Identification and characterization of a salivary adenosine deaminase from the sand fly Phlebotomus duboscqi, the vector of Leishmania major in sub-Saharan Africa
Identification and characterization of a salivary adenosine deaminase from the sand fly Phlebotomus duboscqi, the vector of Leishmania major in sub-Saharan Africa
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DOI：
10.1242/jeb.001289
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发表时间：
2007-03-01
影响因子：
2.8
通讯作者：
Valenzuela, Jesus G.
中科院分区：
文献类型：
--
作者：
Kato, Hirotomo;Jochim, Ryan C.;Valenzuela, Jesus G.
通过对杜氏白蚁唾液腺cDNA文库的测序，鉴定了两个腺苷脱氨酶(ADA)的转录本。腺苷脱氨酶存在于长掌白纹伊蚊的唾液中，但在白纹伊蚊、白纹伊蚊、白纹伊蚊和白纹伊蚊的唾液中不存在，这表明该酶只存在于褐飞虱属的唾液中。在目前的工作中，我们验证了这样的假设，即白头翁的姊妹种白头翁的唾液腺编码ADA的转录本产生了活性的唾液ADA。白头翁的唾液腺匀浆将腺苷转化为肌苷，这表明该种沙蝇的唾液中存在ADA活性；此外，当使用最近采血的沙蝇的唾液腺或被刺穿的唾液腺时，这种酶的活性显著降低，表明这种酶是在这种昆虫的唾液中分泌的。这种酶活性在巴氏杆菌的唾液中是不存在的。与其他白纹伊蚊相比，用高效液相-光电二极管阵列检测，在白纹伊蚊的唾液腺中没有发现AMP或腺苷。为了证实编码ADA的转录物与在沙蝇唾液中观察到的活性有关，我们将该转录物克隆到原核表达载体中，并产生了一个约60 kDa的可溶性活性重组蛋白，能够将腺苷转化为肌苷。用对照质粒转化的细菌提取物没有表现出这种活性。这些结果表明，在这种沙蝇的唾液腺中检测到的活性与编码ADA的杜波斯奇小蜂转录本有关，并且杜波斯齐小蜂的这种活动是独立于其他白纹伊蚊获得的。这是另一个食血节肢动物唾液基因中的基因招募事件，可能与血液摄食有关，由于ADA在免疫中的作用，它也可能在寄生虫传播中发挥作用。
Two transcripts coding for an adenosine deaminase (ADA) were identified by sequencing a Phlebotomus duboscqi salivary gland cDNA library. Adenosine deaminase was previously reported in the saliva of the sand fly Lutzomyia longipalpis but it was not present in the saliva of the sand flies Phlebotomus papatasi, P. argentipes, P. perniciosus and P. ariasi, suggesting that this enzyme is only present in the saliva of sand flies from the genus Lutzomyia. In the present work, we tested the hypothesis that the salivary gland transcript coding for ADA in Phlebotomus duboscqi, a sister species of Phlebotomus papatasi, produces an active salivary ADA.Salivary gland homogenates of P. duboscqi converted adenosine to inosine, suggesting the presence of ADA activity in the saliva of this species of sand fly; furthermore, this enzymatic activity was significantly reduced when using either salivary glands of recently blood-fed sand flies or punctured salivary glands, suggesting that this enzyme is secreted in the saliva of this insect. This enzymatic activity was absent from the saliva of P. papatasi. In contrast to other Phlebotomus sand flies, we did not find AMP or adenosine in P. duboscqi salivary glands as measured by HPLC-photodiode array. To confirm that the transcript coding for ADA was responsible for the activity observed in the saliva of this sand fly, we cloned this transcript into a prokaryotic expression vector and produced a soluble and active recombinant protein of approximately 60 kDa that was able to convert adenosine to inosine. Extracts of bacteria transformed with control plasmids did not show this activity. These results suggest that P. duboscqi transcripts coding for ADA are responsible for the activity detected in the salivary glands of this sand fly and that P. duboscqi acquired this activity independently from other Phlebotomus sand flies. This is another example of a gene recruitment event in salivary genes of blood-feeding arthropods that may be relevant for blood feeding and, because of the role of ADA in immunity, it may also play a role in parasite transmission.