Kawakoshi A, Hyodo S, Yasuda A, Takei Y
Ocean Research Institute, University of Tokyo, Tokyo 164-8639, Japan.
J Mol Endocrinol. 2003 Aug;31(1):209-20. doi: 10.1677/jme.0.0310209.
In teleost fish and tetrapods, the natriuretic peptide (NP) family consists of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and VNP (ventricular natriuretic peptide) that are secreted from the heart, and C-type natriuretic peptide (CNP) that is found in the brain. However, CNP is the only NP identified in the heart and brain of elasmobranchs, suggesting that it is the ancestral type of the NP family and that ANP, BNP and VNP appeared later in the vertebrate phylogeny. To delineate more clearly the molecular evolution of this hormone family, we determined the sequence of NP molecule(s) in evolutionarily the oldest vertebrate group, the cyclostomes. We have cloned a novel NP cDNA from the heart and brain of hagfish, Eptatretus burgeri, using the RACE method and degenerate primers that amplify all known types of NP cDNAs. The novel NP, named EbuNP after the scientific name of this hagfish, appears to be the only NP in the heart and brain, as no other NP cDNAs were amplified even after specific removal of the cloned EbuNP mRNA from the mRNA pool, except for a minor alternatively spliced EbuNP cDNA with a truncated 3'-untranslated sequence. The EbuNP was equally similar to known NPs but was not considered to be a CNP because of the presence of a C-terminal tail sequence. The EbuNP gene was abundantly expressed in the cardiac atrium, ventricle, portal heart and brain but scarcely in the intestine; no expression was observed in the gill and kidney. Mass spectrometry of affinity-purified EbuNP in plasma, heart and brain revealed a 68 amino acid peptide circulating in the blood and stored in the heart, which is cleaved at the typical cleavage signal of a processing enzyme, furin, as observed in mammalian BNP. The C-terminal Gly residue was used for amidation as is the case in eel ANP. The immunoreactive EbuNP was not detected in the brain, suggesting the presence of a different processing form in the brain. These results show that the molecular evolution of the NP family in vertebrates is more complex than previously thought.
在硬骨鱼和四足动物中，利钠肽（NP）家族由心脏分泌的心房利钠肽（ANP）、脑利钠肽（BNP）和心室利钠肽（VNP），以及在脑中发现的C型利钠肽（CNP）组成。然而，CNP是在软骨鱼的心脏和脑中唯一鉴定出的NP，这表明它是NP家族的原始类型，而ANP、BNP和VNP是在脊椎动物系统发育中较晚出现的。为了更清楚地描绘这个激素家族的分子进化，我们确定了进化上最古老的脊椎动物类群——圆口纲动物中NP分子的序列。我们使用RACE方法和能扩增所有已知类型NP cDNA的简并引物，从盲鳗（Eptatretus burgeri）的心脏和脑中克隆了一种新的NP cDNA。这种新的NP，以这种盲鳗的学名命名为EbuNP，似乎是心脏和脑中唯一的NP，因为即使从mRNA池中特异性去除克隆的EbuNP mRNA后，也没有扩增出其他NP cDNA，除了一个具有截短3'非翻译序列的次要可变剪接EbuNP cDNA。EbuNP与已知的NP同样相似，但由于存在C末端尾序列，不被认为是CNP。EbuNP基因在心脏心房、心室、门静脉心脏和脑中大量表达，但在肠道中很少表达；在鳃和肾脏中未观察到表达。对血浆、心脏和脑中亲和纯化的EbuNP进行质谱分析，发现一种68个氨基酸的肽在血液中循环并储存在心脏中，它在加工酶弗林蛋白酶的典型切割信号处被切割，这与在哺乳动物BNP中观察到的情况一样。C末端甘氨酸残基用于酰胺化，就像在鳗鱼ANP中一样。在脑中未检测到免疫反应性EbuNP，这表明脑中存在不同的加工形式。这些结果表明，脊椎动物中NP家族的分子进化比以前认为的更复杂。
