Abstract
DM43, an opossum serum protein inhibitor of snake venom metalloproteinases, has been completely sequenced, and its disulfide bond pattern has been experimentally determined. It shows homology to human alpha(1)B-glycoprotein, a plasma protein of unknown function and a member of the immunoglobulin supergene family. Size exclusion and dynamic laser light scattering data indicated that two monomers of DM43, each composed of three immunoglobulin-like domains, associated to form a homodimer in solution. Analysis of its glycan moiety showed the presence of N-acetylglucosamine, mannose, galactose, and sialic acid, most probably forming four biantennary N-linked chains. DM43 inhibited the fibrinogenolytic activities of bothrolysin and jararhagin and formed 1:1 stoichiometric stable complexes with both metalloproteinases. DM43 was ineffective against atrolysin C or A. No complex formation was detected between DM43 and jararhagin C, indicating the essential role of the metalloproteinase domain for interaction. Homology modeling based on the crystal structure of a killer cell inhibitory receptor suggested the existence of an I-type Ig fold, a hydrophobic dimerization surface and six surface loops potentially forming the metalloproteinase-binding surface on DM43.
Highlights
The natural resistance to envenomation by snakes observed in a few warm-blooded animals as well as in several snakes can be explained, in most cases, by the presence of soluble proteins in the resistant animals’ sera, which can be grouped as inhibitors of either phospholipase A2 or snake venom metalloproteinases
SVMPs are typically found in venom of Viperidae snakes [5]. They are classified as members of the reprolysin subfamily of metalloproteinases [6], which includes ADAMs, proteins composed of a disintegrin and metalloproteinase domain, which function in various physiological processes such as fertilization, cytokine shedding, and neurogenesis [7]
The closest homologs to the SVMP inhibitor DM43 are the antihemorrhagins isolated from the North American opossum
Summary
Materials—N-Isopropyliodoacetamide was from Molecular Probes, Inc. 4-Vinylpyridine was from Sigma. The first half was digested with Asp-N after diluting the urea solution used during the alkylation procedure to 1 M with 0.05 M sodium phosphate (pH 8.0); the second half was desalted by RP-HPLC under the same conditions used for isolating the peptides obtained by lysyl peptidase digestion, dried by vacuum centrifugation, and dissolved in 0.05 M sodium phosphate (pH 8.0) Both aliquots were digested at an enzyme/substrate ratio of 1:200 (w/w) at 37 °C for 18 h. To determine the molecular mass of the DM43 protein moiety, a sample of DM43 chemically deglycosylated by anhydrous trifluoromethanesulfonic acid [32] was analyzed by MALDI-TOF-MS as previously described. Potential interface regions on the surface of the models were analyzed with the program PATCHES [52] using default parameters for dimeric contacts
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