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)在体内外均能刺激大鼠肥大细胞分泌组胺。该酶由两个功能区组成：催化蛋白降解反应的N末端结构域(VVP-N)和促进与蛋白质底物结合的C末端结构域。在本研究中，催化中心的单一锌离子被CuCl2或NiCl1；尽管经Cu2+处理的VVP显示出足够的组胺释放活性，但经Ni2+处理的VVP显示出较低的活性，因为其与靶物质的结合能力降低。同样，VVP-N以剂量和时间依赖的方式诱导组胺释放，但经镍处理的VVP-N的组胺释放活性明显降低。综上所述，该酶可能通过作用于肥大细胞膜上的靶物质(S)上的N末端结构域的催化中心来刺激组胺的释放。为了阐明该蛋白是否是VVP的靶物质，考察了积累GPI锚定蛋白的RAFT制剂的效果。然而，没有观察到对组胺释放的任何抑制作用。这一发现表明，VVP攻击的是膜蛋白，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.