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Characterization of O‐GlcNAc Hydrolase with Phosphomimetic Mutations
Author(s) -
Hinkle Megan,
Crawford Garland
Publication year - 2018
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.2018.32.1_supplement.544.5
Subject(s) - serine , threonine , phosphorylation , biochemistry , context (archaeology) , enzyme , mutant , cytosol , biology , mutagenesis , immunoprecipitation , hydrolase , microbiology and biotechnology , chemistry , gene , paleontology
O‐linked beta N‐acetylglucosamine (O‐GlcNAc) hydrolase, or OGA, is an enzyme involved in 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 with altered enzymatic activity by using site‐directed mutagenesis to convert a single serine or threonine to an aspartic acid. The end goal of this research being to introduce the mutated OGA gene into a mammalian cell system to study the relationship between OGA phosphorylation and its activity . 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. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .