Identifier

etd-11172014-155433

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

O-linked â-N-acetylglucosamine is a regulatory post translational modification. This modification occurs on nearly all functional classes of proteins, in the nucleus and cytoplasm. O-GlcNAc is added to serine or threonine by O-GlcNAc transferase and removed by O-GlcNAcase. Previous attempts to study O-GlcNAc-modified proteins have resulted in low yields, making 3-dimensional structure determination impossible. In this dissertation O-GlcNAc transferase will be co-expressed with domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2) in E. coli, to produce O-GlcNAc-modified protein. The O-GlcNAc-modified protein was expressed in a variety of E. coli cell lines at a variety of conditions, but only small quantities of insoluble protein were produced. A glycosidase was suspected due to the disappearance of the O-GlcNAc modification from the protein. O-(2-acetamido-2-dexoy-dglucopyranosylidene) amino-N-phenylcarbamate (PUGNAc), a â-N-acetylglucosaminidase inhibitor, was added to the culture media and increased the production of O-GlcNAc-modified protein. This was the first evidence that â-N-acetylglucosaminidase (NagZ), an E. coli enzyme, cleaves O-GlcNAc from proteins in vivo. NagZ was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. In E. coli, NagZ cleaves the GlcNAc-â1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm. A NagZ knockout showed no activity towards the O-GlcNAc-peptide, confirming NagZ as the enzyme responsible for cleaving O-GlcNAc from our glycoprotein expressed in vivo. O-GlcNAc-modified protein produced by the NagZ knockout (∆NagZ) co-expression system is highly glycosylated and can be resolubilized from the pellet. The ∆NagZ is a step closer to production of milligram quantities of O-GlcNAc-modified protein for structure determination.

Date

2014

Document Availability at the Time of Submission

Student has submitted appropriate documentation to restrict access to LSU for 365 days after which the document will be released for worldwide access.

Committee Chair

Macnaughtan, Megan

Included in

Chemistry Commons

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