Dissecting OGT’s TPR Domain to Identify Determinants of Cellular Function LC-MS/MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Exploris 480
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Cell Culture, Fibroblast
SUBMITTER: 
Bettine Gibbs
LAB HEAD: Suzanne Walker
PROVIDER: PXD052018 | Pride | 2024-05-06
REPOSITORIES: pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 573498_xb05534_SP_LFQ.mzidML | Mzid | |||
| 573499_xb05532_SP_LFQ.mzidML | Mzid | |||
| 573500_xb05531_SP_LFQ.mzidML | Mzid | |||
| 573501_xb05530_SP_LFQ.mzidML | Mzid | |||
| 573502_xb05548_SP_LFQ.mzidML | Mzid |
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Project description:Abstract: The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we have developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously as-sess OGT activity across >6000 human proteins. We used this assay to examine the contribution of a conserved asparagine ladder within the lumen of OGT’s superhelical tetratri-copeptide repeat (TPR) domain to substrate selection. When these residues were mutated, OGT retained full activity against short peptides, but showed low to no activity against most of the OGT substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes a majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain. This series contains microarray data both comparing the new chemoenzymatic method to antibody-based detection as well as comparing arrays treated with wild-type OGT, 5N5A mutant OGT, or controls not treated with enzyme. Note: all CTD-stained arrays or control array raw files are contained in GSE107911_RAW.tar
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Project description:Abstract: O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.
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Project description:The gene NMB0419 of Neisseria meningitidis strain MC58 encodes a putative tetratricopeptide repeats (TPR) containing protein, which was implicated in respiratory epithelial invasion. We have accordingly sought a role for NMB0419 in meningococcal adherence and invasion of human epithelial cells using Escherichia coli surrogates. In trans expression of NMB0419 promoted adherence of the E. coli strain to human epithelial cells, which showed a unique aggregative adherence pattern that was observed for pathogenic E. coli. This adherence was totally abolished by addition of D-mannose, suggesting that formation of type 1 pili on the surface of E. coli strain was solely responsible for the adherence and was facilitated by the expression of NMB0419. This was further supported by rigid pilus structures seen on the surface of NMB0419 expressing E. coli using electron microscopy. A survey of other meningococcal strains including serogroup A, B, C, W, X, Y and Z (n=3, 42, 10, 1, 1, 1, and 1, respectively) revealed that an NMB0419 homologue was present in all strains, however, encoded varying number of TPR domains from 1 to 7. E. coli with in trans expression of such a homologue (NMA2065) encoding single TPR domain also showed enhanced adherence to the level that was observed for the E. coli strain expressing NMB0419 encoding for 4 TPR domains. Nevertheless, in-frame deletion of the TPR-domain coding sequence from the expression plasmid construct reduced the E. coli adherence to the background level. Revisit of the NMB0419 mutant strain of meningococcus indicated that reduction in adherence was primarily responsible for the reduced epithelial invasion that was previously observed. Study of transcriptome profiles in E. coli using microarray showed that the most significantly up-regulated genes in NMB0419 expressing E. coli belonging to the fim operon encoding all necessary structure proteins and chaperons for type 1 pili, strongly suggesting a gene regulatory role for NMB0419. Although the mechanism yet to be explored, this is the first study to showed that TPR domains are involved in bacterial adherence and that a gene encoding single functional TPR domain produced the same phenotype as a homologue encoding multiple TPR domains did.
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2013-02-07 | E-GEOD-36620 | biostudies-arrayexpress