From the Bombina maxima skin we have purified and characterized a novel trefoil factor protein (Bm-TFF2) and an annexin II related protein (BAIIRP). Bm-TFF2 induces platelet activation while BAIIRP plays an inhibition role. We also studied the difference of signal transduction pathway between Bm-TFF2 and stejnulxin a platelet membrabe glycoprotein GPVI agonist from Trimeresurus stejnegeri venomIn mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel TFF protein, named bomaggin, has been identified and purified from the skin secretions of frog Bombina maxima. It is a single chain protein with an apparent molecular weight of 13,000. It activated human platelets in a dose-dependent manner and activation of integrin anbP3 was involved. Aspirin and apyrase did not largely reduced platelet response to bomaggin (a 30% inhibition), suggesting the aggregation is not substantially dependent on ADP and thrornboxane A2 (TXA2) autocrine feedback. FITC-labeled bomaggin bound to platelet membranes. Selective pharmacological antagonist studies revealed that bomaggin activates platelets not via well-known G-protein coupled receptors, like those of ADP, TXA2 and platelet-activating factor. A tyrosine phosphorylation profile induced by bomaggin in platelets was different from that produced by stejnulxin, a glycoprotein VI agonist, and particularly phospholipase Cy2 (PLCV2) was not phosphorylated. Activation of PLC, then an elevation of cytosolic free Ca2+ and activation of protein kinase C are major molecular events involved in bomaggin induced platelet activation. Containing two TFF domains, bomaggin is composed of 104 amino acids as deduced from its coding cDNA, and its sequence exhibits 28% and 34% sequence identity with human TFF2 and Africa claw toad xP2 respectively. Bm-TFF2 is the first platelet agonist containing two trefoil factor domains from Amphibian.A single chain protein with an apparent molecular mass of 33 kDa was purified from skin of Bombina maxima by a combination of ion exchange and gel filtration chromatography steps. N-terminal amino acid sequence determination indicated that it shares 70%, 64% and 56% identity with those of annexin II from Africa claw toad, red junglefowl and human, respectively. The purified protein from Bombina maxina inhibits stejnulxin (a specific platelet agonist via platelet memdrane glycoproteinVI (GPVI) induced platelet aggregation in a Ca2+ dependent manner. Maximal inhibition rate reaches 48%. Based on the N-terminal amino acid sequence BLAST search results and its Ca2+ dependent activity, the purified protein might be structurally and functionally related to the annexin protein family.
修改评论