Dataset Information

Probing endogenous peptide acceptor substrate specificities of TbSTT3A and TbSTT3B

ABSTRACT: We performed glycoproteomics on endogenous parasite glycoproteins using sequential endoglycosidase-H and peptide-N-glycosidase-F digestions, the latter in the presence of H2[18O]. This allowed us to assess the relative occupancies of native N-glycosylation sites by endoglycosidase-H resistant N-glycans originating from Man5GlcNAc2-PP-dolichol transferred by TbSTT3A, and endoglycosidase-H sensitive N-glycans originating from Man9GlcNAc2-PP-dolichol transferred by TbSTT3B.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Michele Tinti

LAB HEAD: Michael Ferguson

PROVIDER: PXD007267 | Pride | 2017-09-20

REPOSITORIES: Pride

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Publications

Single-subunit oligosaccharyltransferases of Trypanosoma brucei display different and predictable peptide acceptor specificities.

Jinnelov Anders A Ali Liaqat L Tinti Michele M Güther Maria Lucia S MLS Ferguson Michael A J MAJ

The Journal of biological chemistry 20170919 49

Trypanosoma brucei causes African trypanosomiasis and contains three full-length oligosaccharyltransferase (OST) genes; two of which, TbSTT3A and TbSTT3B, are expressed in the bloodstream form of the parasite. These OSTs have different peptide acceptor and lipid-linked oligosaccharide donor specificities, and trypanosomes do not follow many of the canonical rules developed for other eukaryotic N-glycosylation pathways, raising questions as to the basic architecture an ...[more]

PMID: 28928222

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Project description:Objectives: Hashimoto’s thyroiditis (HT) is one of the most common autoimmune disorders; however, its underlying pathological mechanisms remain unclear. Although aberrant glycosylation has been implicated in the N-glycome of immunoglobulin G (IgG), changes in serum proteins have not been comprehensively characterized. This study aimed to investigate the glycosylation profile of serum samples of HT patients that were depleted of highly abundant proteins by and propose the potential functions of glycoproteins for the further study of pathological mechanisms of HT . Methods: A lectin microarray containing 70 lectins was used to detect and analyze glycosylation of serum proteins using serum samples (N=27 HT; N=26 healthy control [HC]) depleted of abundant proteins. Significant differences in glycosylation status between HT patients and the HC group were verified by lectin blot analysis. A lectin-based pull-down assay combined with mass spectrometry was used to investigate potential glycoproteins combined with differentially present lectins, and an enzyme-linked immunosorbent assay (ELISA) was used to identify the expression of targeted glycoproteins in 131 patients with papillary thyroid carcinoma (PTC), 131 patients with benign thyroid nodules (BTN) patients, 130 patients with HT, and 128 HCs. Results: Compared with the HC group, the majority of the lectin binding signals in HT group were weakened, while the Vicia villosa agglutinin (VVA) binding signal was increased. The difference in VVA binding signals verified by lectin blotting was consistent with the results of the lectin microarray. A total of 113 potential VVA-binding glycoproteins were identified by mass spectrometry and classified by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses . Using ELISA, we confirmed that lactoferrin (LTF) and mannan-binding lectin-associated serine protease 1 (MASP-1) levels were elevated in the serum of patients with HT and PTC. Conclusions: Following depletion of abundant proteins, remaining serum proteins in HT patients exhibited lower glycosylation levels than those observed in HCs. An increased level of potential VVA-binding glycoproteins may play an important role in HT development. LTF and MASP-1 expression was significantly higher in the serum of HT and PTC patients, suggesting key roles played by glycosylation in pathological mechanisms underlying HT and PTC.

Dataset Information

Probing endogenous peptide acceptor substrate specificities of TbSTT3A and TbSTT3B

Publications

Single-subunit oligosaccharyltransferases of <i>Trypanosoma brucei</i> display different and predictable peptide acceptor specificities.

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