Elsevier

Gene

Volume 314, 18 September 2003, Pages 89-102
Gene

Shadoo, a new protein highly conserved from fish to mammals and with similarity to prion protein

https://doi.org/10.1016/S0378-1119(03)00707-8Get rights and content

Abstract

We report evidence from cDNA isolation and expression analysis as well as analyses of genome, expressed sequence tag (EST), cDNA and expression databases for a new gene named SPRN (shadow of prion protein). SPRN comprises two exons, with the open reading frame (ORF) contained within exon 2, and codes for a protein of 130–150 amino acids named Shadoo (Japanese shadow), predicted to be extracellular and GPI-anchored. The SPRN gene was found in fish (zebrafish, Fugu) and mammals (mouse, rat, human). Conservation of order and transcription orientation of two proximal genes between fishes and mammals strongly indicates gene orthology. Sequence comparison shows: a highly conserved N-terminal signal sequence; Arg-rich basic region containing up to six tetrarepeats of consensus XXRG (where X is G, A or S); a hydrophobic region of 20 residues with strong homology to PrP; a less conserved C-terminal domain containing a conserved glycosylation motif; and a C-terminal peptide predicted to be a signal sequence for glycophosphotidylinositol (GPI)-anchor attachment. Fish Shadoos (Sho) show well conserved sequences (identity 54%) over 106 amino acids (zebrafish length), and conservation among the mammalian sequences is very high (identity 81–96%). The fish and mammalian sequences are also well conserved, particularly for zebrafish, to beyond the end of the hydrophobic sequence (identity 41–53%, 78 amino acids, zebrafish length). The overall structure appears closely related to prion proteins (PrPs), although the C-terminal domains of Shos are quite different from those of PrPs, for which conformational changes in mammals are implicated in disease. The structural similarity is particularly interesting given recent reports of three new genes with similarities to PrPs found in Fugu (PrP-like, PrP-461/stPrP-1 and stPrP-2) and other fish, but for which direct evolution to higher vertebrate PrPs is unlikely and for which no other mammalian homologues have been found. Database information indicates expression of SPRN in embryo, brain and retina of mouse and rat, hippocampus of human, and in embryo and retina of zebrafish, and we directly confirmed a strikingly specific expression of the mammalian (human, mouse, rat) transcripts in whole brain. This result together with some common structural features led to the suggestive hypothesis of a possible functional link between mammalian PrP and Sho proteins.

Introduction

Comparative genomics is a powerful method for identification of new genes, allowing initial characterization of their distribution among the kingdoms of life, and tracking of the sequence and regulatory changes underpinning their conserved or diverged functions (Hedges and Kumar, 2002). Application of the method is now enormously facilitated by the ability to rapidly search for and integrate genome, expressed sequence tag (EST), cDNA and expression information from databases; based on analysis of the data, deductions can be made on gene structure, location and regulation, and on translated protein sequences, including cellular location and possible function.

We report an application of this approach which, starting from the initial lead of the sequence of a zebrafish cDNA we isolated, enabled us to discover other homologous proteins in fish and mammals, and to start to characterize the gene and protein using the substantial information which already exists in the databases. Striking sequence similarities of this protein to prion protein (PrP), as well as its expression almost entirely specific to brain, allow the hypothesis that it may be functionally related to PrP and important for understanding prion disease (Prusiner, 1998). We propose to call this new gene SPRN (shadow of PrP) and the protein Shadoo (Japanese shadow).

PrP is the remarkable protein associated with lethal neurodegenerative diseases grouped as transmissible spongiform encephalopathies (Collinge, 2001). Although normally expressed in many tissues other than brain, recent work indicates expression at high levels only in discrete subpopulations of cells, particularly nerve and immune cells of the neuroimmune, neuroendocrine and peripheral nervous systems (Ford et al., 2002). However, its function is still unclear. Although the first cDNA encoding mammalian PrP was cloned and characterized in hamster, and immediately confirmed in mouse and human, almost 20 years ago (Oesch et al., 1985), it was more difficult to find homologues in birds (Harris et al., 1991), reptiles (Simonic et al., 2000) and amphibians (Strumbo et al., 2001). Identification of homologues using conventional experimental methods has been difficult because despite the conservation of all, or almost all, the structural motifs found in mammalian PrPs (Fig. 1), PrPs from different vertebrate classes exhibit remarkable differences in their primary sequences.

As shown in Fig. 1, the PrP sequence is very unusual in having several regions with distinct amino acid composition, and potential 3-D structure and contributions to function, within a quite short protein of ∼250 residues (mammals) encoded by a single exon (Lee et al., 1998). Both conserved and variable features between amphibians and mammals are apparent. Most notably, PrPs show extremely high conservation in the middle hydrophobic sequence (although this is shorter in frog PrP), in the presence of one disulfide bond and two N-glycosylation sites in the C-terminal domain, in the presence of an N-terminal basic region, and in N- and C-terminal signal sequences for extracellular export and glycophosphotidylinositol (GPI)-anchor attachment. However, the N-terminal repeat region is highly variable in repeat length and sequence among eutherian and marsupial mammal and avian/turtle PrPs Windl et al., 1995, Wopfner et al., 1999, Simonic et al., 2000, and is entirely absent in frog PrP (Strumbo et al., 2001). Solution NMR structures for mammalian PrPs show only the C-terminal domain (from residue ∼126) to be folded, with the whole N-terminal region (23–125) being flexibly disordered (Zahn et al., 2000).

