technique I use after digestion is western blotting, but the enzyme is also very useful when the digestion reaction is combined with metabolic labeling and immunoprecipitation. This combination allows one to follow the glycosylation of a particular protein along the secretory pathway. The protein extraction buffer that I use works well with the buffers the enzyme needs. The extraction buffer I use is 1% Nonidet P-40 (Calbiochem, San Diego, CA, USA) in phosphate buffer saline (PBS – Gibco, Grand Island, NY) with a protease inhibitor cocktail (Boehringher Mannheim, Mannheim, Germany). The proteic concentration of the samples to be digested varies from 0.5 to 20 ug. In my experiments, I obtained very good signal from my western blots using an average of 1-2 ug protein in the starting sample. The enzymatic digestion protocol goes as follows: the cell lysates are denatured in the EndoH denaturing buffer (5% SDS and 10% 2-mercatptoethnaol) for 5 minutes at 100oC, cooled and mixed with 1/10 concentrated EndoH reaction buffer (G5: 0.5 M sodium citrate, pH 5.5). Samples are then digested with 500 units of EndoH for 3 hours at 37oC. After this time, the reaction is stopped by the addition of the sample buffer (BioRad, Hercules, CA) containing 2-mercaptoethanol and incubation at 100oC for 5 minutes. For the glycoproteins that I worked with (Tyrosinase, Tyrosinase-related protein 1, Dopachrome tautomerase etc.), this amount of enzyme gives the same results after 1, 3 or 24 h incubation time. It is critical to keep in mind when calculating the volumes of the reaction that the denaturing buffer and the G5 buffer are both 10X. Also, all of the calculations have to be made taking into account the maximum loading volume of 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. After long incubations, it is wise to use a screw cap tube or to put parafilm over the top of regular eppendorf tubes to avoid evaporation.
In conclusion, EndoH is very important in several applications involving in N-glycosylated proteins. It is easy to adjust the protocol to specific technical needs.
Gertrude-Emilia Costin, PhD
Senior Research Scientist
Avon Products, Inc.
R&D, New Technology Department
Suffern, NY