Exocytotic histamine release caused by the bacterial metalloprotease : identification of the target protein on the mast cell membrane

创伤弧菌分泌的锌金属蛋白酶（VVP）在体内外均能刺激肥大细胞释放组胺。该蛋白酶由两个功能结构域组成：N端结构域（VVP-N）催化蛋白水解反应，C端结构域促进与蛋白底物的结合<2->。虽然Cu^&lt;2+&gt;处理的VVP显示出足够的组胺释放活性，但Ni^&lt;2+&gt;处理的VVP由于与靶物质结合的潜力降低而显示出较低的活性。同样，VVP-N以剂量和时间依赖的方式诱导组胺释放，然而，Ni^2+处理的VVP-N显示出更少的组胺释放活性。总之，蛋白酶可能通过N-末端结构域的催化中心作用于肥大细胞膜上的靶物质而刺激组胺释放。为了明确该蛋白质是否是VVP的靶物质，检查了其中GPI锚定蛋白质积累的筏制备物的效果。然而，未观察到对组胺释放的任何抑制作用。这一发现表明，VVP攻击膜蛋白而不是GPI锚定蛋白。

The zinc metalloprotease (VVP) secreted by Vibrio vulnificus stimulates exocytotic histamine release from rat mast cells both in vitro and in vivo. This protease consists of two functional domains : the N-terminal domain (VVP-N) catalyzing the proteolytic reaction and the C-terminal domain promoting the association with a protein substrate.In the present study, the single zinc ion in the catalytic center was substituted by treatment with CuCl_2 or NiCl_<2->. Although Cu^<2+>-treated VVP showed sufficient histamine-releas ing activity, Ni^<2+>-treated VVP showed the less activity because of the reduced potential to attach to the target substance. Likewise, VVP-N induced histamine release in a dose- and time-dependent manner, however, Ni^<2+>-treated VVP-N revealed much less histamine-releasing activity. Taken together, the protease may stimulate histamine release through the action of the catalytic center of the N-terminal domain on the target substance(s) on the mast cell membrane.The GPI-anchored protein has been reported to function as the receptor for exocellular stimulus. To clarify whether this protein is the target substance of VVP, the effect of the raft preparation in which the GPI-anchored protein accumulates was examined. However, any inhibitory effect on the histamine release was not observed. This finding indicates that VVP attacks the membrane protein other than the GPI-anchored protein.