García David, Ordenes Patricio, Benítez José, González Arlette, García-Robles María A, López Vasthi, Carvajal Nelson, Uribe Elena
Departamento de Ciencias Biológicas y Químicas, Facultad de Ciencias, Universidad San Sebastián, Lientur 1457, Concepción, Chile.
Departamento de Biología Celular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
Histochem Cell Biol. 2016 Mar;145(3):305-13. doi: 10.1007/s00418-015-1389-0. Epub 2015 Dec 17.
Agmatine, a precursor for polyamine biosynthesis, is also associated with neurotransmitter, anticonvulsant, antineurotoxic and antidepressant actions in the brain. This molecule results from the decarboxylation of L-arginine by arginine decarboxylase, and it is hydrolyzed to urea and putrescine by agmatinase. Recently, we have described a new protein that also hydrolyzes agmatine, agmatinase-like protein (ALP), which was identified through immunohistochemical analysis in the hypothalamus and hippocampus of rats. However, its sequence differs greatly from all known agmatinases and does not contain the typical Mn(2+) ligands associated with the urea hydrolase family of proteins. ALP has a LIM-like domain close to its carboxyl terminus, and the removal of which results in a truncated variant with a tenfold increased k cat value and a threefold decreased K m value for agmatine. Analysis of the gene database revealed several transcripts, denominated LIMCH1 isoforms, with extreme 3' sequences identical to ALP. Limch1 gene products have been described as members of a multi-domain family of proteins with the biggest isoform containing a calponin homology (CH) domain at its N-terminus. Here, we cloned two LIMCH1 transcripts, one of 3177 bp and the other of 2709 bp (ALP contains 1569 bp) and analyzed LIMCH1 expression and distribution in rat brain using RT-PCR, Western blot and immunohistochemical analyses. LIMCH1 was detected mainly in the hypothalamic and hippocampal regions, which is similar to the distribution of ALP and agmatine in brain. In addition, we cloned and expressed both isoforms in E. coli and confirmed that they were catalytically active on agmatine with kinetic parameters similar to ALP. LIM domain-truncated variants of both isoforms moderately increased the k cat and catalytic efficiency. Thus, we propose that LIMCH1 is useful to regulate the intracellular concentrations of the neurotransmitter/neuromodulator, agmatine.
胍丁胺是多胺生物合成的前体，也与大脑中的神经递质、抗惊厥、抗神经毒性和抗抑郁作用有关。该分子由精氨酸脱羧酶使L-精氨酸脱羧产生，然后被胍丁胺酶水解为尿素和腐胺。最近，我们描述了一种也能水解胍丁胺的新蛋白质，即胍丁胺酶样蛋白（ALP），它是通过免疫组织化学分析在大鼠下丘脑和海马中鉴定出来的。然而，其序列与所有已知的胍丁胺酶有很大差异，并且不包含与尿素水解酶家族蛋白质相关的典型锰（2+）配体。ALP在其羧基末端附近有一个类似LIM的结构域，去除该结构域会产生一个截短变体，其对胍丁胺的催化常数（k cat）值增加了10倍，米氏常数（K m）值降低了3倍。对基因数据库的分析揭示了几种转录本，命名为LIMCH1异构体，其3'端序列与ALP完全相同。Limch1基因产物被描述为一个多结构域蛋白质家族的成员，最大的异构体在其N端含有一个钙调蛋白同源（CH）结构域。在这里，我们克隆了两个LIMCH1转录本，一个3177 bp，另一个2709 bp（ALP含有1569 bp），并使用逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹和免疫组织化学分析来分析LIMCH1在大鼠脑中的表达和分布。LIMCH1主要在下丘脑和海马区域被检测到，这与ALP和胍丁胺在脑中的分布相似。此外，我们在大肠杆菌中克隆并表达了这两种异构体，并证实它们对胍丁胺具有催化活性，其动力学参数与ALP相似。两种异构体的LIM结构域截短变体适度增加了k cat和催化效率。因此，我们认为LIMCH1有助于调节神经递质/神经调质胍丁胺的细胞内浓度。
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