Abstract
Cardiotoxins (CTXs), most constituents of cobra venoms, was found to cause inflammatory action. In the present study, we found that toxin γ can rapidly bind to human polymorphonuclear neutrophils (PMNs) and subsequent induce inflammatory response and its possible binding targets were isolated and identified from cell surface-bound fractions. Toxin γ itself did not activate significant PMNs oxidative and chemotactic ability comparing to a well-established chemoattractant fMLP under the cytotoxic dosage. However, the PMNs were dramatically attracted by a modified Boyum assay in which the target cell, such as human PMNs, differentiated macrophages and mouse cardiomyocyte cell line were first treated with toxin respectively and the releasing chemicals were used as the assaying attractants, indicating that CTX exert its inflammatory ability, if not all, via a autocrine and/or paracrine mechanism. When PMNs were exposed to toxin γ, the binding constant was estimated to be 1 μM by a direct binding assay using FITC-conjugated toxin γ and the time course of such binding revealed a rapid and saturated interaction with a T1/2 in 5 minutes. We isolated four putative Tγ-binding proteins from PMNs cell surface-bound fractions and identified them respectively to be lactoferrin, Cathepsin G, elastase 2 and one novel protein without certain identity with a MW near 60 kDa, by the methods of MS/MS peptide search and/or protein N-terminal sequencing. Interestingly, all these proteins are serine proteinases and the caseinolytic activity of the serine proteinases and metalloproteinases can also be detected in the cell-free supernatant, whereas the corresponding control has no effect. These findings provide an insight into the inflammatory properties of CTX and suggest a novel mechanism for illustrating neutrophils action in response to CTX.