Dataset Information

Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.

ABSTRACT: GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and GalNAc-T2) in liver cells and explore how the GalNAc-T repertoire quantitatively affects the O-glycoproteome. We demonstrate that the majority of the O-glycoproteome is covered by redundancy, whereas distinct subsets of substrates are modified by non-redundant functions of GalNAc-T1 and GalNAc-T2. The non-redundant O-glycoproteome subsets and specific transcriptional responses for each isoform are related to different cellular processes; for the GalNAc-T2 isoform, these support a role in lipid metabolism. The results demonstrate that GalNAc-Ts have different non-redundant glycosylation functions, which may affect distinct cellular processes. The data serves as a comprehensive resource for unique GalNAc-T substrates. Our study provides a new view of the differential regulation of the O-glycoproteome, suggesting that the plurality of GalNAc-Ts arose to regulate distinct protein functions and cellular processes.

SUBMITTER: Schjoldager KT

PROVIDER: S-EPMC4693523 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.

Schjoldager Katrine T KT Joshi Hiren J HJ Kong Yun Y Goth Christoffer K CK King Sarah Louise SL Wandall Hans H HH Bennett Eric P EP Vakhrushev Sergey Y SY Clausen Henrik H

EMBO reports 20151113 12

GalNAc-type O-glycosylation is found on most proteins trafficking through the secretory pathway in metazoan cells. The O-glycoproteome is regulated by up to 20 polypeptide GalNAc-Ts and the contributions and biological functions of individual GalNAc-Ts are poorly understood. Here, we used a zinc-finger nuclease (ZFN)-directed knockout strategy to probe the contributions of the major GalNAc-Ts (GalNAc-T1 and GalNAc-T2) in liver cells and explore how the GalNAc-T repertoire quantitatively affects ...[more]

PMID: 26566661

Similar Datasets

Project description:Characterizing protein GalNAc-type O-glycosylation has long been a major challenge, and as a result, our understanding of this glycoproteome is particularly poor. Recently, we presented a novel strategy for high throughput identification of O-GalNAc glycosites using zinc finger nuclease gene-engineered "SimpleCell" lines producing homogeneous truncated O-glycosylation. Total lysates of cells were trypsinized and subjected to lectin affinity chromatography enrichment, followed by identification of GalNAc O-glycopeptides by nLC-MS/MS, with electron transfer dissociation employed to specify sites of O-glycosylation. Here, we demonstrate a substantial improvement in the SimpleCell strategy by including an additional stage of lectin affinity chromatography on secreted glycoproteins from culture media (secretome) and by incorporating pre-fractionation of affinity-enriched glycopeptides via IEF before nLC-MS/MS. We applied these improvements to three human SimpleCells studied previously, and each yielded a substantial increase in the number of O-glycoproteins and O-glycosites identified. We found that analysis of the secretome was an important independent factor for increasing identifications, suggesting that further substantial improvements can also be sought through analysis of subcellular organelle fractions. In addition, we uncovered a substantial nonoverlapping set of O-glycoproteins and O-glycosites using an alternative protease digestion (chymotrypsin). In total, the improvements led to identification of 259 glycoproteins, of which 152 (59%) were novel compared with our previous strategy using the same three cell lines. With respect to individual glycosites, we identified a total of 856 sites, of which 508 (59%) were novel compared with our previous strategy; this includes four new identifications of O-GalNAc attached to tyrosine. Furthermore, we uncovered ? 220 O-glycosites wherein the peptides were clearly identified, but the glycosites could not be unambiguously assigned to specific positions. The improved strategy should greatly facilitate high throughput characterization of the human GalNAc-type O-glycoproteome as well as be applicable to analysis of other O-glycoproteomes.

Dataset Information

Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.

Publications

Deconstruction of O-glycosylation--GalNAc-T isoforms direct distinct subsets of the O-glycoproteome.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure