Molecular characterization of four midgut aminopeptidase N isozymes from the cabbage looper, Trichoplusia ni.
Molecular characterization of four midgut aminopeptidase N isozymes from the cabbage looper, Trichoplusia ni.
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DOI：
10.1016/j.ibmb.2005.02.002
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发表时间：
2005-06
影响因子：
3.8
通讯作者：
Ping Wang;Xin Zhang;Jie Zhang
中科院分区：
文献类型：
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作者：
Ping Wang;Xin Zhang;Jie Zhang
利用cDNA克隆技术从甘蓝粉纹夜蛾（Trichoplusia ni）中鉴定出4种氨肽酶N（APN）同工型TnAPN 1、TnAPN 2、TnAPN 3和TnAPN 4。推测的氨基酸序列表明TnAPN 1、TnAPN 2、TnAPN 3和TnAPN 4分别是以110、106、114和108 kDa的前蛋白形式合成的。T. APN包括在其N-末端存在信号肽和在C-末端存在前肽用于GPI锚、锌结合/谷锌蛋白基序HEX 2 HX 18 E、谷锌蛋白氨肽酶基序GAMENWG和存在糖基化位点。在去除信号肽和C-末端前肽后，TnAPN 1、TnAPN 2、TnAPN 3和TnAPN 4的预测分子量分别为106、102、110和104 kDa。不同幼虫组织的酶活性测定表明，氨肽酶活性主要集中在中肠，且每毫克中肠组织蛋白的比酶活性在T.无论饲料中蛋白质和氨基酸的组成如何，酶活性测定和RT-PCR分析T.在幼虫组织中发现APN基因在Malphigian管中表达，这是首次发现APN也在Malphigian管中合成。在Malphigian小管中APN基因表达和酶活性的发现表明昆虫Malphigian小管与哺乳动物对应物肾脏的生化和功能相似，已知APN在肾脏中发挥重要功能。
Four aminopeptidase N (APN) isoforms, TnAPN1, TnAPN2, TnAPN3 and TnAPN4, were identified from the cabbage looper, Trichoplusia ni, by cDNA cloning. The deduced amino acid sequences of the four APNs indicate that TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are synthesized as preproteins of 110, 106, 114 and 108kDa, respectively. Sequence features of the T. ni APNs include the presence of a signal peptide at their N-termini and a prepeptide at the C-termini for the GPI anchor, the zinc binding/gluzincin motif HEX2HX18E, the gluzincin aminopeptidase motif GAMENWG and the presence of glycosylation sites. After removal of the signal peptide and the C-terminal prepeptide, the predicted molecular weights of TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are 106, 102, 110 and 104kDa, respectively. Enzymatic activity assays of various larval tissues showed that aminopeptidase activities were mainly localized in the midgut and the specific enzyme activity per mg of midgut tissue proteins was constant in T. ni larvae regardless of the composition of dietary proteins and amino acids. Both enzyme activity assays and RT-PCR analyses for the expression of the APN genes in T. ni larval tissues demonstrated that APN genes were expressed in Malphigian tubules in addition to the midgut, which was the first observation that APNs were also synthesized in insect Malphigian tubules. The finding of APN gene expression and enzyme activity in the Malphigian tubules indicated the biochemical and functional similarity of the insect Malphigian tubules to the mammalian counterpart, the kidney, in which APNs are known to play important functions.