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    • Previous reports have implicated the guanylin GC

    2022-07-01

    Previous reports have implicated the guanylin/GC-C signalling system as being important in osmoregulation in fish, especially in euryhaline species such as the eel which migrate between freshwater (FW) and seawater (SW) environments at different stages of their life cycle (Comrie et al., 2001a, Comrie et al., 2001b, Yuge et al., 2003, Yuge et al., 2006, Cramb et al., 2005). Juvenile, sexually immature “yellow” eels can live in a variety of inland FW habitats for up to 30 years or more before metamorphosing into sexually maturing adult “silver” eels prior to the migration back to the Sargasso sea to breed. Although yellow eels are capable of surviving in SW, it is possible that certain morphological and physiological changes that accompany this FW metamorphosis are pre-adaptive responses, which optimise the osmoregulatory capacity of fish after entering the SW environment. In this study, Northern blotting and quantitative real-time polymerase chain reaction (qRT-PCR) techniques were employed to investigate the intestinal and renal expression of guanylin, uroguanylin and renoguanylin peptides and GC-C1 and GC-C2 receptor mRNAs in both yellow and migratory silver eels following transfer from FW to SW. The experiments were designed to ask the question, is the metamorphic silvering event, or the subsequent transfer from FW to SW, associated with changes in the expression of any components of the guanylin peptide signalling system within the European eel (Anguilla anguilla)?
    Materials and methods
    Results
    Discussion RT-PCR and cloning techniques have shown that a guanylin peptide–guanylate cyclase signalling system is expressed within the intestine and kidney of the European eel. Three peptides, guanylin, uroguanylin and the eel-specific peptide renoguanylin are expressed along with two guanylate cyclase type C isoforms, designated GC-C1 and GC-C2. In terms of the peptides, the pre-prouroguanylin sequence was the most divergent sharing only 38.7% and 43.1% amino Zoledronic Acid identities with pre-proguanylin and pre-prorenoguanylin, respectively, with the latter two exhibiting the highest homology level of 56.6%. This suggests that the initial duplication event common to all vertebrates resulted in the generation of the pre-prouroguanylin gene and a common ancestral gene for the pre-proguanylin and pre-prorenoguanylin genes. Molecular phylogenetic analysis also supports this theory (Takei and Yuge, 2007). The subsequent genome duplication event isolated to the teleost fish lineage is likely to have been responsible for the genesis of the guanylin–renoguanylin genes with the uroguanylin duplicate either still to be identified, or more likely, lost due to subsequent mutations or genome rearrangements. The greatest homology between the eel pre-prohormones and also with sequences from other fish, amphibians and mammals is found within the active C-terminal peptide region where there are distinct differences between guanylin and uroguanylin peptides from all species (Forte, 2004). The active guanylin peptides are normally 15 amino acids in length but the active peptide region for uroguanylins is usually extended at the C-terminal by one or two additional amino acids, although the C-terminal extension in the catfish produces a peptide of 20 amino acids in length (Forte, 2004, Cramb et al., 2005, Takei and Yuge, 2007). The addition of amino acids to the C-terminal of uroguanylins may serve to stabilise their active conformation after cleavage from the prohormone (Schulz et al., 2005). Within the active C-terminal region of the peptides, at position nine in the amino acid sequence, guanylin peptides have a hydrophobic phenylalanine (F) or tyrosine (Y) residue while all uroguanylin peptides possess an asparagine (N) residue. In mammals, it is thought that this amino acid substitution makes uroguanylin peptides more resistant to inactivation by extracellular proteases with chymotrypsin-like activity (Hamra et al., 1996, Forte, 2003, Sindic and Schlatter, 2005). These proteases, found at high concentrations in the renal tubules (Carvalho et al., 2008), hydrolyse peptide bonds on the C-terminal side of aromatic residues such as phenylalanine or tyrosine, as present in guanylin. These structural differences within the active peptide region of guanylin and uroguanylin peptides are postulated to be functionally significant in mammals, ensuring that systemic uroguanylin and not guanylin, which is hydrolysed and inactivated by renal proteases, can bind to the apically located GC-C receptor of renal tubular epithelial cells and stimulate fluid secretion. As these structural differences are retained in fish, it is likely that the functional differences reported in mammals may also be operative in the eel. Although the highest level of amino acid homology between the guanylin-like peptides occurs at the carboxy terminal there are a few amino acids that are conserved throughout all prohormones from all species. The N-terminus of the pre-prohormone of opossum guanylin was sequenced by Hamra et al. (1996) and this study identified a cleavage site (between residue 26, serine and amino acid 27, valine) for post-translational processing to produce a mature prohormone. Similar cleavage sites between serine and valine or glycine and valine residues have been proposed for mammalian guanylin and uroguanylin prohormones (Miyazato et al., 1996, Forte et al., 2000). In all of the teleost pre-proguanylins and pre-prouroguanylins sequenced to date, a valine residue at position 27 has been conserved (unpublished observation). This suggests that like the peptide prohormones in mammals, the signal sequence cleavage site is also at this position in teleosts. In addition to the four cysteine residues within the active C-terminal peptide region of the prohormone, two additional cysteine residues (at aa.s 64 and 77 in Fig. 1) are conserved in the prohormone sequences published for all species. It has been reported that these two cysteine residues form a third disulphide bond, stabilising the prohormone structure and allowing the well characterised disulphide bonds which are formed within the active peptide to adopt the correct conformation (Lauber et al., 2004). Also conserved within the sequences of all prohormones from all species are two proline residues (at aa.s 70 and 75 in Fig. 1). Together with the cysteine residues, these four conserved amino acids within the prohormone sequence may be important for the correct folding of the prohormone especially as proline residues are thought to cause turns or “kinks” in amino acid chain.