A wide variety of nuclear and cytoplasmic proteins are modified by the attachment of a single N-acetylglucosamine attached to Ser/Thr residues (O-GlcNAc). This post-translational modification is highly dynamic and abundant. Many of the known sites of O-GlcNAc attachment resemble sites of prolinedirected protein kinases. On many proteins such as cytokeratins and the large subunit of RNA polymerase II, O-GlcNAc appears to have a reciprocal relationship with protein phosphorylation. Our working hypothesis is that OGlcNAc, like phosphorylation, regulates protein function. The rat liver O-GlcNAc transferase (OGT) is a hetero-trimer composed of two catalytic 110-kDa subunits and one 78-kDa (regulatory) subunit. We generated polyclonal antisera against synthetic peptides based on internal peptide sequence of the 110-kDa subunit. The 110-kDa subunit is present in all tissues examined, and appears to be sufficient for enzyme activity. By Western analysis using an anti-phosphotyrosine mAb we have shown that the OGT contains phosphorylated tyrosine. We also found that the OGT can be labeled by Galactosyltransferase, suggesting that the OGT is itself modified by O-GlcNAc. We have cloned the gene encoding the OGT using a combination of PCR and standard library screening techniques. The gene encodes a protein with a predicted molecular weight of ~112-kDa, this is in close agreement with the apparent weight of 110-kDa. It is interesting to note that in all tissues examined there are two transcripts for the OGT. Both transcripts are quite long, 5.6-Kb and 6.7-Kb suggesting the possibility that protein expression is highly regulated. Our data show that the OGT is highly conserved, and present in all eukaryotes examined to date. Using both the anti-pi 10 antibodies and the DNA sequence we are characterizing the expression, and biosynthesis of the OGT in various tissues. As well as examining the possible interactions of the OGT with other proteins.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology