Oligosaccharide residues of Loxosceles intermedia (brown spider) venom proteins: dependence on glycosylation for dermonecrotic activity

Loxosceles spp. (brown spider) envenomation has been reported to provoke de rmonecrosis and haemorrhage at the bite site (a hallmark of accidents) and, to a lesser extent, thrombocytopenia, hemolysis and disseminated intravasc ular coagulation in some cases. Using lectin-immunolabeling, lectin-affinit y chromatography, glycosidase and proteinase K treatments we were able to i dentify several venom N-glycosylated proteins with high-mannose oligosaccha ride structures, complex-type glycoconjugates such as fucosylated glycans, but no galactose or sialic acid residues as complex sugars or glycosaminogl ycan residues. Working with enzymatically or chemically deglycosylated veno m we found that platelet aggregation (thrombocytopenic activity) as well as the fibronectinolytic and fibrinogenolytic (haemorrhagic) effects of the v enom were sugar-independent when compared to glycosylated venom. Neverthele ss, zymograph analysis in co-polymerized gelatin gels showed that enzymatic N-deglycosylation of loxolysin-B, a high mannose 32-35 kDa glycoprotein of the venom with gelatinolytic metalloproteinase activity, caused a reductio n of approximately 2 kDa in its molecular weight and a reduction of the gel atinolytic effect to a residual activity of 28% when compared to the glycos ylated molecule. indicating a post-translational glycosylation-dependent ge latinolytic effect. Analysis of the dermonecrotic effect of the chemically or enzymatically N-deglycosylated venom detected only residual activity whe n compared with the glycosylated control. Thus, the present report suggests that oligosaccharide moieties play a role in the destructive effects of br own spider venom and opens the possibility for a carbohydrate-based therapy . (C) 1999 Elsevier Science Ltd. All rights reserved.