Molecular characterization of the skate peripherin/rds gene: relationship to its orthologues and paralogues

Abstract

Purpose: A great deal of information about functionally significant domains of a protein may be obtained by comparison of primary sequences of gene homologues over a broad phylogenetic base. This study was designed to identify evolutionarily conserved domains of the photoreceptor disc membrane protein peripherin/rds by analysis of the homologue in a primitive vertebrate, the skate.

Methods: A skate retinal cDNA library was screened using a mouse peripherin/rds clone. The 5' and 3' untranslated regions of the skate peripherin/rds (srds) cDNA were isolated by the rapid amplification of cDNA ends (RACE) approach. The gene structure was characterized by PCR amplification and sequencing of genomic fragments. Northern and Western blot analyses were used to identify srds transcript and protein, respectively.

Results: A new homologue of peripherin/rds was identified from the skate retinal cDNA library. SRDS is a glycoprotein with a predicted molecular mass of 40.2 kDa. The srds gene consists of two exons and one small intron and transcribes into a single 6-kb message. Phylogenetic analysis places SRDS at the base of peripherin/rds family and near the division of that group and the branch leading to rds-like and rom-1 genes. SRDS protein is 54.5% identical with peripherin/rds across species. Identity is significantly higher (73%) in the intradiscal domains. Sequence comparison revealed the conservation of all residues that have been shown, on mutation, to associate with retinitis pigmentosa and showed conservation of most residues associated with macular dystrophies. Comparison with ROM-1 and other rds-like proteins revealed the presence of a highly conserved domain in the large intradiscal loop.

Conclusions: Srds represents the skate orthologue of mammalian peripherin/rds genes. Conservation of most of the residues associated with human retinal diseases indicates that these residues serve important functional roles. The high degree of conservation of a short stretch within the large intradiscal loop also suggests an important function for this domain.

FIGURE 1

Presence of a peripherin/rds-like protein in the skate retina. (A) Western blot analysis of peripherin/rds from skate, mouse, rat, and cow retinas was performed with the rds-D₂ Ab. Note that the peripherin/rds detected in skate retina migrates at a position of 42 kDa, higher than the protein homologues detected in mouse, rat, and cow (approximately 36 kDa). (B) Enzymatic deglycosylation of peripherin/rds. Retinal extracts from skate and mouse were digested with endoglycosidase F (EF) before SDS-PAGE separation and Western blot analysis. Note that after deglycosylation, the migration of peripherin/rds in skate retina shifted to a position similar to that of the mouse homologue, suggesting a similar core polypeptide size for peripherin/rds in mouse and skate retinas.

FIGURE 2

Peripherin/rds transcript in skate retina. Northern blot analysis of total RNA isolated from retinas of skate, mouse, rat, and cow and from bovine pigment epithelium was hybridized with a bovine peripherin/rds cDNA probe. Each lane contained 5 µg of total RNA. More than one transcript was observed in all the species studied except for the skate, in which a single band of ~6 kb was strongly labeled with the probe. No signal was detected in RNA isolated from the bovine pigment epithelium. RNA size standards are indicated to the left.

FIGURE 3

Isolation and sequence analysis of the srds cDNA clones. (A) Positional comparison of the srds cDNA clones to the mouse cDNA sequence. The three groups of the original 17 clones and the two RACE clones are shown. Filled boxes: translated sequences; horizontal lines: untranslated sequences. (B) Compiled nucleotide and predicted amino acid sequence of the srds cDNA. The four predicted transmembrane domains are highlighted with shaded boxes in the polypeptide sequence. Arrow: the putative transcription start point (Tsp) used as +1. Open circles: conserved cysteine residues within the second intradiscal loop, which are predicted to be involved in the secondary structure of peripherin/rds and its association with other proteins, are present in the skate homologue. The conserved site for N-linked glycosylation (N229) is located in the second intradiscal loop (shaded circle). Right: names of the primers used; arrows: direction of the primer. Sequence of the primers is marked by a solid line above the nucleotide sequence. Solid- and dotted-line boxes: potential polyadenylation signals. The srds cDNA sequence has been entered into the GenBank database under accession number AF162436.

FIGURE 4

Organization of the srds gene. (A) Map shows the introns in the mouse peripherin/rds cDNA and the positions of the primers used to identify the presence of introns in the skate homologue. (B) PCR amplification and analysis of DNA fragments from skate genomic DNA reveals the presence of a 208-bp intervening sequence in fragments amplified by primer pairs 160/189 and 188/189, but not in the fragment amplified by primer pair 188/161. (C) Sequence of the intron-exon junctions of the srds gene. Uppercase letters: exon sequences; lowercase letters: intron sequences.

FIGURE 5

Phylogenetic analysis of the peripherin/rds and ROM-1 family of proteins. The tree was constructed using a neighbor-joining analysis of the amino acid sequence of all known members of the family. Bootstrapping was performed to estimate support for each node of the tree. Bootstrap values shown at the nodes represent the percentage of 1000 bootstrap replicates in which the node occurred. Values below 50% are not included.

FIGURE 6

Conservation of residues associated with human retinal diseases. The protein sequences from nine known peripherin/rds homologues are aligned. For clarity, the full amino acid sequences of the nonskate proteins have not been shown, with the exception of residues that align with positions of the human peripherin/rds that are mutated in certain retinal degenerative diseases. (⋆) Residues identical in all homologues with those of SRDS; (…) conserved residues. The unmarked residues are not conserved. Boxes: the putative positions of transmembrane domains. Shaded residues in all nine homologues are those associated with RP; unshaded residues are associated with macular dystrophies in humans. Vertical arrows: mutations that cause variable phenotypes among family members. Mutations reported here are base substitutions causing missense mutations or in-frame deletions. Shaded circle: N229 potential glycosylation site. Underscored sequences: a highly conserved region in the large intradiscal loop between the third and fourth transmembrane domains and a region of the C terminus that has been found to have membrane fusogenic properties in mammals.