Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, Protopterus annectens, during aestivation or exposure to ammonia

Abstract

This study aimed to obtain the full sequence of carbamoyl phosphate synthetase III (cps III) from, and to determine the mRNA expression of cps III in, the liver of P. annectens during aestivation in air, hypoxia or mud, or exposure to environmental ammonia (100 mmol l(-1) NH(4)Cl). The complete coding cDNA sequence of cps III from the liver of P. annectens consisted of 4530 bp, which coded for 1,510 amino acids with an estimated molecular mass of 166.1 kDa. The Cps III of P. annectens consisted of a mitochondrial targeting sequence of 44 amino acid residues, a GAT domain spanning from tyrosine 45 to isoleucine 414, and a methylglyoxal synthase-like domain spanning from valine 433 to arginine 1513. Two cysteine residues (cysteine 1337 and cysteine 1347) that are characteristic of N-acetylglutamate dependency were also present. The critical Cys-His-Glu catalytic triad (cysteine 301, histidine 385 and glutamate 387) together with methionine 302 and glutamine 305 affirmed that P. annectens expressed Cps III and not Cps I. A comparison of the translated amino acid sequence of Cps III from P. annectens with CPS sequences from other animals revealed that it shared the highest similarity with elasmobranch Cps III. A phylogenetic analysis indicates that P. annectens CPS III could have evolved from Cps III of elasmobranchs. Indeed, Cps III from P. annectens used mainly glutamine as the substrate, and its activity decreased significantly when glutamine and ammonia were included together in the assay system. There were significant increases (9- to 12-fold) in the mRNA expression of cps III in the liver of fish during the induction phase (days 3 and 6) of aestivation in air. Aestivation in hypoxia or in mud had a delayed effect on the increase in the mRNA expression of cps III, which extended beyond the induction phase of aestivation, reiterating the importance of differentiating effects that are intrinsic to aestivation from those intrinsic to hypoxia. Furthermore, results from this study confirmed that environmental ammonia exposure led to a significant increase in the mRNA expression of cps III in the liver of P. annectens, alluding to the important functional role of urea not only as a product of ammonia detoxification but also as a putative internal cue for aestivation.

Aestivation induces changes in transcription and translation of coagulation factor II and fibrinogen gamma chain in the liver of the African lungfish,Protopterus annectens

“…As lungfishes are the closest living sister group of land vertebrates, they would logically possess some genes/proteins that are closer to those of other fishes (e.g. carbamoyl phosphate synthetase III, Loong et al, 2012a;argininosuccinate synthase, Chng et al, 2014) and others that have greater similarity to those of tetrapods (e.g. F2 and Fgg, this study; argininosuccinate lyase, Chng et al, 2014; Na + /K + -ATPase α-subunit isoforms, Hiong et al, 2014;L-gulono-γ-lactone oxidase, Ching et al, 2014;betaine-homocysteine Smethyltransferase, Ong et al, 2015).…”

Excretory nitrogen metabolism and defence against ammonia toxicity in air‐breathing fishes

“…aethiopicus and P . dolloi possess carbamoyl phosphate synthetase III instead (Chew et al , 2003 a ; Loong et al , 2005, 2012 b , c ), and that the transition of carbamoyl phosphate synthetase III to carbamoyl phosphate synthetase I occurred among amphibians (Ip et al , 2012 c ). Despite being ammonotelic in water, P .…”

“…Indeed, results from a subsequent study confirmed that environmental ammonia exposure led to a significant increase in the mRNA expression of cps III in the liver of P . annectens , alluding to the important functional role of urea not only as a product of ammonia detoxification but also as an internal signal in the induction of aestivation (Loong et al , 2012 b ).…”

Differential Gene Expression in the Brain of the African Lungfish, Protopterus annectens, after Six Days or Six Months of Aestivation in Air

“…Among the four African species, P. annectens is known to be the most dependent on aestivation; it normally aestivates for periods of 7–8 months in the wild, and a captive lungfish has emerged from its cocoon after periods of up to seven years [3], [4], [5], [6]. Recently, it has been reported that African lungfishes can be induced to aestivate in completely dried mucus cocoon in plastic boxes or in mud cocoon in the laboratory [7], [8], [9], [10], [11], [12].…”

“…Only until very recently, did Loong et al [11], [12] report on the differential gene expression, and the up-regulation of mRNA expression of carbamoyl phosphate synthetase III and ornithine-urea cycle capacity, in the liver of P. annectens during the induction phase (the first 6 days) of aestivation in air. More importantly, there are very few recent studies [13], [14] on the brain of aestivating African lungfish in spite of its possible role in coordinating a whole-body aestivation-specific response during the induction phase of aestivation.…”