Proteome-Wide Analysis of Single-Nucleotide Variations in the N-Glycosylation Sequon of Human Genes

Mazumder, Raja; Morampudi, Krishna S.; Motwani, Mona; Vasudevan, Sona; Goldman, Radoslav

doi:10.1371/journal.pone.0036212

Cited by 32 publications

(38 citation statements)

References 64 publications

(73 reference statements)

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“…Out of 2299 proteins that do not have structures, 2247 proteins are predicted to be glycosylated at the gain of glycosylation site by the SFAT tool (total NXS/T sites: 12,623; sites predicted as yes: 11,651 and sites predicted as no: 972). Based on GO analysis using Panther tools [21], the over-represented GO biological processes in this dataset include immune response, response to stimulus, signaling and blood coagulation, which agrees with the our results obtained previously [4]. …”

Section: Resultssupporting

confidence: 90%

Structure-based Comparative Analysis and Prediction of N-linked Glycosylation Sites in Evolutionarily Distant Eukaryotes

Lam

Goldman

Karagiannis

et al. 2013

Genomics, Proteomics & Bioinformatics

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The asparagine-X-serine/threonine (NXS/T) motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated proteins allows us to carry out structural analysis of the N-linked glycosylation sites (NGS). Our analysis shows that there is enough structural information from diverse glycoproteins to allow development of rules which can be used to predict NGS. A Python-based tool was developed to investigate asparagines implicated in N-glycosylation in five species: Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana and Saccharomyces cerevisiae. Our analysis shows that 78% of all asparagines of NXS/T motif involved in N-glycosylation are localized in the loop/turn conformation in the human proteome. Similar distribution was revealed for all the other species examined. Comparative analysis of the occurrence of NXS/T motifs not known to be glycosylated and their reverse sequence (S/TXN) shows a similar distribution across the secondary structural elements, indicating that the NXS/T motif in itself is not biologically relevant. Based on our analysis, we have defined rules to determine NGS. Using machine learning methods based on these rules we can predict with 93% accuracy if a particular site will be glycosylated. If structural information is not available the tool uses structural prediction results resulting in 74% accuracy. The tool was used to identify glycosylation sites in 108 human proteins with structures and 2247 proteins without structures that have acquired NXS/T site/s due to non-synonymous variation. The tool, Structure Feature Analysis Tool (SFAT), is freely available to the public at http://hive.biochemistry.gwu.edu/tools/sfat.

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Section: Resultssupporting

confidence: 90%

Structure-based Comparative Analysis and Prediction of N-linked Glycosylation Sites in Evolutionarily Distant Eukaryotes

Lam

Goldman

Karagiannis

et al. 2013

Genomics, Proteomics & Bioinformatics

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“…It is clear that to get a comprehensive view of the glycoproteome, we need to factor in gain/loss of glycosylation sites due to SNVs [11]. Such novel sites can be present in some populations or in cancer cells.…”

Section: Resultsmentioning

confidence: 99%

“…Several services are available to identify the effect of nsSNVs on individual protein function which is done mainly through sequence conservation analysis and/or through mapping of sequence and structure features to individual proteins [8–10]. To the best of our knowledge none of them allow proteome-wide analysis of integrated data similar to the work on proteome-wide analysis on the effect of nsSNVs on N-glycosylation sites [11]. SNVDis is a web tool that addresses this by integrating variation data with curated sequence feature annotations in a protein-centric fashion.…”

Section: Introductionmentioning

confidence: 99%

SNVDis: A Proteome-wide Analysis Service for Evaluating nsSNVs in Protein Functional Sites and Pathways

Karagiannis

Simonyan

Mazumder

2013

Genomics, Proteomics & Bioinformatics

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Amino acid changes due to non-synonymous variation are included as annotations for individual proteins in UniProtKB/Swiss-Prot and RefSeq which present biological data in a protein- or gene-centric fashion. Unfortunately, proteome-wide analysis of non-synonymous single-nucleotide variations (nsSNVs) is not easy to perform because information on nsSNVs and functionally important sites are not well integrated both within and between databases and their search engines. We have developed SNVDis that allows evaluation of proteome-wide nsSNV distribution in functional sites, domains and pathways. More specifically, we have integrated human-specific data from major variation databases (UniProtKB, dbSNP and COSMIC), comprehensive sequence feature annotation from UniProtKB, Pfam, RefSeq, Conserved Domain Database (CDD) and pathway information from Protein ANalysis THrough Evolutionary Relationships (PANTHER) and mapped all of them in a uniform and comprehensive way to the human reference proteome provided by UniProtKB/Swiss-Prot. Integrated information of active sites, pathways, binding sites, domains, which are extracted from a number of different sources, provides a detailed overview of how nsSNVs are distributed over the human proteome and pathways and how they intersect with functional sites of proteins. Additionally, it is possible to find out whether there is an over- or under-representation of nsSNVs in specific domains, pathways or user-defined protein lists. The underlying datasets are updated once every 3 months. SNVDis is freely available at http://hive.biochemistry.gwu.edu/tool/snvdis.

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“…Although absent in ␣1␤3␥2-GABA A R, the four minor regions include: ␤8-␤8 loop (D, outer-surface counterpart of ␤4-␤5 loop, different within ␣ and subtypes), ␤8-␤9 lower-loop F (E, unique to 5HT3R ␤), upper ␤10 (F, GABA A R ) and lower ␤10 sheet (G, GABA A R 1, 2, ). Further, single-nucleotide variation analysis of human proteome found only two gains (one in ␣3, one in ) and zero loss of NXS/T motifs in GABA A R subunits (59), suggesting functional essentiality of N-glycosylation for hGABA A R. Subunit-Western blot saw N-glycosylation shifts in several GABA A R subunits (sites unknown) in human Schizophrenia brains, despite constant protein levels (60). Antibody N-glycans proved powerful to enhance antibody-target binding (61).…”

Section: Mid-ecd N-glycosylations May Underpin Subunit-biased Hlgic Amentioning

confidence: 98%

Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias

Zhang

2016

Molecular & Cellular Proteomics

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Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins-at high efficiency and peptide reproducibility-poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergentfacilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/Dexchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. Molecular & Cellular

show abstract

Proteome-Wide Analysis of Single-Nucleotide Variations in the N-Glycosylation Sequon of Human Genes

Cited by 32 publications

References 64 publications

Structure-based Comparative Analysis and Prediction of N-linked Glycosylation Sites in Evolutionarily Distant Eukaryotes

Structure-based Comparative Analysis and Prediction of N-linked Glycosylation Sites in Evolutionarily Distant Eukaryotes

SNVDis: A Proteome-wide Analysis Service for Evaluating nsSNVs in Protein Functional Sites and Pathways

Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias

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