Unique mitochondrial localization of arginase 1 and 2 in hepatocytes of air-breathing walking catfish, Clarias batrachus and their differential expression patterns under hyper-ammonia stress

Bodhisattwa Banerjee, Debaprasad Koner, Priyanka Lal, Nirmalendu Saha

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14 Scopus citations

Abstract

Arginase (ARG) catalyzes the final step of ornithine-urea cycle (OUC) leading to a conversion of L-arginine to L-ornithine and urea. Several isoforms of ARG have been reported in vertebrates, out of which the two predominant isoforms are the cytosolic ARG1 and the mitochondrial ARG2. The air-breathing walking catfish (Clarias batrachus) is frequently being challenged by different environmental insults such as hyper-ammonia, dehydration and osmotic stresses in their natural habitats throughout the year. The present study investigated the active presence of ARG1 and ARG2 isoforms in hepatocytes along with unique localization of both the isoforms inside the mitochondria, and also their specific expression patterns under hyper-ammonia stress (5 mM NH4Cl) in isolated hepatocytes of walking catfish. Initially, full length sequences of both arg1 and arg2 genes were obtained by RACE-PCR. Studies on molecular characterization demonstrated the presence of all the conserved amino acids required for stability and activity of binuclear metal center in both the isoforms. Phylogenetic analysis of the amino acid sequences of ARG isoforms showed a differentiation of the ARG1 and ARG2 into two distinct clusters with their respective isoforms from other species. Most interestingly, both the isoforms of ARG in hepatocytes were found to be localized inside the mitochondria as evidenced by the presence of mitochondrial target peptide (mTP) in N-terminal of the derived amino acid sequences, and exclusive localization of ARG activity in the mitochondrial fraction. This was additionally confirmed by Western blot analysis of ARGs in mitochondrial and cytosolic fractions, and by immunocytochemical analysis in isolated hepatocytes. Although the possible reasons associated with the presence of both the isoforms of ARGs inside the mitochondria is not clearly understood, perhaps this mitochondrial localization of ARG is functionally advantageous in this catfish for the synthesis of N-acetyl-l-glutamate, the allosteric regulator for the first OUC enzyme, the carbamoyl phosphate synthetase III, and for supplying ornithine required for citrulline synthesis intramitochondrially. Furthermore, the ammonia stress, due to exposure to high external ammonia, led to greater synthesis of urea-N probably as a consequence of induction of ureogenesis, as evidenced by a larger accumulation of urea-N in hepatocytes and higher secretion in culture media parallel to the increased concentration of ammonia-N in hepatocytes. Ammonia stress also led to specific coordinated patterns of induction of both the arg genes in isolated hepatocytes of walking catfish.

Original languageEnglish
Pages (from-to)13-22
Number of pages10
JournalGene
Volume622
DOIs
StatePublished - 30 Jul 2017
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2017 Elsevier B.V.

Funding

This study was supported by a project sanctioned to NS by the SERB-DST (SR/SO/AS-88/2013), New Delhi, DBT (BT/152/NE/TBP/2011), New Delhi, the DSA program to the Department of Zoology (F.4-1/2011/SAP-II) and the UPE-Bioscience project to the North-Eastern Hill University, Shillong by the University Grants Commission (F.4-11/2008/UPE-I), New Delhi.

FundersFunder number
SERB-DSTSR/SO/AS-88/2013
Department of Biotechnology, Ministry of Science and Technology, IndiaF.4-1/2011/SAP-II, BT/152/NE/TBP/2011
University Grants CommissionF.4-11/2008/UPE-I

    Keywords

    • Arginase isoforms
    • Clarias batrachus
    • Hyper-ammonia stress
    • Mitochondrial localization
    • Primary hepatocytes

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