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Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools.

ABSTRACT: Protein glycosylation fundamentally impacts biological processes. Nontemplated biosynthesis introduces unparalleled complexity into glycans that needs tools to understand their roles in physiology. The era of quantitative biology is a great opportunity to unravel these roles, especially by mass spectrometry glycoproteomics. However, with high sensitivity come stringent requirements on tool specificity. Bioorthogonal metabolic labeling reagents have been fundamental to studying the cell surface glycoproteome but typically enter a range of different glycans and are thus of limited specificity. Here, we discuss the generation of metabolic 'precision tools' to study particular subtypes of the glycome. A chemical biology tactic termed bump-and-hole engineering generates mutant glycosyltransferases that specifically accommodate bioorthogonal monosaccharides as an enabling technique of glycobiology. We review the groundbreaking discoveries that have led to applying the tactic in the living cell and the implications in the context of current developments in mass spectrometry glycoproteomics.

SUBMITTER: Cioce A

PROVIDER: S-EPMC7955280 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools.

Cioce Anna A Malaker Stacy A SA Schumann Benjamin B

Current opinion in chemical biology 20201027

Protein glycosylation fundamentally impacts biological processes. Nontemplated biosynthesis introduces unparalleled complexity into glycans that needs tools to understand their roles in physiology. The era of quantitative biology is a great opportunity to unravel these roles, especially by mass spectrometry glycoproteomics. However, with high sensitivity come stringent requirements on tool specificity. Bioorthogonal metabolic labeling reagents have been fundamental to studying the cell surface g ...[more]

PMID: 33125942

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Project description:MotivationOver the last decades, vast numbers of sequences were deposited in public databases. Bioinformatics tools allow homology and consequently functional inference for these sequences. New profile-based homology search tools have been introduced, allowing reliable detection of remote homologs, but have not been systematically benchmarked. To provide such a comparison, which can guide bioinformatics workflows, we extend and apply our previously developed benchmark approach to evaluate the 'next generation' of profile-based approaches, including CS-BLAST, HHSEARCH and PHMMER, in comparison with the non-profile based search tools NCBI-BLAST, USEARCH, UBLAST and FASTA.MethodWe generated challenging benchmark datasets based on protein domain architectures within either the PFAM + Clan, SCOP/Superfamily or CATH/Gene3D domain definition schemes. From each dataset, homologous and non-homologous protein pairs were aligned using each tool, and standard performance metrics calculated. We further measured congruence of domain architecture assignments in the three domain databases.ResultsCSBLAST and PHMMER had overall highest accuracy. FASTA, UBLAST and USEARCH showed large trade-offs of accuracy for speed optimization.ConclusionProfile methods are superior at inferring remote homologs but the difference in accuracy between methods is relatively small. PHMMER and CSBLAST stand out with the highest accuracy, yet still at a reasonable computational cost. Additionally, we show that less than 0.1% of Swiss-Prot protein pairs considered homologous by one database are considered non-homologous by another, implying that these classifications represent equivalent underlying biological phenomena, differing mostly in coverage and granularity.Availability and implementationBenchmark datasets and all scripts are placed at (http://sonnhammer.org/download/Homology_benchmark).Contactforslund@embl.deSupplementary informationSupplementary data are available at Bioinformatics online.

Dataset Information

Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools.

Publications

Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools.

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