Cloning of two LIMCH1 isoforms: characterization of their distribution in rat brain and their agmatinase activity

Abstract

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.

Reduced bioavailable manganese causes striatal urea cycle pathology in Huntington's disease mouse model

“…The level of ARG2 protein was almost as high in hippocampus as in striatum, and ARG2 was significantly increased by Mn exposure across all brain regions where it was detected. AGMAT was undetectable in these mouse brain tissues, and LIMCH1 was unchanged by Mn-exposure and genotype, corroborating findings on ALP [21] (Fig. 2, C, Supplemental Fig.…”

Metabolic strategies for the degradation of the neuromodulator agmatine in mammals

“…The ability of ALP to generate putrescine ( Fig. 1) for polyamine synthesis, has also been demonstrated using the same complementation assay that was employed to demonstrate biological activity of mammalian AGMs in a yeast strain (see above and compare Figs 3 and 4) [72,79,81].…”

“…Recently, a novel enzyme with AGM activity was cloned from a rat brain cDNA library, the AGM-Like Protein (ALP) [78][79][80][81][82]. However, its amino acid sequence bears no resemblance to known AGMs (from any source) nor to other ureohydrolases; no motif that may define the active site of this enzyme has yet been identified [69].…”

“…Using a polyclonal antibody raised against ALP was shown to be localized mainly in astrocytes and neurons in rat hypothalamus and hippocampus [80]. Recombinant ALP expressed in E. coli and purified, is the first (and to date only) mammalian enzyme with in vitro AGM activity (k cat ~1.0 s -1 ; K m ~3.0 mM for agmatine [78,79]. The ability of ALP to generate putrescine ( Fig.…”

“…LIMCH1 isoforms I (3918 bp) and II (2871 bp); their 3'-regions are identical to that of their shorter counterpart [79]. Interestingly, in isoform I the N-terminal region is characteristic of a calponin homology (CH) domain.…”