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Analysis of the substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms

ABSTRACT: Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a pre-assembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p. These two OST complexes have different protein substrate specificities in vivo. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We showed that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Diploid Cell

SUBMITTER: Chia-wei Lin

LAB HEAD: Prof. Markus Aebi

PROVIDER: PXD024590 | Pride | 2021-05-19

REPOSITORIES: Pride

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			Action	DRS
	20180112_09_JE_OST6_1_F292945.mzid.gz	Mzid
	20180112_09_JE_OST6_EndoH.raw	Raw
	20180112_10_JE_OST3_EndoH.raw	Raw
	20180112_OST3_1_F292940.mzid.gz	Mzid
	20180711_14_JE_OST3_1_EndoH.raw	Raw

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Project description:Bacterial pathogens use various strategies to interfere with host cell functions. Among these strategies, bacteria induce transcriptional changes, in order to modify the set of proteins synthetized by the host cell, or target pre-existing proteins by modulating their post-translational modifications or by triggering their degradation. Protein levels variations in host cells during infection integrates both transcriptional and post-transcriptional regulations induced by pathogens. Here, we focused on host proteome alterations induced by the bacterial pathogen Listeria monocytogenes, and more specifically the Listeria toxin Listeriolysin O (LLO). In order to characterize host proteome alterations induced by LLO, we performed a shotgun proteomics analysis on HeLa cells treated or not with the LLO toxin. To this end, we used SILAC labelling (stable isotope labelling by amino acids in cell culture) to compared in a quantitative manner the protein content from two differentially treated cell populations: one control population and one population incubated with a sublytic dose of LLO (3 nM). We decided to expose cells to LLO during only 20 min to limit protein level changes resulting from transcriptional changes. We performed two independent experiments (experiment #1 and experiment #2) with swapped SILAC labelling to rule out putative labelling-dependent effects. A total of 2009 and 1684 proteins was quantified in each independent experiment, respectively, and 1360 proteins were quantified in both experiments. Among these 1360 proteins, we identified a total of 91 proteins for which protein levels were consistently decreased in cells treated with LLO (i.e. with a log2 control/LLO ratio >0.5 in both experiments). To assess whether the host proteasome was involved in the decrease of these 91 proteins, we repeated our proteomics analysis on SILAC-labelled HeLa cells pre-treated with proteasome inhibitors before exposure to LLO. Two independent experiments were similarly performed with swapped SILAC labelling (experiment #3 and experiment #4). Interestingly, we observed that the vast majority of host proteins identified as strongly downregulated in response to LLO also displayed significant decreased levels in the presence of proteasome inhibitors, indicating that the majority of observed decreases in host protein levels induced by LLO are not due to proteasome-mediated degradation. In follow-up experiments, we identified that LLO decreases in particular the protein level of two E2 ubiquitin enzymes, UBE2K and UBE2N, leading to major changes in the host ubiquitylome. This toxin-induced proteome remodeling involves post-transcriptional regulations, as no modification in the transcription levels of the corresponding genes was observed. These decreases in host protein levels were observed in different epithelial cell lines but not in macrophages. In addition, we could show that Perfringolysin O, another bacterial pore-forming toxin similar to LLO, also induces host proteome changes. Taken together, our data show that different bacterial pore-forming toxins induce deep proteome remodeling, that may impair host epithelial cell functions.

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Analysis of the substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms

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