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]

Afiliação do(s) autor(es)

^[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

• Tipo de documento: Artigo Científico
• Fonte: JOURNAL OF PROTEOMICS; v. 248, SEP 30 2021.
• Citações Web of Science: 0

Resumo

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)

• Processo FAPESP: 18/15549-1 - Modificações pós-traducionais nos processos biológicos e no diagnóstico da Doença de Chagas: novas abordagens metodológicas e implicações biológicas
• Beneficiário: Giuseppe Palmisano
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores - Fase 2
• Processo FAPESP: 13/07937-8 - Redoxoma
• Beneficiário: Ohara Augusto
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 20/04923-0 - Glicosilação do SARS-CoV-2 para identificação das características estruturais da COVID-19
• Beneficiário: Giuseppe Palmisano
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/04032-5 - Dissecando a patogênese da Doença de Chagas através de abordagens glicômicas e glicoproteômicas
• Beneficiário: Simon Ngao Mule
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 18/18257-1 - EMU concedido no processo 14/06863-3: sistema de cromatografia líquida configurado para análise de carboidratos, aminoácidos, peptídeos e glicoproteínas
• Beneficiário: Giuseppe Palmisano
• Modalidade de apoio: Auxílio à Pesquisa - Programa Equipamentos Multiusuários