Aestivation Induces Changes in the mRNA Expression Levels and Protein Abundance of Two Isoforms of Urea Transporters in the Gills of the African Lungfish, Protopterus annectens

Abstract

The African lungfish, Protopterus annectens, is ammonotelic in water despite being ureogenic. When it aestivates in mucus cocoon on land, ammonia is detoxified to urea. During the maintenance phase of aestivation, urea accumulates in the body, which is subsequently excreted upon arousal. Urea excretion involves urea transporters (UT/Ut). This study aimed to clone and sequence the ut isoforms from the gills of P. annectens, and to test the hypothesis that the mRNA and/or protein expression levels of ut/Ut isoforms could vary in the gills of P. annectens during the induction, maintenance, and arousal phases of aestivation. Two isoforms of ut, ut-a2a and ut-a2b, were obtained from the gills of P. annectens. ut-a2a consisted of 1227 bp and coded for 408 amino acids with an estimated molecular mass of 44.7 kDa, while ut-a2b consisted of 1392 bp and coded for 464 amino acids with an estimated molecular mass of 51.2 kDa. Ut-a2a and Ut-a2b of P. annectens had a closer phylogenetic relationship with Ut/UT of tetrapods than Ut of fishes. While the mRNA expression pattern of ut-a2a and ut-a2b across various tissues of P. annectens differed, the transcript levels of ut-a2a and ut-a2b in the gills were comparable, indicating that they might be equally important for branchial urea excretion during the initial arousal phase of aestivation. During the maintenance phase of aestivation, the transcript level of ut-a2a increased significantly, but the protein abundance of Ut-a2a remained unchanged in the gills of P. annectens. This could be an adaptive feature to prepare for an increase in the production of Ut-a2a upon arousal. Indeed, arousal led to a significant increase in the branchial Ut-a2a protein abundance. Although the transcript level of ut-a2b remained unchanged, there were significant increases in the protein abundance of Ut-a2b in the gills of P. annectens throughout the three phases of aestivation. The increase in the protein abundance of Ut-a2b during the maintenance phase could also be an adaptive feature to prepare for efficient urea excretion when water becomes available.

Keywords: Ut-a2a; Ut-a2b; ammonia; nitrogenous waste; urea excretion.

Figure 1

A multiple amino acid alignment of the isoforms of urea transporter (Ut-a2a and Ut-a2b) from Protopterus annectens with Latimeria chalumnae Ut-a2 (XP_006007026.1), Squalus acanthias Ut-a2 (AAF66072.1), Opsanus beta Ut-a2 (AAD53268.2), Rhinella marina Ut-a2 (BAE16706.1), Homo sapiens UT-A2 (CAA65657.1) and UT-B1 (CAB60834.1), and Desulfovibrio vulgaris Ut (Q72CX3). Identical amino acid residues are indicated by asterisks, strongly similar amino acids are indicated by colons and weakly similar amino acids are indicated by periods. The conserved LPXXTXPF motifs are underlined. The potential urea binding sites are indicated by open triangles. The dotted box denotes the UT-B signature ALE domain. Potential N-glycosylation and phosphorylation sites are indicated by open and shaded arrows, respectively. The predicted transmembrane domains (TM1-10) of Ut-a2a and Ut-a2b of P. annectens are indicated by open boxes and were predicted using MEMSATS and MEMSAT-SVA provided by PSIPRED protein structure prediction server.

Figure 2

A dendrogram of urea transporters (Ut/UT) including Ut-a2a and Ut-a2b of Protopterus annectens. Strongylocentrotus purpuratus Ut is used as outgroup for the dendrogram. Numbers presented at each branch point represent bootstrap values from 100 replicates.

Figure 3

Gene expression of (A) urea transporter a2a (ut-a2a) and (B) urea transporter a2b (ut-a2b) in the eye (E), brain (Br), gills (Gi), heart (H), liver (Li), spleen (Sp), pancreas (P), gut (Gu), kidney (K), Lung (Lu), muscle (M), and skin (Sk) of P. annectens kept in fresh water.

Figure 4

Absolute quantification of mRNA (×10² copies of transcripts per ng total RNA) of urea transporter a2a (ut-a2a) in the gills of Protopterus annectens kept in fresh water on day 0 (FW; control), after 6 days (d; induction phase), or 6 months (mon; maintenance phase) of aestivation, or after 1 or 3 d of arousal (Ar) from 6 mon of aestivation. Results represent means ± S.E.M (N = 4). Means not sharing the same letter are significantly different (P < 0.05).

Figure 5

Absolute quantification of mRNA (×10² copies of transcripts per ng total RNA) of urea transporter a2b (ut-a2b) in the gills of Protopterus annectens kept in fresh water on day 0 (FW; control), after 6 days (d; induction phase) or 6 months (mon; maintenance phase) of aestivation, or after 1 or 3 d of arousal (Ar) from 6 mon of aestivation. Results represent means ± S.E.M (N = 4).

Figure 6

Protein abundance of urea transporter a2a (Ut-a2a) in the gills of Protopterus annectens kept in fresh water on day 0 (FW; control), after 6 days (d; induction phase) or 6 months (mon; maintenance phase) of aestivation, or after 1 or 3 d of arousal (Ar) from 6 mon of aestivation. (A) An example of immunoblot of Ut-a2a (left) and Ut-a2a pre-incubated with immunising peptide for the peptide competition assay (PCA; right). (B) The protein abundance of Ut-a2a expressed as arbitrary densitometric units per 200 μg protein. Results represent mean ± S.E.M. (N = 3). Means not sharing the same letter are significantly different (P < 0.05).

Figure 7

Protein abundance of urea transporter a2b (Ut-a2b) in the gills of Protopterus annectens kept in fresh water on day 0 (FW; control), after 6 days (d; induction phase) or 6 months (mon; maintenance phase) of aestivation, or after 1 or 3 d of arousal (Ar) from 6 mon of aestivation. (A) An example of immunoblot of Ut-a2b (left) and Ut-a2b pre-incubated with immunising peptide for the peptide competition assay (PCA; right). (B) The protein abundance of Ut-a2b expressed as arbitrary densitometric units per 200 μg protein. Results represent mean ± S.E.M. (N = 3). Means not sharing the same letter are significantly different (P < 0.05).