Topological analysis of peripherin/rds and abnormal glycosylation of the pathogenic Pro216→Leu mutation

Abstract

Peripherin/ rds is an integral membrane glycoprotein found in the rim regions of vertebrate photoreceptor cell discs. The protein is believed to be involved in both formation and maintenance of the characteristic flattened morphology of the outer segment discs and its essential nature is demonstrated by the wide range of retinal degenerative disorders in which the protein has an involvement. Little structural data has been determined for peripherin/ rds, but a topological model of the protein has been proposed. In this paper, we present the first direct evidence for the topology of the protein through the use of scanning glycosylation mutagenesis. Both the topological data and the observation that only the Asn(229) site is efficiently glycosylated in this in vitro transcription/translation system support the common hypotheses. Additionally, expression of the Pro(216)-->Leu mutant demonstrates an abnormal glycosylation pattern, which may explain the mechanism by which this mutation precipitates a retinal degenerative phenotype.

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology

“…4) corresponding to apparent molecular weights of *37 and *40 kD being the dominant species. The apparent molecular weight of 37 kD is consistent with the apparent molecular weight of the monomer reported in the literature (Wrigley et al 2002). The same two bands are present upon analysis with Western blot at apparent molecular weights of *37 and *40 kD.…”

“…From previous studies it is known that an additional glycosylation site is created by this mutation (Wrigley et al 2002), and it was suggested that the attachment of two bulky carbohydrate chains to the protein may result in an altered conformation in the protein. The SDS-PAGE analysis presented here indicates that the glycosylation status of wild-type p/RDS and the P216L mutant expressed in S2 cells is the same; therefore, it is not possible to completely test this hypothesis.…”

“…To fully evaluate the implications of our CD data, we compared the fractions of secondary structure of wild-type p/RDS to the topology model from our earlier work (Wrigley et al 2002). Neither the V5 epitope nor the hexahistidine tag is expected to contribute secondary structure (Li et al 2006;Narayana et al 1997).…”

“…In vivo, p/RDS is located in the outer segment of rod cells, where it is involved in the flattening of the membrane discs (Wrigley et al 2000) that contain rhodopsin, the protein responsible for the initial events in phototransduction [i.e., absorption of a photon and initiation of the biochemical pathway that ultimately lead to an hyperpolarisation of the rod cell outer membrane; for a review see (Palczewski 2006)]. P/RDS has the hallmark characteristics of a tetraspanin: i.e., four transmembrane a-helices and an LEL domain with the CysCys-Gly motif that sets it apart from other proteins with four transmembrane a-helices (Wrigley et al 2002). The main difference between p/RDS and other tetraspanins is the size of the LEL domain, which is almost twice as long as that of other tetraspanins such as CD81 (140 vs. 87 amino acid residues).…”

Retinal Degeneration Slow (RDS) Glycosylation Plays a Role in Cone Function and in the Regulation of RDS·ROM-1 Protein Complex Formation

“…Its N-and C-terminal domains are cytosolic, whereas its two extracellular loop domains (D1 and D2) project into the enclosed discs in rods and the extracellular space between lamellae in cones (24). The C-terminal domain is known to direct the targeting of RDS complexes to the OS, engage in protein-protein interactions, and form an amphipathic helix that is thought to play a role in maintaining the unique membrane topology of the OS itself by potentially regulating membrane fusion or curvature (25)(26)(27)(28)(29).…”