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First published online November 2, 2007
Journal of Experimental Biology 210, 3883-3896 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.007898
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Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspects

Ivar Rønnestad1,*, Paulo J. Gavaia2, Carla S. B. Viegas2, Tiziano Verri3, Alessandro Romano3, Tom Ole Nilsen1, Ann-Elise O. Jordal1, Yuko Kamisaka1 and M. Leonor Cancela2

1 University of Bergen, Department of Biology, N-5020 Bergen, Norway
2 University of Algarve –CCMAR, Campus de Gambelas, 8005-139 Faro, Portugal
3 University of Salento (formerly University of Lecce), Department of Biological and Environmental Sciences and Technologies, I-73100 Lecce, Italy


Figure 1
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  		Fig. 1. Nucleotide and predicted amino acid sequence of Atlantic cod PepT1 (codPepT1). The numbers on the right refer to positions of the nucleotides (lower row) and amino acids (upper row). The stop codon is indicated by ***. A polyadenylation signal is underlined. Positions for degenerated (broken underline) and species specific (solid underline and double underline) primers are indicated in the nucleotide sequence. In the amino acid sequence, putative transmembrane domains are underlined in red and named I to XII. Potential extracellular N-glycosylation sites (dark green boxed areas) and potential cAMP/cGMP-dependent protein kinase phosphorylation sites at the cytoplasmic surface (light grey boxed areas) are indicated.

 

Figure 2
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Figure 2
Figure 2
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  		Fig. 2. Amino acid alignment of human, macaque, rat, mouse, dog, rabbit, sheep, cow, pig, chicken, turkey, zebrafish and Atlantic cod PepT1. Multiple sequence alignment was generated using Clustal W 1.82 using the tool at the www.ebi.ac.uk/clustalw web site with the following (default) parameters: Matrix=Gonnet 250, Gap Open=10, End Gaps=–1, Gap Extension=0.2, Gap Distances=4. Putative transmembrane segments (Ia to XII) were predicted using the TMHMM 2.0 program (TMHMM Server v. 2.0 at http://www.cbs.dtu.dk/services/TMHMM-2.0/), which is part of the Simple Modular Architecture Research Tool (SMART; at http://www.expasy.org/prosite/), and are indicated by grey-boxed double-headed broken arrows (with darker grey designating the core stretch of amino acids within each putative TMHMM-predicted transmembrane segment that is shared by at least two of the aligned sequences). Within each sequence, the topological location of each TMHMM2-predicted transmembrane helix is specifically underlined. Potential phosphorylation (solid triangle, protein kinase C; solid circle, protein kinase A) and N-glycosylation (Y) sites are indicated and correspondently highlighted in dark grey, light grey and dark green, respectively, along the sequences where found. The proposed `PTR2 family proton/oligopeptide symporters signature 1' motif (PROSITE pattern: PS01022; amino acid residues 77–101 in Atlantic cod PepT1) and `PTR2 family proton/oligopeptide symporters signature 2' motif (PROSITE pattern: PS01023; amino acid residues 170–182 in Atlantic cod PepT1) are highlighted in black. Individual amino acid residues identified by site-directed mutagenesis in PepT1 proteins from various mammalian species and found to be either involved in transport activity or responsible for incorrect synthesis and/or transport of the protein to the plasma membrane are indicated in red (for details, see Table 1).

 

Figure 3
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  		Fig. 3. Unrooted phylogenetic tree depicting the evolutionary relationship of vertebrate PepT1 transporters. The unrooted tree was constructed using the neighbor-joining (NJ) method (Saitou and Nei, 1987Go) based on the alignment of the complete amino acid sequences of known vertebrate PepT1 transporters. Bootstrap values (1000 replicates) indicating the occurrence of nodes are reported above each branch in the figure.

 

Figure 4
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  		Fig. 4. Tissue distribution of Atlantic cod PepT1 mRNA in adult fish. RT-PCR performed on equal amounts of total RNA (1.5 µg) isolated from adult fish tissues using Atlantic cod PepT1-(top row) and EF1{alpha}-specific (bottom row) primers. Control, no RNA (H2O instead) in RT (negative control). DNA markers indicate 10 kb SmartLadder (Eurogentec, Seraing, Belgium).

 

Figure 5
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  		Fig. 5. Spatial distribution of Atlantic cod PepT1 mRNA in the digestive tract of adult fish. (A) A representative picture of Atlantic cod digestive tract. 1, stomach; 2, pyloric area; 3, pyloric caeca (inner segments); 4, pyloric caeca (outer segments); 5–10, six adjacent intestinal segments in the remainder of the digestive tract, starting after the pyloric area (segment 5) and ending with the anus (segment 10), and based on divisions of the three loops present in the dissected gut. The last segment (e.g. segment 10) also comprises the hindgut. Arrows indicate the incisions between the segments. (B) RT-PCR performed on equal amounts of total RNA (1.5 µg) isolated from the different segments of the digestive tract of Atlantic cod using PepT1-(top row) and EF1{alpha}-specific (bottom row) primers. Progressive numbers (1 to 10) indicate the segments of the digestive tract indicated in A. Control (C), no RNA (H2O instead) in RT (negative control); M, DNA markers (10 kb SmartLadder; Eurogentec).

 

Figure 6
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  		Fig. 6. PepT1 gene expression on neighbor sections of Atlantic cod intestine by in situ hybridization. (A) The sense probe showed no staining (negative control). (B) The antisense probe revealed mRNA expression in the epithelial layer of the intestine. (C) Epithelium from boxed area in B enlarged. Scale bars, 100 µm (A,B), 20 µm (C).

 




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