Purification and properties of ghrelin from the intestine of the goldfish, Carassius auratus
Purification and properties of ghrelin from the intestine of the goldfish, Carassius auratus
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DOI：
10.1016/j.peptides.2008.12.016
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发表时间：
2009-04-01
期刊：
影响因子：
3
通讯作者：
Matsuda, Kouhei
中科院分区：
文献类型：
--
作者：
Miura, Tohru;Maruyama, Keisuke;Matsuda, Kouhei
在金鱼中，腹膜内（IP）或脑室内（ICV）给药合成生长激素释放肽，其由12或19个氨基酸残基组成，由其前体cDNA推导，在第三个N-末端丝氨酸残基（Ser（3））处具有辛酸修饰，刺激生长激素释放和食物摄入。然而，从其前体产生的天然ghrelin尚未在该物种中被鉴定。因此，我们采用酸提取、阳离子交换和反相高效液相色谱结合免疫亲和纯化的方法从金鱼肠道中纯化ghrelin。为了确认在每个纯化步骤的馏分中的ghrelin活性，我们检查了每个馏分对大鼠生长激素促分泌素受体（GHS-R）表达细胞中细胞内Ca 2+动员的影响。我们将金鱼ghrelin分为11种分子形式，由14-，17-，18-和19-氨基酸残基组成，第3组酰化，其中17-残基形式占主导地位。然后，我们合成了在Ser（3）处具有辛酸修饰（辛酰基ghrelin 17）和没有酰化（脱酰基ghrelin 17）的17-残基形式，并检测了它们的生物活性。辛酰ghrelin 17，但不脱酰基ghrelin 17，增加大鼠GHS-R表达细胞的细胞内Ca 2+浓度，其效力类似于由12个残基组成的合成ghrelin（辛酰ghrelin 12）和辛酰大鼠ghrelin。IP和ICV给予辛酰基生长素释放肽17和辛酰基生长素释放肽12，但不去酰基生长素释放肽17，增加金鱼的食物摄入。目前的研究结果表明，天然金鱼ghrelin由11个分子变体组成，主要形式是17个残基的肽。这种具有酰化作用的主要形式与金鱼摄食的调节有关。(c)2008年爱思唯尔公司All rights reserved.
In goldfish, intraperitoneal (IP) or intracerebroventricular (ICV) administration of synthetic ghrelin consisting of 12- or 19-amino-acid residues, deduced from its precursor cDNA, with an octanoic acid modification at the third N-terminal serine residue (Ser(3)), stimulates growth hormone release and food intake. However, native ghrelin generated from its precursor has not yet been identified in this species. Therefore, we purified ghrelin from the goldfish intestine using acid extraction, cation-exchange and reverse-phase high-performance liquid chromatography combined with immune-affinity purification. In order to confirm ghrelin activity in the fractions at each purification step, we examined the effect of each fraction on intracellular Ca2+ mobilization in rat growth hormone secretagogue-receptor (GHS-R)-expressing cells. We characterized the goldfish ghrelin as 11 molecular forms consisting of 14-, 17-, 18- and 19-amino-acid residues with acylation at Set 3, and the 17-residue form was predominant. We then synthesized 17-residue forms with octanoic acid modification (octanoyl ghrelin17) and without acylation (des-acyl ghrelin17)at Ser(3), and examined their biological activity. Octanoyl ghrelin17, but not des-acyl ghrelin17, increased the intracellular Ca2+ concentration in rat GHS-R-expressing cells with a potency similar to those of synthetic ghrelin consisting of 12 residues (octanoyl ghrelin12) and octanoyl rat ghrelin. IP and ICV administration of octanoyl ghrelin17 and octanoyl ghrelin12, but not des-acyl ghrelin17, increased food intake in goldfish. The present findings indicate that native goldfish ghrelin consists of 11 molecular variants, the major form being a 17-residue peptide. This dominant form with acylation is implicated in the regulation of food intake in goldfish. (c) 2008 Elsevier Inc. All rights reserved.