cDNA Cloning and Sequence Analysis of Preproendothelin-1 (PPET-1) from Salmon,Oncorhynchus keta

Introduction

The family of endothelin (ET) ligands comprises ET-1, ET-2/vasoactive intestinal contractor, and ET-3. These ligands are 21-residue peptide hormones with varied and important roles in the regulation of mammalian physiological processes (1–12). ET-like immunoreactivity has been observed not only in mammals but also in fish (13, 14). The biological actions of exogenously administered mammalian ET-1 peptide are similar in fish and mammals (15, 16). Recently, trout (Oncorhynchus mykiss) ET-1 peptide was purified and its myotropic activity analyzed (17). Zebrafish (Danio rerio) ET-1 was identified as a morphogen that is expressed ventrally in the pharyngeal arches and required for correct patterning of pharyngeal cartilage development (18).

In the present article, the rapid amplification of cDNA ends (RACE) method (10) was used to screen ET-related peptides from specimens of salmon stomach. The cloned cDNA sequence and deduced amino acid sequence of a salmon precursor of the ET-related peptide were analyzed.

Materials and Methods

Reagents and software used are the same as those in the report by Quan et al. (10). The stomach was excised from a fresh-caught salmon preserved on ice. Isolation of mRNA, cDNA amplification, subcloning of RACE-PCR products, sequencing of the cloned products, and sequence analysis were performed as previously described (10). Two kinds of guess PCR primers, 5′-GSP (5′-GGBGTRTTGACCCA-GATKATRTCC-3′, where B = G, T, or C; R = A or G; and K = G or T) and 5′-NGSP (5′-GATGATRTCCARGTGR-CAGAART-3′), which were designed on the basis of the conserved region in the mature peptide (10), were used in 5′-RACE PCR. For 3′-RACE PCR, 3′-GSP (5′- CCTATGGGAAAAGAAACAGCGGTA-3′) and 3′-NGSP (5′-CAGTGCGCCACATCAGGAAC-3′), which were designed on the basis of the sequences of the 5′-RACE products shown in Figure 1, were used. Several clones of 5′-and 3′- RACE products were obtained, and the resulting sequences were the same as an identical cDNA shown in Figure 1.

Comparison of Salmon PPET-1 Sequence with Zebrafish, Frog, Mouse, and Human PPET-1 Sequences.

Salmon PPET-1 is the first ET-1 precursor sequence identified in Salmoniformes, Teleostei. Deduced amino acid sequences of salmon PPET-1 and zebrafish PPET-1 (18) are aligned in Figure 2. The salmon ET-1 (C⁴⁹–W⁶⁹) and zebrafish ET-1 sequences are different; one of 21 amino acid differs between the two sequences. Salmon big ET-1 (C⁴⁹–M⁸⁷) has a high homology with zebrafish big ET-1, with 36 of 39 amino acids identical (Figs. 2 and 3). The salmon PPET-1 sequence has low identity with zebrafish (96 [39%] of 244 amino acids; Fig. 2) (18), frog (Xenopus laevis) (24%) (10), mouse (29%) (4), and human (26%) (20) PPET-1 sequences. The dibasic amino acid pairs flanking the 5′- and 3′- regions of salmon big ET-1 (K⁴⁷–R⁴⁸ and K⁸⁸–R⁸⁹) are identical to those in other PPET-1s, indicating that the generation of big ET-1 is conserved from fish to mammal (Fig. 3). Further studies based on this cDNA sequence, including expression and bioactivity experiments, are needed to better understand the biological characteristics of the ET-1 peptide in salmon.