Analyses of characterization and structure of NO synthase and NO-carrier protein from blood-sucking insects

吸血昆虫NO合成酶和NO载体蛋白的特性和结构分析

• 批准号: 10670227
• 负责人: YUDA Masao
• 金额: $ 2.37万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670227/
• 关键词: Coagulation; inhibitor; Rhodnius; factor IX; Prolixin-S; 吸血性昆虫; 唾液腺; 生理活性物質

Rhodnius prolixus is a blood-sucking Hemipteran insect which is known as a vector of American trypanosomiasis. This insect has several physiologically active molecules in its saliva that assist its blood-sucking behavior. We have already reported purification and the cDNA sequence of a salivary anticoagulant, prolixin-S, from this insect and demonstrated that prolixin-S was an inhibitor of the intrinsic Xase. We also reported that prolixin-S bound nitric oxide (NO) in its heme group as a vasodilator and a platelet aggregation inhibitor.We further studied on its inhibitory mechanisms of the blood coagulation. We found prolixin-S specifically bound to factor IX/IXa in the presence of CaィイD12+ィエD1 ions. Light scattering and surface plasmon resonance (SPR) studies showed that prolixin-S interfered with factor Ix/IXa binding to the phospholipid membrane, indicating that prolixin-S inhibit Xase activity of factor IXa by interference with its Xase complex formation. Furthermore, reconstitution experiments showed that prolixin-S binding to factor IX strongly inhibited factor IXa generation by factor XIa. We also found that prolixin-S inhibited factor IXa generation by factor VIIa/tissue factor complex and factor IXalfa generation by factor Xa. These results suggests that prolixin-S inhibits both intrinsic and extrinsic coagulations by sequential inhibition of all coagulation pathways in which factor IX participates. It was also suggested that prolixin-S may bind to factor IX/IXa by recognizing conformational change of the Gla domain induced by CaィイD12+ィエD1 binding. To gain further understanding, we are now trying to locate the interactive sites using synthesized Gla-residue peptide.

Rhodnius prolixus 是一种吸血半翅目昆虫，被称为美洲锥虫病的媒介。这种昆虫的唾液中含有多种生理活性分子，有助于其吸血行为。我们已经报道了这种昆虫唾液抗凝剂 Prolixin-S 的纯化和 cDNA 序列，并证明 Prolixin-S 是内在 Xase 的抑制剂。我们还报道了prolixin-S在其血红素基团中结合一氧化氮(NO)作为血管扩张剂和血小板聚集抑制剂。我们进一步研究了其抑制凝血的机制。我们发现在存在 CaィイD12+ィエD1 离子的情况下，prolixin-S 与因子 IX/IXa 特异性结合。光散射和表面等离子共振 (SPR) 研究表明 Prolixin-S 干扰因子 Ix/IXa 与磷脂膜的结合，表明 Prolixin-S 通过干扰其 Xase 复合物形成来抑制因子 IXa 的 Xase 活性。此外，重构实验表明prolixin-S与因子IX的结合强烈抑制因子XIa产生因子IXa。我们还发现 prolixin-S 抑制因子 VIIa/组织因子复合物产生因子 IXa 和抑制因子 Xa 产生因子 IXalfa。这些结果表明prolixin-S通过顺序抑制因子IX参与的所有凝血途径来抑制内在和外在凝血。还提出 Prolixin-S 可能通过识别由 CaiiD12+iiD1 结合诱导的 Gla 结构域的构象变化而与因子 IX/IXa 结合。为了获得进一步的了解，我们现在正尝试使用合成的 Gla 残基肽来定位相互作用位点。

项目成果

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Yuji Kaneko 等人：“重组硝基蛋白-2 一氧化氮复合物对血管平滑肌的影响”Biosci。

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Jianxin Sun et al.: "Characterization and cDNA cloning of a hemoprotein in the salivary glands of the blood-sucking insect, Rhodinius prolixus"Insect Biochem. Mol. Biol.. 28. 191-200 (1999)

孙建新等人：“吸血昆虫 Rhodinius prolixus 唾液腺中血红素蛋白的表征和 cDNA 克隆”Insect Biochem。

Yuji Kaneko, Masao Yuda, Takayoshi Iio, Tomohiko Murase and Yasuo Chinzei: "Kinetic analysis on nitric oxide binding of recombinant Prolixin-S, a nitric oxide transport protein from the blood-sucking bug, Rhodnius prolixus"Biochemica et Biophysica. Acta..

Yuji Kaneko、Masao Yuda、Takayoshi Iio、Tomohiko Murase 和 Yasuo Chinzei：“重组 Prolixin-S（一种来自吸血虫 Rhodnius prolixus 的一氧化氮转运蛋白）一氧化氮结合的动力学分析”Biochemica et Biophysicala。

P.Waidhet et al.: "Purification and characterization of a thrombin inhibitor from the salivary glands of a malarian vector mosquito,Anopheles stephensi"Biochem.Biophy.Acta.. 1381. 227-233 (1999)

P.Waidhet 等人：“从疟疾媒介蚊子 Anopheles stephensi 的唾液腺中纯化和表征凝血酶抑制剂”Biochem.Biophy.Acta.. 1381. 227-233 (1999)