Functional analysis of DM64, an antimyotoxic protein with immunoglobulin‐like structure from Didelphis marsupialis serum

Abstract

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human a 1 B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA 2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA 2 . Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.

Overlooked Issues of Snakebite Management: Time for Strategic Approach

“…All reptile PLIs isolated so far belongs to any of these three different groups. A fourth class contains only DM64, the unique PLI from Didelphis aurita that share sequence homology with members of the immunoglobulin super family [138]. PLIs are acidic oligomers of 75 and 180 kDa and composed of three to six subunits (either identical or different) ranging from 20 to 50 kDa [137].…”

A liver specific gene that is expressed in growth hormone transgenic mice and in normal female mice as a function of age

“…In this regard, CRISP-3 was found to bind to A1BG in human blood by Udby and colleagues [34]. A similar opossum protein, DM64, is found in the serum and has been shown to inhibit myotoxins by binding to them non-covalently [35]. A related protein in opossum, DM43, is a metalloproteinase inhibitor thought to function in a similar fashion [36].…”

Natural Inhibitors of Snake Venom Metallopeptidases

“…Amino acid sequences of DM43 and DM43b share 81 % identity and 88 % similarity. A third homologous inhibitor of 64 kDa has also been purified from opossum serum (Rocha et al 2002). Surprisingly, instead of inhibiting metallopeptidases, DM64 neutralizes myotoxicity induced by venom proteins bearing PLA 2 structure.…”