Structural characterization of the second intra‐discal loop of the photoreceptor tetraspanin RDS

Abstract

Vertebrate photoreceptors contain a unique tetraspanin protein known as 'retinal degeneration slow' (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases, and more than 70% of these mutations are located in the second intra-discal (D2) loop, highlighting the importance of this region. Here we examined the conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with ROM-1, a non-glycosylated homolog of RDS. The RDS D2 loop was expressed in Escherichia coli as a fusion protein with maltose binding protein (MBP). The fusion protein, referred to as MBP-D2, was purified to homogeneity. Circular dichroism spectroscopy showed that the wild-type (WT) D2 loop consists of approximately 21% a-helix, approximately 20% b-sheet and approximately 59% random coil. D2 loop fusion proteins carrying disease-causing mutations in RDS (e.g. R172W, C214S, N244H/K) were also examined, and conformational changes were observed (compared to wild-type D2). In particular, the C150S, C214S and N244H proteins showed significant reductions in a-helicity. However, the thermal stability of the mutants was unchanged compared to wild-type, and all the mutants were capable of interacting with ROM-1, indicating that this functional aspect of the isolated D2 loop remained intact in the mutants despite the observed conformational changes. An I-TASSER model of the RDS D2 loop predicted a structure consistent with the circular dichroism experiments and the structure of the conserved region of the D2 loop of other tetraspanin family members. These results provide significant insight into the mechanism of RDS complex formation and the disease process underlying RDS-associated retinal degeneration.

Novel molecular mechanisms for Prph2‐associated pattern dystrophy

“…35,36 The tetraspanin D2 loop has been extensively characterized and contains both a highly conserved region and a hypervariable region. 13,38 These six cysteines form three intramolecular disulfide linkages, Cys165-Cys250, Cys214-Cys222, and Cys166-Cys213, predicted based on alignment with other crystallized F I G U R E 8 ER stress and autophagy are not implicated in the observed mutant phenotype. The six highly conserved D2 loop cysteines involved in intramolecular disulfide bonding are essential for maintaining the secondary and tertiary structure of the D2 loop.…”

“…The six highly conserved D2 loop cysteines involved in intramolecular disulfide bonding are essential for maintaining the secondary and tertiary structure of the D2 loop. 13,38 These six cysteines form three intramolecular disulfide linkages, Cys165-Cys250, Cys214-Cys222, and Cys166-Cys213, predicted based on alignment with other crystallized F I G U R E 8 ER stress and autophagy are not implicated in the observed mutant phenotype. A-B, Total mRNA was harvested from three independent retinal extracts per indicated genotypes at P30 and each was tested for XBP1 mRNA cleavage/activation on an agarose gel (A) or underwent by qRT-PCR quantification for the common ER stress markers Activating transcription factor 4 (ATF4), Binding immunoglobulin protein (aka GRP-78) (BIP), and C/EBP homologous binding protein (CHOP) (B).…”

NGS Analysis for Molecular Diagnosis of Retinitis Pigmentosa (RP): Detection of a Novel Variant in PRPH2 Gene

“…Similarly to our variant, most of them are missense, are localized within a specific D2 loop region spanning from Lys193 to Glu226, and have been described as pathogenic for late-onset adRP [20,21,22]. However, PRPH2 variants have also been involved in a wide range of autosomal dominant retinal disorders, including RP, cone-rod dystrophy, adult vitelliform macular dystrophy, cone dystrophy, and pattern dystrophy [20,21,22]. Such a high genetic heterogeneity further complicates the genotype–phenotype correlations.…”

“…PRPH2 is able to interact with itself and its homologue Rod Outer Segment Membrane protein 1 (ROM-1). PRPH2 and ROM-1 can interact together, forming homo- and hetero-tetramers which are further connected by disulphide bounds to constitute high-order oligomers and allow disc rim formation [21]. One of the most important domains of PRPH2 is the large D2 loop domain that extends for 142 amino acids (from the 125 th to the 163 rd residue of protein) and is normally located within the intradiscal part of the rim [20].…”

Estudios estructurales de las proteínas de hemidesmosomas: integrina α6β4 y tetraspanina cd151

“…Dicho trabajo no analiza si el EC2 de CD151 fusionado a GST retiene una estructura nativa, por lo que no se puede descartar que el efecto observado sobre la infección por VIH-1 pueda deberse a motivos de secuencia cortos que no requieren de una correcta estructura tridimensional de este dominio. Por otro lado, recientemente se ha descrito la expresión soluble del dominio EC2 de la tetraspanina RDS (Retinal degeneration slow) (también conocida como periferina o Tspan-22) (Chakraborty et al, 2013). RDS se encuadra en el mismo subgrupo, 2b, que CD151 (Seigneuret et al, 2001).…”