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Using mass spectrometry to explore the neglected glycan moieties of the antiophidic proteins DM43 and DM64

Authors

  • Ileana Rodriguez León,

  • Ana Gisele da Costa Neves-Ferreira,

  • Surza Lucia Gonçalves da Rocha,

  • Monique Ramos de Oliveira Trugilho,

  • Jonas Perales,

  • Richard Hemmi Valente

    Corresponding author
    • Laboratory of Toxinology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil
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  • Colour Online: See the article online to view Figs. 1–4 in colour.

Correspondence: Dr. Richard Hemmi Valente, FIOCRUZ, Pavilhão Ozório de Almeida, Sala 05, Avenida Brasil, 4365, 21040-900 – Rio de Janeiro – RJ, Brazil

E-mail: rhv4u@ioc.fiocruz.br; richardhemmi@gmail.com

Fax: + 55-21-25621410

Abstract

The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.

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