Abstract
Purpose::
The human ABCR protein has been linked to several inherited macular degenerations including Stargardt disease, cone-rod dystrophy and fundus flavimacultitis. A unique feature of ABCR is the presence of two long extracellular loops, characteristic of the ABCA subclass of ABC transporters. The high degree of sequence conservation between vertebrate ABCR proteins suggests important, but unknown, functional roles for these domains. Mutations in the extracellular domains (ECDs) have been identified in patients with Stargardt and other retinal degenerative diseases. At the present time, the structures or functions of these domains remain unknown. The purposes of this study were to clone, express, purify and characterize these extracellular domains as isolated polypeptides.
Methods::
Using standard recombinant DNA techniques the region corresponding to ECD1 (aa 62-646) and ECD2 (aa 1395-1680) was amplified from hABCR cDNA and cloned into a T7 expression system. The polypeptides were expressed as affinity tagged fusion proteins and purified to homogeneity. Circular dichroic studies were carried out to determine the solution conformation of ECD1. CED spectra were measured on a Jasco 810 circular dichrosim spectrophotometer. Spectra were obtained for native and heat denatured ECD1. Secondary structure quantitation was determined via the KD2 method from DICHROWEB.
Results::
ECD1 displayed a unique secondary structure, with a high beta sheet content. The structure was extraordinarily heat resistant, signifying that the conformation is highly stable. CD derived conformation compared well with sequence-based structural prediction using the Garnier (GOR IV) method.
Conclusions::
This represents the first account of the structural parameters of the extracellular loops of the ABCA subfamily. Our results suggest that the ECDs of ABCR have unique and highly stable tertiary structure. These studies help pave the way for analysis of the structural modification of the ECDs of ABCR as a consequence of disease associated mutations.
Keywords: protein structure/function • proteins encoded by disease genes • retinal degenerations: hereditary