Characterization of the N-Glycosylation of DM43 and DM64 for mass spectrometry and analysis of its contribution for the antiophidian activity of these proteins.
In tropical and sub-tropical countries, envenomations inflicted by snake bites are a public health hazard. The global mortality ranges from 50,000 to 100,000 per year. However,for every death, there are unreported sequelae for survivors including chronic ulceration, renal failure, neurological damage and amputations, among others. Treatment relies upon animalantiserum mostly produced in horse or sheep against antigenic toxins present in snake venoms. Nevertheless, antivenom is ineffective in the treatment of local tissue damage and itsadministration may provoke serious adverse reactions. Some animals, such as snakes and a few mammals, present natural resistance to snake envenomation. Mostly, this resistance is due to the presence of soluble neutralizing proteins in their sera. The South American opossum Didelphis aurita is resistant to several Viperidae snake venoms. From its serum two proteins with antihemorrhagic (DM43) and antimyotoxic (DM64) activities were previously isolated. DM43 and DM64 are homodimeric acidic glycoproteins showing 21% and 15% (w/w) carbohydrate content, respectively. The aim of this work was to implement and develop different glycoprotein analytical techniques in order to characterize N-glycosylation sites, using these inhibitors as model. The different generated peptides or glycopeptides, after enzimatic digestions, were characterized by mass spectrometric analysis. Oligosaccharidestructures and the biological activity of the inhibitors after either partial or complete deglycosylation were also analyzed. Using MALDI-TOF/TOF in combination with twosequential digestions by three different proteases, with and without previous digestion with peptide-N-Glycosidase F, four N-glycosylation sites in DM43 (Asn23, Asn156, Asn160 andAsn175) were confirmed. Additionally, four putative N-glycosylation sites in DM64 (Asn46, Asn179, Asn183 and Asn379) were experimentally determined. Complex-type oligosaccharides containing galactose and sialic acid were assigned to both proteins. We also determined thatpartial deglycosylation using two exoglycosidases did not have any measurable influence on the inhibitors´ biological activities. On the other hand, the deglycosylation of DM43 using PNGase F, under native conditions, originated different deglycosylated species and lead to a 2-fold decrease in its inhibitory activity. This is the first time that the importance of part of the carbohydrate content of these inhibitors, for their biological activity, is demonstrated. Although, Asn23 (DM43) and Asn379 (DM64) do not seem to be involved in any kind ofinteraction, the other N-glycosilation sites might have a relevant role in keeping the inhibitors´ natural homodimeric structures and/or in the formation / mantainance of the non-covalent complexes composed of an inhibitor monomer with a toxin molecule.
Advisor:Richard Hemmi Valente; Jonas Enrique Aguilar Perales
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Didelphis Glicosilação Immunity Natural Glycosylation Mass Spectrometry Snake Venoms
Date of Publication:03/17/2008