As suggested by the amphibian PrP structure, this protein appears to have gained new structural features during evolution (Fig. 1), and, therefore, potentially new functions. The recent reports of, at least, three new genes in fish with similarities to PrP are, therefore, of great interest. Suzuki et al. (2002) reported isolation and characterization of a cDNA coding for a protein of 180 amino acids in Fugu rubripes. This sequence was called PrP-like, based mainly on conservation of the hydrophobic sequence (albeit four residues shorter), and other features in common with PrP, including its basic nature, N-terminal signal sequence, possible GPI-anchor attachment, and single-exon coding region. However, overall sequence homology between PrPs and Fugu PrP-like (and a similar database sequence for Tetraodon) is low, with the latter lacking repeats, disulfide and glycosylation sites, and having a different C-terminal domain. Nonetheless, orthology was suggested between Fugu PrP-like and human PrP by conservation of adjacent genes (Suzuki et al., 2002). The fish gene region did not, however, contain the Doppel gene PRND, which is reported so far only in mammals and is adjacent to PRNP; PRND and PRNP are regarded as paralogues arising from gene duplication (Moore et al., 1999). Suzuki et al. (2002) also reported a (database) PrP-like sequence for Danio rerio (zebrafish) which although homologous by sequence alignment to Fugu PrP-like (not shown) shows significant divergence (see Fig. 1); as the genomic context of this sequence has yet to be elucidated it is unclear whether the Fugu/Tetraodon and Danio PrP-like genes are true homologues.

More recently, two other genes with similar structural features to PrPs of higher vertebrates (amphibians to mammals) have been reported in fish Rivera-Milla et al., 2003, Oidtmann et al., 2003. The cDNA of a F. rubripes gene (Genbank AF531159) reported by Rivera-Milla et al. (2003) encoded a protein of 461 amino acids (PrP-461) with a greatly expanded repeat region, but apparently similar C-terminal domain to those in PrPs, including disulfide bridge and one of the conserved N-glycosylation sites. A homologous F. rubripes cDNA (AY141106) of slightly different length (450 residues; stPrP-1) as well as that for Atlantic salmon Salmo salar (AY141107) with an even more expanded repeat region (605 residues) were obtained by Oidtmann et al. (2003), while the Tetraodon homologue was reported by both groups from genomic data. This stPrP-1 gene in Fugu is at a different genomic location from the PrP-like gene, while the locations of the Tetraodon and salmon genes are undetermined. However, Oidtmann et al. (2003) reported the cDNA for a second Fugu gene, stPrP-2 (AY188583), closely related to stPrP-1 but with a hydrophobic region disrupted by charged residues, which was located adjacent to PrP-like. These findings provide a puzzle as while in terms of structural similarity stPrP-1 is closest to PrPs, in terms of genomic localization stPrP-2 and PrP-like seem more closely linked to PrPs (Oidtmann et al., 2003).

During our own searches for fish PrP candidates in EST databases, we identified an interesting zebrafish sequence fragment, and now report the isolation and cloning of the cDNA. This sequence encodes a novel protein, different from the PrP-like, stPrP-1 and stPrP-2 sequences, but again with intriguing similarities to PrPs, as summarized in Fig. 1. Database searches indicate that this protein sequence is well conserved between fish (zebrafish, Fugu) and mammals (human, mouse, rat). Where available, data for chromosomal location, adjacent genes, gene structure and overall protein structure and sequence all show high homology (see Table 1). Extending expression data already in the databases, we also demonstrate expression of the corresponding mammalian mRNAs, which is almost entirely in brain.

Section snippets

RT-PCR and PCR assays

Oligonucleotides were purchased from MWG-Biotech (Germany). Total RNA was extracted from zebrafish head and hepatopancreas, from mouse and human adult brain and from several rat tissues (Chomczynsky and Sacchi, 1987), genomic DNA from zebrafish hepatopancreas following the standard procedure (Sambrook et al., 1989), and plasmid DNA using QIAprep Spin Miniprep Kit (QIAGEN, Germany). Reverse transcriptions were primed by random primers, anchored oligo-dT primer (AdT) or fish specific primers,

Isolation of Zebrafish Sho protein cDNA and features of the encoded protein

Repeated screenings of EST databases aimed at finding fish sequences coding for proteins with structural motifs peculiar to known PrPs were done. Whole protein and/or selected regions of the amino acid sequences of known PrPs from different vertebrate species were used as protein queries for translated BLAST searches. Results were analyzed by translating the whole cDNA fragments corresponding to promising amino acid sequences exhibiting some similarities to the query. From a number of

Sequence regions

Inspection of Fig. 3 shows that Sho proteins have the following major features:

  • (a)

    An N-terminal peptide sequence (aa 1–24) consistent with an endoplasmic reticulum targeting signal for extracellular export.

  • (b)

    A basic RG-rich region starting from Lys-25 with up to six tetrarepeats of consensus XXRG, where X is G, A or S. For mammals, the pattern is GGRG GARG SARG (G/-)VRG GARG ASRV; this pattern is well conserved in zebrafish but is distorted in Fugu by an insertion of 14 residues in the middle. This

Acknowledgements

JEG and JAMG are supported by the ANU IAS block grant, and TS is supported by EC grant QLK5-2002-00866 and FIRST. We thank DNASTAR for an extended trial of their Lasergene software. The authors thank Dr. G.A. Niemi for providing the zebrafish retina cDNA library.

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