Purification and characterization of an O-GlcNAc selective N-acetyl-beta-D-glucosaminidase from rat spleen cytosol.

D.L Dong,G.W Hart

doi:10.1016/s0021-9258(17)32170-1

D.L Dong, G.W Hart

Open Access

https://doi.org/10.1016/s0021-9258(17)32170-1

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Abstract

Glycosylation of nuclear and cytoplasmic proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is an abundant, ubiquitous, and transient post-translational modification. To characterize enzymes involved in removal of these sugars, a neutral and cytoplasmic N-acetyl-beta-D-glucosaminidase (O-GlcNAcase) with strong selectivity for O-GlcNAc-synthetic glycopeptides has been purified over 22,000-fold from rat spleen homogenate. The purified O-GlcNAcase has two major polypeptides of apparent M(r) = 54,000 (alpha subunit) and M(r) = 51,000 (beta subunit). Enzyme activity sediments at M(r) = 106,000 on sucrose gradients, indicating that the native O-GlcNAcase is an alpha beta heterodimer. The O-GlcNAcase also shows substantially stronger relative activity against O-GlcNAc-synthetic glycopeptides than other hexosaminidases. Unlike acidic lysosomal hexosaminidases, O-GlcNAcase is not inhibited by GalNAc or its analogs, has no other detectable glycosidase activities, and does not cross-react with antibodies against acidic hexosaminidases. Subcellular fractionation and latency studies demonstrate the cytoplasmic and nucleoplasmic localization of the enzyme and its ubiquitous presence in tissues. These studies suggest that O-GlcNAcase is involved in the regulated removal of O-GlcNAc from O-GlcNAc-bearing glycoproteins in the nucleoplasmic and cytoplasmic compartments of cells.

Highlights

Glycosylation ofnuclear and cytoplasmic proteins by fied, and the number is growing rapidly
We have identified, purified, and extensively characterizeda neutral andcytoplasmicN-acetyl-P-D-glucosaminidasefrom rat spleen that does not cleavep-nitrophenyl-GalNAc andis highly selective for 0-GlcNAc bearing glyco
In presured in the presence or absence of Triton X-100.Obviously, even ence of 50 m~ GalNAc, cytosol fraction revealed at pH 6.4, lysosomes showed more than 2-fold acidic hex- higher 0-GlcNAcase activity than all othecrompartments, and osaminidase activity in thepresence of Triton X-100than in the the addition of Triton X-100did not increase its activity

Summary

To whom correspondence should be addressed

Goat antisera to P-hexosaminidase A and B and to the denatured a-chain were kindly provided by Dr Richard Proia, National Institutes mM GalNAc was included in assays This probably explains the low-foldof purification and low yield (total activity was decreased t o 62% a t ammonium sulfate step) during these early of Health [48].The partial purified acidic HEX A from the rat spleen homogenate and the 0-GlcNAcase were incubated with 2 pl of antibody in final 50 pl of phosphate-buffered saline for 1 h on ice, followed by incubation with 25 pl of protein-A Sepharose suspension (50% w/v) for 1 h on ice. The protein-A was precipitated by centrifugation, and washed twice with 100 pl of phosphate-buffered saline. The dialyzed 3 6 5 0 % ammonium sulfate pellet was loaded onto a DE52 cellulose column, washed, and elutedas described under “ExperimentalProcedures.”As shown in thetop panel of Fig. 1,at each step of washing and eluting withNaCl, a large suspended in 150 pl of phosphate-buffered saline, and the supernatant combined with the first wash were separately assayed for hexosaminidase activity usingpNP-0-GlcNAc as substrate amsethod I1 above. In the absence of 50 m~ GalNAc at pH 4.5, three

RESULTS

DISCUSSION

Findings

Noantibody anti-alpha