Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential

Abstract

Background: Cone degeneration is the hallmark of the inherited retinal disease retinitis pigmentosa. We have previously identified a trophic factor "Rod-derived Cone Viability Factor (RdCVF) that is secreted by rods and promote cone viability in a mouse model of the disease.

Results: Here we report the bioinformatic identification and the experimental analysis of RdCVF2, a second trophic factor belonging to the Rod-derived Cone Viability Factor family. The mouse RdCVF gene is known to be bifunctional, encoding both a long thioredoxin-like isoform (RdCVF-L) and a short isoform with trophic cone photoreceptor viability activity (RdCVF-S). RdCVF2 shares many similarities with RdCVF in terms of gene structure, expression in a rod-dependent manner and protein 3D structure. Furthermore, like RdCVF, the RdCVF2 short isoform exhibits cone rescue activity that is independent of its putative thiol-oxydoreductase activity.

Conclusion: Taken together, these findings define a new family of bifunctional genes which are: expressed in vertebrate retina, encode trophic cone viability factors, and have major therapeutic potential for human retinal neurodegenerative diseases such as retinitis pigmentosa.

Figure 1

RdCVF and RdCVF2 gene structure conservation. At top, panels a and b are the gene structures for RdCVF and RdCVF2 genes. The RdCVF-L mRNA (NM_145598, mouse chromosome 8, minus strand, from 70'033'763 to 70'027'717) is composed of three exons (1–3) of 348, 687 and 1751 bp. The RdCVF-S mRNA (BC017153, from 70'033'785 to 70'032'615) is composed of one exon (1172 bp). The RdCVF2-L mRNA (AK015847, mouse chromosome 13, plus strand, from 50'202'630 to 50'206'797) is composed of two exons (1–2) of 603 and 564 bp. The RdCVF2-S mRNA (BC016199, from 50'202'667 to 50'205'571) is composed of one exon (2904 bp). Coding and non-coding regions are depicted in dark grey) and light grey respectively. At middle, panels a and b, the genomic region surrounding the stop codon at the end of the first coding exon and the corresponding orthologous sequences in 12 other vertebrate genomes are aligned. The black triangles indicate the end of the first RdCVF(2)-L coding exon. Conserved stop codons are colored in red. At bottom, panels a and b, lengths of the coding (CDS) and terminal untranslated regions (UTR) are given.

Figure 2

Sequence and structure similarities of mouse RdCVF and RdCVF2 proteins with thioredoxin superfamily members. Panel a shows the multiple sequence alignment of the thioredoxin (TXN) tryparedoxin (TRYX-I) nucleoredoxin (NXN) and the existing and predicted RdCVF and RdCVF2 proteins. The name, organism and accession number (in brackets) of each protein sequence are given (left). Identical (white text on black) small (A, D, G, P, S, T ; white text on green) hydrophobic (A, C, F, G, I, L, M, S, T, V, W, Y ; black text on yellow) polar (D, E, H, K, N, Q, R, S ; blue text) and charged (D, E, K, R ; white text on red) conserved residues are shown according to a conservation threshold of 85%. A consensus sequence is given below the multiple alignment in which s, h, p and c correspond to small, hydrophobic, polar and charged residues respectively. The secondary structures (β sheet and α helix) of the Crithidia fasciculata tryparedoxin I structure (1EWX) are given below the consensus sequence. The blue dashed rectangles indicate the three RdCVF(2) specific insertions. The green dashed rectangle shows the "cap" region absent in RdCVF(2)-S. The position of the human thioredoxin cleavage product (TRX80) is indicated (red triangle). Panel b displays the structure of the Crithidia fasciculata TRYX-I (1EWX) (left) mouse RdCVF-L (center) and mouse RdCVF2-L (right) models. Regions of TRYX-I backbone conserved in RdCVF(2)-L are colored in red. The "cap" region and the three specific insertions are depicted in green and blue respectively. The putative catalytic site (C₄₄XXC₄₇) is shown in yellow with a space-filling representation.

Figure 3

Spatial proximity of the three specific insertions and specifically-accessible hydrophobic surface in the RdCVF-S and RdCVF2-S proteins. Panel a displays the space-filling representation of RdCVF-L (left) and RdCVF2-L (right) using the same color code as Figure 2 panel b. Importantly, the RdCVF(2)-L models were rotated as indicated to focus on the three specific insertions. Note that catalytic site and "cap" region are on opposite ends of the protein. Panel b shows the RdCVF-S and RdCVF2-S models. Hydrophobic, small, polar and charged residues are colored in yellow, green, blue and red respectively. The models were rotated to focus on the large hydrophobic surface specifically-accessible in the short isoforms.

Figure 4

Validation of the RdCVF2 expression in retina. Panel a shows the expression of the short and long forms of RdCVF2 mRNA in the wild-type and rd1 retina at post-natal day 35. The RT-PCR product lengths are (RdCVF2-S: 176 bp, RdCVF2-L: 170 bp. Panel b: northern blotting analysis of RdCVF2 'exon 1) and G6PDH control. Panel c: In situ hybridization on sections of wild-type and rd1 mice retina with digoxigenin-labeled RdCVF2-S and L riboprobes (AS : antisens, S : sens). Panel d shows expression time-courses for both isoforms of RdCVF2 and rhodopsin in wild-type and rd1 mice during the first postnatal month. GC, Ganglion cells; INL, inner nuclear layer; ONL, outer nuclear layer. Original magnification : 40 ×.

Figure 5

Cone viability assay of RdCVF-S and RdCVF2-S. Panel a displays cells from cone-enriched cultures labeled with the viability dye calcein-AM and incubated with conditioned media from COS-1 cells transfected with: empty vector pcDNA3, pcDNA-RdCVF-S or pcDNA-RdCVF2-S. Panel b shows the rescue activity of RdCVF-S and RdCVF2-S when compared to that of empty vector. Statistical analysis (Tuckey test) shows that the results are statistically significant (p < 0.001).