Preparation and characterization of the extracellular domain of human Sid-1 - PubMed (original) (raw)

Preparation and characterization of the extracellular domain of human Sid-1

Ashley J Pratt et al. PLoS One. 2012.

Abstract

In C. elegans, the cell surface protein Sid-1 imports extracellular dsRNA into the cytosol of most non-neuronal cells, enabling systemic spread of RNA interference (RNAi) throughout the worm. Sid-1 homologs are found in many other animals, although for most a function for the protein has not yet been established. Sid-1 proteins are composed of an N-terminal extracellular domain (ECD) followed by 9-12 predicted transmembrane regions. We developed a baculovirus system to express and purify the ECD of the human Sid-1 protein SidT1. Recombinant SidT1 ECD is glycosylated and spontaneously assembles into a stable and discrete tetrameric structure. Electron microscopy (EM) and small angle x-ray scattering (SAXS) studies reveal that the SidT1 ECD tetramer is a compact, puck-shaped globular particle, which we hypothesize may control access of dsRNA to the transmembrane pore. These characterizations provide inroads towards understanding the mechanism of this unique RNA transport system from structural prospective.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: This work was partly supported by Novartis Research Foundation. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Recombinant SidT1 ECD is a glycoprotein.

A, Heterogeneity in purified SidT1 ECD samples. Double arrows indicate the presence of two bands on SDS-PAGE. B, MALDI-TOF mass spectrum reveals a mixture of two major species of 36.3 and 37.4 kDa. C, N-Glycosidase F treatment of the protein resulted in increased electrophoretic mobility, suggesting N-linked glycosylation. Lambda phosphatase treatment had no noticeable effect.

Figure 2. Mature SidT1 ECD forms an oligomer.

Size exclusion elution profiles from a Superdex 200 column are displayed. A, Concentrated protein applied directly to the column, with no pre-incubation period, elutes as three species with sizes consistent with monomeric, dimeric and tetrameric forms of the protein. B, Following an incubation period of two hours, the protein elutes as a homogeneous tetrameric species.

Figure 3. SidT1 SAXS analysis.

A, Solution SAXS profile for glycosylated SidT1 ECD. Inset displays Guiner region (data <q = 0.028, beyond light gray line, were omitted in subsequent modeling). B, P(r) plot of SidT1 ECD reveals a unimodal curve, consistent with a globular protein species. The maximum particle diameter, Dmax, is 110 Å. C, Kratky plot of SAXS data shows very little flexibility/looseness, indicated by the convergence of the curve. The Porod exponent is 3.9.

Figure 4. 3D model of the SidT1 ECD.

A, Averaged 3D reconstruction of SAXS data using Gasbor software and P4 symmetry reveals a puck-like shape. B, Transmission electron micrograph showing negatively-stained SidT1 ECD particles.

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Preparation and characterization of the extracellular domain of human Sid-1 - PubMed (original) (raw)