The functional effect of pathogenic mutations in Rab escort protein 1

Abstract

Choroideremia (CHM) is a chorioretinal degeneration with an X-linked pattern of inheritance. Affected males experience progressive atrophy of the choroid, retinal pigment epithelium and retina leading to eventual blindness. The CHM gene encodes Rab escort protein 1 (REP-1). REP-1 is involved in trafficking of Rab proteins in the cell. To date, the majority of reported mutations in the CHM gene cause a complete loss of REP-1 function. Here we report pathogenic mutations: a novel missense mutation, L550P; a truncation c.1542T>A, STOP; and two deletions (c.525_526delAG and c.1646delC) in the CHM gene and their phenotypic effect. To analyze the effect of mutations, the 3D structure of human REP-1 and the proteins associated with REP-1 function were modeled using sequence homology with rat proteins. In silico analysis of the missense mutation L550P suggests that the proline residue at position 550 destabilizes the beta-structural elements, and the REP-1 tertiary structure. Truncation and deletion mutants are associated with a partial or total loss of the REP-1 essential activity and protein-protein interactions as predicted by the analysis of the structure and stability of these protein products. The presumptive loss of protein was confirmed by Western Blot analysis of protein from mononuclear cells and fibroblasts (FB) from CHM patients.

Figure 1

Fundus photographs of CHM males showing extensive chorioretinal degeneration. Panels from left to right: Panel A: CHM c.1542T>A, STOP (76 year old male); Panel B: c.1646delC (49 year old male); Panel C: c.525_526delAG deletions (44 year old male) and Panel D: missense L550P mutation (44 year old male).

Figure 2

The structure of human REP-1 is modeled by homology to rat protein. The superposition of rep-1 structures from rat (gray) and human (red) and the sequence alignment are shown on the Panel A and on Panel D, respectively. Panels B and C show the model of C-terminal extension connected through the 4 proline motif (Poly-P) to the rest of REP-1 and the location of the mutation L550P in the human protein, respectively. Rab-7 protein structure is shown in forest green. The side chain of residue L550 is shown by yellow (Panel C). Two fragments of protein sequence (part of loop 106–213 and C-terminal extension 557–653), which do not have a definitive structure in the rat (PDB file: 1vg0), are shown by orange (Panels A and D). Rat rep-1 ligands (geranylgeranyl and GDP) and metal ions are shown by cyan ball and stick models.

Figure 3

Analysis of structural changes in REP-1 caused by genetic mutations in CHM patients is presented. The expected protein products and the parts of polypeptide chain removed from the protein due to mutation are shown by red and yellow, respectively. The last residues at the C-termini of expected protein products are represented by blue space-filling models. Mutations c.525_526delAG (Panel A), c.1512T>A, STOP (Panel B) and c.1646delC (Panel C) are shown respectively from left to right. Sequence tracings of partial fragments of the REP-1 gene are shown on Panel D. The mother is a heterozygous for the missense mutation c. 1679T>C (p. L550P) in the REP-1 gene (top), an unaffected brother does not carry the mutation (middle), and the proband has the mutation (bottom). Mutation sites are indicated by arrows. The β-sheet fragments of wild type (Panel E) and L550P mutant (Panel F) rep-1 formed by amino acid residues 98–101, 454–458 and 549–551 are shown by light grey and magenta, respectively. Leucine (Panel E) and proline (Panel F) residues located in position 550 are shown in red. The hydrogen-bonding pattern is shown by blue lines connecting the corresponding donor-acceptor pairs of atoms in polypeptide chains Western blots of the monocyte fractions (CD14+) or dermal fibroblasts from the CHM patients are shown in Panel G. Lanes from c1 to c5 are controls. REP-1 protein bands for PMBCs in lanes from 1 to 4 correspond to Ex13 c.1542T>A, STOP; Ex14-L550P; Ex14 c.1646delC and Ex5 c525_526delAG mutations, respectively. Western blot for REP-1 from dermal FB for the Ex14-L550P mutation is shown in lane 5. The alignment of conserved parts of rep-1 sequences from different species is demonstrated in the Panel H. The position of the amino acid residue L550 is shown by an arrow.

Figure 4

CHM-linked mutations affect the structure and function of REP-1. Three structural domains of REP-1 are shown in red (domain 1: residues 1–92, 370–446, 554–653), blue (domain 2: residues 93–270 and 447–553) and forest green (domain 3: residues 271–369), respectively. The missing crystal structure of rat protein fragments of loop 106–213 and the C-terminal extension 557–653 are indicated by dotted lines. The structures of Rab-7, RabGGTα and RabGGTβ are represented by forest green, purple and magenta, respectively. Color bars of corresponding colors located above the diagram show the domain structure and schematically represent areas involved in protein-protein interactions. Divided into fragments, the blue ribbon represents the CHM gene structure and its corresponding exon borders. CHM-linked mutations causing truncations of rep-1 from the Retina International Mutation Database (http://www.retina-international.com/sci-news/repmut.htm) and from the review [31] are shown in black letters. Mutations described in this work are shown in blue.