Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Glycosite mapping and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

ABSTRACT: Mucin-domain glycoproteins are densely O-glycosylated and play key roles in a host of biological functions. In particular, the T-cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, 3, 4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans, which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamic (MD) simulations of TIM-3 and 4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Alexandra Steigmeyer

LAB HEAD: Stacy A. Malaker

PROVIDER: PXD039583 | Pride | 2023-09-27

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

A Combinatorial Enzymatic Approach for Identifying O-glycopeptides

Project description:Proteins glycosylation is primarily characterized as N-glycosylation or O-glycosylation. Recognition of the consensus N-glycosylation sequon (N-X-S/T/C) has enabled the mapping of the glycosite occupancy of intact glycopeptides, whereas O-glycosylation consensus sequons do not exit, making the characterization of O-glycosites challenging because of the different degrees of occupancy within a protein. Most O-glycosylation occurs via O-GalNAcylation, which is critical to diverse biological functions. However, only a small fraction of the O-glycosites and their glycan structures have been identified. We introduced a robust and reliable O-glycoproteomic workflow to achieve the long-standing goal of glycobiology—identifying novel O-glycosylation profiles on a large-scale. We developed and implemented a novel O-glycoproteomic workflow through the use of complementary digestion with O-glycoprotease (IMPa), trypsin, Asp-N, and Glu-C for enrichment of O-glycopeptides. The N-terminal of O-glycosylated serine or threonine residues cleaved using IMPa (95% of total PSMs) and over 99% of O-glycopeptides were enriched by the RAX column. Our O-glycoproteomic workflow involving peptide identification (using sceHCD-based MS/MS) and annotation (using FDR evaluation) enabled the identification of O-glycosylation of 189 O-glycosites carrying 379 glycan structures on 79 O-glycoproteins in human plasma. Of the O-glycan forms, 91.7% were distinctively and abundantly observed in core 1 and 2 structures. Importantly, we assessed five well-characterized native proteins purified from human plasma (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) to validate the identification of O-glycosites with high confidence and confirm the presence of numerous novel glycosites. We believe that this combinatorial approach is broadly applicable for comprehensive characterization of O-glycoproteomes in biomedical research.

2024-04-25 | PXD042701 | Pride

Deciphering O-glycoprotease substrate preferences with O-Pair Search

Project description:O-glycoproteases are an emerging class of enzymes that selectively digest glycoproteins at positions decorated with specific O-linked glycans. O-glycoprotease substrates range from any O-glycoprotein (albeit with specific O-glycan modifications) to only glycoproteins harboring specific O-glycosylated sequence motifs, such as those found in mucin domains. Their utility for multiple glycoproteomic applications is driving the search to both discover new O-glycoproteases and to understand how structural features of characterized O-glycoproteases influence their substrate specificities. One challenge of characterizing O-glycoprotease specificity restraints is the need to characterize O-glycopeptides with site-specific analysis of O-glycosites. Here, we demonstrate how O-Pair Search, a recently developed O-glycopeptide-centric identification platform that enables rapid searches and confident O-glycosite localization, can be used to determine substrate specificities of various O-glycoproteases de novo from LC-MS/MS data of O-glycopeptides. Using secreted protease of C1 esterase inhibitor (StcE) from enterohemorrhagic Escherichia coli and O-endoprotease OgpA from Akkermansia mucinophila, we explore numerous settings that effect O-glycopeptide identification and show how non-specific and semi-tryptic searches of O-glycopeptide data can produce candidate cleavage motifs that can be used to define new protease cleavage settings that lower search times and improve O-glycopeptide identifications. We use this platform generate the first consensus motif for the recently characterized immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa and show that IMPa is a favorable O-glycoprotease for characterizing densely O-glycosylated mucin-domain glycoproteins.

2023-02-15 | PXD035775 | Pride

Comprehensive analysis of platelet glycoprotein Ibα glycosylation.

Project description:Background: Platelet glycoprotein (GP) Ibα is the major ligand-binding subunit of the GPIb-IX-V complex that binds von Willebrand Factor (VWF). GPIbα is heavily glycosylated, and its glycans have been proposed to play key roles in platelet clearance, VWF binding, and as target antigens in immune thrombocytopenia syndromes. Despite its importance in platelet biology, the glycosylation profile of GPIbα is not well characterized. Objectives: The aim of this study was to comprehensively analyze GPIbα amino acid sites of glycosylation (glycosites) and glycan structures. Methods: GPIbα ectodomain that was recombinantly expressed or that was purified from human platelets was analyzed by Western blot, mass spectrometry (MS) glycomics, and MS glycoproteomics to define glycosites and the structures of the attached glycans. Results: We identified a diverse repertoire of N- and O-glycans, including sialoglycans, Tn antigen, T antigen, and ABH blood group antigens. In the analysis of the recombinant protein, we identified 62 unique O-glycosites. In the analysis of the endogenous protein purified from platelets, we identified at least 48 unique O-glycosites and 1 N-glycosite. The GPIbα mucin domain is densely O-glycosylated. Glycosites are also located within the macroglycopeptide domain and mechanosensory domain (MSD). Conclusions: This comprehensive analysis of GPIbα glycosylation lays the foundation for further studies to determine the functional and structural roles of GPIbα glycans.

