Identification of ammonia-induced changes in the astrocyte O-GlcNAcome

Question Glutamine accumulation in astrocytes is a hallmark of the pathogenesis of hepatic encephalopathy (HE) (Häussinger et al. 1994, Gastroenterology 107, 1475-1480) and recent studies suggest a central role of the glutamine-dependent O-GlcNAcylation of so far unknown proteins for astrocyte dysfunction in HE (Görg et al. 2019, J Hepatol 71, 930-941). The aim of the present study was to identify comprehensive changes in the O-GlcNAcome in ammonia-exposed astrocytes in vitro and to identify potential consequences of the O-GlcNAcylation for the functions of the respective proteins.

Methods O-GlcNAcomics were performed by mass spectrometry on Click-iTTM chemistry-purified O-GlcNAcylated proteins. Individual O-GlcNAcylated proteins were identified by Click-iTTM and Western blot and heme oxygenase 1 (HO1) interacting proteins were detected by chemical crosslinking with 3,3-dithio-bis-(sulfosuccinimidyl)propionate and Western blot analysis. Purified recombinant HO1 was O-GlcNAcylated in vitro using recombinant O-GlcNAc-Transferase (OGT).

Results Using Click-iT^TM chemistry and mass spectrometry we identified 871 proteins of the astrocytic O-GlcNAcome. The O-GlcNAcylation of 51 and 17 of these proteins was enhanced or reduced in NH₄Cl (5mM, 72h)-exposed cultured rat astrocytes, respectively. By Click-iT^TM and Western blot analysis we further identified HO1 to be O-GlcNAcylated in NH₄Cl-exposed astrocytes. After crosslinking interacting proteins in cultured astrocytes, we detected a large number of anti-HO1 immunoreactive proteins by Western blot in NH4Cl-exposed astrocytes with molecular weights between 25-250kDa. Some of these matched with molecular weights of HO1 oligomers and HO1 oligomers also formed upon in vitro O-GlcNAcylation of purified rat HO1 using recombinant OGT. Bioinformatic analyses predicted serine¹⁶⁰ as a potential O-GlcNAcylation site in HO1 and showed that it is located at the center of a putative HO1 dimerization site.

Conclusions In the present study we identified a variety of proteins whose O-GlcNAcylation is altered by ammonia in cultured astrocytes. O-GlcNAcylation of HO1 triggers HO1 oligomerization which is suggested to enhance HO1 catalytic activity. As HO1 is central for the induction of astrocyte senescence in ammonia-exposed astrocytes, HO1 O-GlcNAcylation may be of relevance for the pathogenesis of HE.

Supported by DFG through SFB974 “Communication and Systems Relevance in Liver Injury and Regeneration”.

Publication History

Article published online:
04 January 2021