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Characterization of O‐GlcNAc Hydrolase with Phosphomimetic Mutations in HeLa Cells
Author(s) -
Hinkle Megan Leigh,
Crawford Garland
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.799.2
Subject(s) - serine , threonine , phosphorylation , biochemistry , mutant , biology , mutagenesis , enzyme , chemistry , microbiology and biotechnology , gene
O‐linked beta N‐acetylglucosamine (O‐GlcNAc) hydrolase, or OGA, is an enzyme involved in a post‐translational modification. This enzyme hydrolyzes a single O‐GlcNAc from serine or threonine residues on target proteins located in the cytosol of metazoan organisms. OGA is the only protein known to carry out this function in the context of cytosolic proteins. This exclusivity of function makes OGA a likely target of post‐translational modifications in order to regulate its activity. Our aim is to generate a phosphomimetic OGA using site‐directed mutagenesis to convert a single serine or threonine to an aspartic acid and to introduce the mutated OGA gene into HeLa cells to study the relationship between OGA phosphorylation and its activity. While multiple serine and threonine residues of OGA have been predicted by bioinformatics and experimental approaches to be phosphorylated, the phosphorylation of OGA at serine 364 is of particular interest. This residue has been identified to be phosphorylated in 45 separate experiments and the introduction of OGA with a mutation from a serine to an aspartic acid residue at this point shuts down bacterial growth. Our aim is to study the effect of the phosphomimetic mutation at serine 364 in a mammalian cell system. Analysis by Western blot will reveal the extent to which the mutants alter OGA expression levels and O‐GlcNAc modification levels in the cell. Additionally, interactions of OGA with known binding partners, such as OGT, the enzyme which adds the O‐GlcNAc sugar to target proteins, can be studied with co‐immunoprecipitation. This work lays the foundation for identifying potential phosphorylation states of OGA and would aid in elucidating the regulatory pathways of this enzyme. Support or Funding Information SEED grant, Office of the Provost, Mercer University This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .