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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

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Mule, Simon Ngao [1] ; Rosa-Fernandes, Livia [1] ; Coutinho, Joao V. P. [1] ; Gomes, Vinicius De Morais [2, 1] ; Macedo-da-Silva, Janaina [1] ; Santiago, Veronica Feijoli [1] ; Quina, Daniel [1] ; de Oliveira, Gilberto Santos [1] ; Thaysen-Andersen, Morten [3] ; Larsen, Martin R. [4] ; Labriola, Leticia [2] ; Palmisano, Giuseppe [1]
Total Authors: 12
[1] Univ Sao Paulo, Inst Biomed Sci, Dept Parasitol, GlycoProte Lab, Sao Paulo - Brazil
[2] Univ Sao Paulo, Inst Chem, Dept Biochem, Sao Paulo - Brazil
[3] Macquarie Univ, Dept Mol Sci, Sydney, NSW - Australia
[4] Univ Southern Denmark, Dept Biochem & Mol Biol, Odense - Denmark
Total Affiliations: 4
Document type: Journal article
Source: JOURNAL OF PROTEOMICS; v. 248, SEP 30 2021.
Web of Science Citations: 0

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of Nglycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of formerly N-glycosylated peptides (FNGPs). LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases. Significance: We describe a novel method termed limited deglycosylation assay (LDA), to probe conformational changes of glycoproteins on a systems-wide scale. This method improves the current toolbox of structural proteomics by combining site and conformational-specific PNGase F enzymatic activity with large scale quantitative proteomics. X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoEM techniques are the major techniques applied to elucidate macromolecule structures. However, the size and heterogeneity of the oligosaccharide chains poses several challenges to the applications of these techniques to glycoproteins. The LDA method presented here, can be applied to a range of pathophysiological conditions and expanded to investigate PTMs-mediated structural changes in complex proteomes. (AU)

FAPESP's process: 18/15549-1 - Post-translational modifications in Chagas Disease biological processes and diagnostics: novel methodological approaches and biological applications
Grantee:Giuseppe Palmisano
Support type: Research Grants - Young Investigators Grants - Phase 2
FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
Grantee:Ohara Augusto
Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC
FAPESP's process: 20/04923-0 - SARS-CoV-2 glycosylation: a blueprint structural insight for understanding COVID-19 pathogenesis
Grantee:Giuseppe Palmisano
Support type: Regular Research Grants
FAPESP's process: 17/04032-5 - Dissecting the pathogenesis of Chagas Disease by deep glycomics and glycoproteomics approaches
Grantee:Simon Ngao Mule
Support type: Scholarships in Brazil - Doctorate
FAPESP's process: 18/18257-1 - Multi-user equipment approved in grant 14/06863-3: HPLC system configured for analysis of carbohydrates, amino acidis, peptides and glycoproteins
Grantee:Giuseppe Palmisano
Support type: Multi-user Equipment Program