2023-02-09 | PXD035030 | Pride

Expression analysis of Tim-3 knockdown RAW264.7 cell

Project description:Investigation of whole genome gene expression level changes in a Tim-3 knockdown RAW264.7 cell, compared to control siRNA transfected RAW264.7 cell. The Tim-3 knockdown of this cell render it hyper-reactive to TLR stimulation. Tim-3 knockdown analyzed in this study are further described in Li Y, Feng J, Geng S, Geng S, Wei H, Chen G, Li X, Wang L, Wang R, Peng H, Han G, Shen B, Li Y. 2011. The N- and C-terminal carbohydrate recognition domains of galectin-9 contribute differently to its multiple functions in innate immunity and adaptive immunity. Mol Immunol. 48(4):670-7. (PID 21146220) A six chip study using total RNA recovered from Tim-3 knockdown and control RAW264.7 cells. There are 135,000 probes in a chip,each chip measures the expression level of 27526 genes of mouse from Nimblegen. Fold-change screening between the two groups obtained from the experiment. The threshold used to screen up or down regulated genes is Fold changeM-oM-<M-^-oM-<M-^]2.0

2012-12-05 | E-GEOD-42729 | biostudies-arrayexpress

microRNA expression in Tim-3 knockdown RAW264.7 cell

Project description:RAW264.7 cell was stably transfected with Tim-3 siRNA(SI) or negative control(NC) and were analyzed for microRNA expression Differerently expressed microRNA in Tim-3 knockdown RAW264.7 cells were summarized and were used for further analysis RAW264.7 cells stably transfected with Tim-3 siRNA(SI) or negative control(NC) and were used to collect microRNA without any other treatment

2016-01-14 | E-GEOD-76839 | biostudies-arrayexpress

PRL-1 selectively dephosphorylates TIMELESS

Project description:The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways. Yet a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1 dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to changing day-night cycles.

2020-11-11 | PXD020986 | Pride

Optimized mucin-selective enrichment strategy to probe the mucinome

Project description:Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, we recently introduced a new mucinase (SmE) and demonstrated it has complementary recognition patterns to StcE. Here, we optimized our previous enrichment method to increase throughput and lower sample input amounts while maintaining mucin selectivity and enhancing glycopeptide signal. We also compare the ability of different mucinases, lectins, and mucin binding domains to enrich mucins from various cell lines and human serum. Overall, we demonstrate that our improved method StcE effectively isolates more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, which will allow for enhanced analysis of the mucinome.

2024-04-10 | PXD046534 | Pride

Gene expression change induced by TIM-3/galectin-9 interaction in primary AML

Project description:Analysis of gene expression change after galectin-9 stimulation in primary CD34+TIM-3+ AML cells Total RNA obtained from purified CD34+TIM-3+ primary AML cells with or without recombinant human galectin-9 stimulation (20hrs) was subjected to transcriptome analysis

2014-10-10 | E-GEOD-62223 | biostudies-arrayexpress

A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

Project description:Analysis of mucin type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these en-zymes, known O-glycoproteases have specificity constrains, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain type of glycoproteins or protein sequence bias, that limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. The peptide substrate sequence selectivity and its impact on IMPa activity was interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were specifically cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomics workflow for glycoprofiling of individual purified glycoproteins granulocyte colony-stimulating factor (G-CSF) and receptor-type tyrosine-protein phosphatase C (CD45) without the need for glycopeptide enrichment. In these examples, IMPa enabled identification of O-glycosites and the range of complex O-glycan structures at each site.

2022-01-05 | PXD029534 | Pride

The E3 Ligase CUL3 physically interacts with TANGO10 protein in vivo

Project description:List of protein scores for putative TANGO10 interactors from mass spectrometry analysis of FLAG-tagged TANGO10 using either elav-GAL4 or tim-GAL4 at both ZT10 and ZT 22, as determined using Proteome Discoverer software (ThermoFisher). Fly heads from GAL4 heterozygotes were used as controls. The hits from FLAG-tagged twenty-four (TYF) proteomics were also excluded from the list to increase TANGO10-specificity. Proteins listed are those present in at least one elav-GAL4 sample and at least one tim-GAL4 sample but no negative controls.

2021-11-02 | PXD029326 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data