title: Using mass spectrometry to explore the neglected glycan moieties of the antiophidic proteins DM43 and DM64.
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.
authors: Laboratory of Toxinology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil.
authors: Ileana Rodriguez
authors: IR
authors: León
authors: Ana Gisele da Costa
authors: AG
authors: Neves-Ferreira
authors: Surza Lucia Gonçalves
authors: SL
authors: da Rocha
authors: Monique Ramos de Oliveira
authors: MR
authors: Trugilho
authors: Jonas
authors: J
authors: Perales
authors: Richard Hemmi
authors: RH
authors: Valente
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism; D017837:Bothrops / Q000378:metabolism*; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048293:Didelphis / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D001798:Blood Proteins; D003435:Crotalid Venoms; D006023:Glycoproteins; D011134:Polysaccharides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N