Assay for cytoplasmic ENGase
ENGase (endo-β-N-acetylglucosaminidase (EC 22.214.171.124)) is an endoglycosidase that hydrolyzes the β1,4-glycosidic bond in the N,N’-diacetylchitobiose core of N-glycans. The activity of cytoplasmic ENGase activity was described in various animal cells, and the gene encoding this enzyme was identified [Kato, et al., Glycobiology 2002; Suzuki, et al., Proc. Natl. Acad. Sci. USA, 2002]. This enzyme activity was known to be involved in the catabolism of free oligosaccharides accumulated in the cytosol [Katoh, et al., Trends Glycosci. Glycotechnol. 2009; Suzuki, Trends Glycosci. Glycotechnol., 2009].
1 M Tris-HCl buffer (pH 7.5/8.0)
5 M NaCl
0.5 M EDTA (pH adjusted with 1 N NaOH to 8.0)
CompleteTM protease inhibitor cocktails (Roche)
200 mM Mes-NaOH buffer (pH 6.0)
Man9GlcNAc2-PA (from Takara Bio-Clontech, ; 4120)
0.1 M Ammonium acetate buffer (pH 4.0) (buffer A)
0.1 M Ammonium acetate buffer (pH 4.0)/0.5% n-butanol (buffer B)
HPLC with a fluorescence detector
Preparation of Cytoplasmic fraction for enzyme source
Culture cells of your interest. Collect cells.
Perform all the subsequent procedures either on ice or at 4oC. Suspend cells at a density of 5 x 107 cells/ml in 10 mM Tris-HCl buffer (pH 7.5)/1 mM EDTA/250 mM sucrose/1 mM DTT with various protease inhibitors (1 x completeTM protease inhibitor cocktail (Roche)/1 mM AEBSF (Pefabloc SC, Roche)).
Homogenize cells using Potter-Elvehjem homogenizer or equivalent.
Clear the solution first with regular centrifuge at 14,000 rpm for 10 min
Take supernatant to a new tube.
Centrifuge the sup further using ultracentrifuge at 100,000 x g for 1 hour.
Soluble (cytosol) fraction thus obtained can be used for ENGase assay. For later use, the sample can be aliquoted, stored at -80oC. Avoid repeated freeze-thawing.
Assay for ENGase assay [Li et al, Bioorg. Med. Chem. 2008]
Mix 1 ml of the cytosol fraction with 3 μl of 200 mM Mes-NaOH buffer (pH 6.0) and start the reaction by adding 1 μl of the substrate (Man9GlcNAc2-PA; 2 pmol) to the solution.
Incubate at 37oC for 6 hours (incubation time has to be optimized for your enzyme source).
Stop the reaction by heating sample at 95oC for 5 min.
Add 1.5 volume of ethanol (final concentration; 60%) to the sample, and centrifuge at 14,000 rpm for 15 min at 4oC.
Evaporate the sup to dryness using centrifugal evaporator.
Dissolve the sample with 5 ml of distilled water for HPLC analysis.
Prepare buffer A (0.1 M ammonium acetate buffer, pH 4.0) and buffer B (0.1 M ammonium acetate buffer, pH 4.0/0.5 % n-butanol).
Elution conditions are as follows:
Column: ODS column (TSK gel ODS-80-TM; 7.5x 75 mm; Tosoh Corp.)
Column temperature: 30oC
Flow rate: 1.0 ml/min
Elution condition: linear gradient of 5% (0.025% n-butanol) to 100% (0.5% n-butanol) buffer B for 55 min. Wash the column with the starting buffer (5% B solution) between the analyses.
Fluorescence detection: λex=320 nm; λex=400 nm.
Precaution has to be taken if samples with high a-mannosidase activity are applied. It won’t be a concern for most of the mammalian-derived tissues/cells.
Figure & Legends
For enzyme source, CHO-K1 cells were used and incubation was carried out at 30oC for 6 hours. Similar results can be obtained for other mammalian-derived culture cells. GL Sciences HPLC system (PU611 double pumps/CO630 column over) with a fluorescence detector (LaChrom, Hitachi High Technologies, Tokyo) was used.
Kato, T., Fujita, K., Takeuchi, M., Kobayashi, K., Natsuka, S., Ikura, K., Kumagai, H., and Yamamoto, K. Glycobiology 12, 581-587, 2002. [PMID : 12244070]
Katoh, T., Ashida, H., and Yamamoto, K. Trends Glycosci. Glycotechnol. 21, 163-177, 2009. [PMID : not available]
Suzuki,T., Yano, K., Sugimoto, S., Kitajima, K., Lennarz, W.J., Inoue, S., Inoue, Y., and Emori, Y. Proc. Natl. Acad. Sci. USA 99, 9691-9696, 2002. [PMID : 12114544]
Suzuki, T. Trends Glycosci. Glycotechnol. 21, 219-227, 2009. [PMID : not available]
B. Li, K. Takegawa, T. Suzuki, K. Yamamoto, and L.-X. Wang (2008) Synthesis and inhibitory activity of oligosaccharide thazolines as a class of mechanism-based inhibitors for endo-b-N-acetylglucosaminidase. Bioorg. Med. Chem. 16, 4670-4675. [PMID : 18304822]