Regulation of ABCC6 trafficking and stability by a conserved C-terminal PDZ-like sequence

Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE). The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein. © 2014 Xue et al

Impact of the C-terminus on ABCC6 degradation.

<p>ABCC6 degradation was evaluated after treatment with a proteasome inhibitor (lactacystin) or a combination of lysosomal protease inhibitors, (leupeptin and pepstatin) and assessed by western blotting. <i>A,B,</i> inhibition of the proteasome by lactacystin results in an accumulation of the ER-resident, band B protein in the mutant ABCC6. <i>A,</i> increasing lactacystin concentrations from 0–10 µM, results in an accumulation of the band B form of the mutant ABCC6 protein as seen by western blotting. No increase in the formation of the band C, complexly glycosylated protein is seen for either wildtype or mutant ABCC6 with lactacystin treatment. <i>B,</i> immunofluorescence of the ABCC6 proteins reveals the wildtype and mutant accumulate after proteasome inhibition, but the mutant fails to redistribute to the cell surface. ABCC6 is shown in green, phalloidin is shown in red and DAPI is shown in blue. <i>C,D,</i> lysosomal inhibition results in an increase in the complexly glycosylated, band C protein for both wildtype and mutant ABCC6. <i>C,</i> a dose response of leupeptin/pepstatin treatment is shown from 0–100 µM leupeptin treatment in the presence of 1 µg/ml pepstatin. Increasing pepstatin concentrations resulted in an increase in the band C ABCC6 protein. <i>D,</i> immunofluorescence of HEK293 cells treated with leupeptin/pepstatin is shown. Treatment with leupeptin/pepstatin resulted in an increase in the quantities of ABCC6 intracellularly. ABCC6 is shown in green, phalloidin is shown in red and DAPI is shown in blue.</p

Structural characterization of wildtype and Δ6-COOH NBD2.

<p>To evaluate potential changes in ABCC6 NBD2 resulting from the C-terminal deletion, NBD2 was expressed and purified for <i>in vitro</i> analysis. <i>A,</i> CD spectroscopy was used to evaluate changes in the secondary structure of the mutant NBD2. Spectra were collected from 260 to 198 nm and corrected for buffer absorbance. The traces were smoothed using a window of 5 nm. The wildtype NBD2, <i>black circles</i>, shows a mixed α/β secondary structure qualitatively consistent with known structures of NBD proteins. The Δ6-COOH mutant NBD2, <i>open circles</i>, shows no significant differences in CD spectra. <i>B,</i> analytical gel filtration was used to evaluate changes in hydrodynamic radii of the wildtype and mutant NBD2 proteins. The wildtype protein eluted as a single symmetrical peak at ∼12.2 mls, consistent with a protein of ∼25,000 Da MW. The mutant proteins eluted similarly, with a peak at 12.2 mls. No discernible differences in either CD or GFC could be detected between the wildtype and mutant proteins.</p

Regulation of cell surface stability by the C-terminus.

<p>Cell surface stability was evaluated by selective biotinylation of the ABCC6 protein using the BirA ligase and acceptor peptide in the extracellular N-terminus (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097360#pone-0097360-g001" target="_blank">Figure 1A</a>). <i>A,</i> western blots of the wildtype and Δ6-COOH ABCC6 protein with the BLAP tag are shown. The inclusion of the BLAP tag in the N-terminus had no detectable effect on the trafficking of the wildtype or mutant ABCC6 proteins evaluated by western blotting. <i>B,</i> fluorophore-conjugated streptavidin was applied to the culture media and cell surface expression of the BLAP ABCC6 protein was evaluated by fluorescence microscopy. Consistent with western blotting, no detectable differences were seen between the BLAP tagged and untagged ABCC6 proteins. The wildtype protein expressed robustly in HEK 293 cells, while the mutant protein was only labeled in a small fraction of cells transfected. <i>C,</i> fluorescence analysis of the timecourse of ABCC6 internalization and degradation from the cell surface is shown. The BLAP tagged proteins were sequentially labeled with fluorophore-conjugated streptavidin. Initial staining, time zero, was performed using AlexaFluor-488, <i>green</i>, and secondary labeling was performed using AlexaFluor-555, <i>red.</i> The internalization and degradation of ABCC6 could be seen over the course of 4–18 hours as the loss of green signal. <i>D,</i> western blots of cell surface labeled ABCC6 are shown. Streptavidin was incubated extracellularly on intact HEK293 and the BLAP-tagged ABCC6 protein was bound and washed. The lystes were subjected to SDS-PAGE and western blotting. The conjugated ABCC6-streptavidin complex could be distinguished readily from the total ABCC6 protein, allowing for the evaluation of plasma-membrane ABCC6 protein. Proteins were labeled, washed and incubated for zero to eight hours before lysis. Negative controls included expression of the BLAP-ABCC6 protein without streptavidin treatment (C1) and mock-transfected HEK293 cells treated with BirA and streptavidin (C2). Both negative controls showed no staining, consistent with specific detection of labeled BLAP-tagged ABCC6 in the experimental samples.</p

Alteration of ABCC6 trafficking by the PDZ-like C-terminus.

<p>To evaluate the potential role of the PDZ-like sequences at the C-terminus of ABCC6, wildtype and mutant proteins were expressed in HEK293 cells and evaluated by western blotting and immunofluorescence. <i>A,</i> a cartoon illustrating the domain organization and topology of ABCC6 is shown. ABCC6 contains three transmembrane domains and two nucleotide-binding domains. The single glycosylation site is represented as Ψ in the extracellular N-terminus. The insertion site for the biotin ligase acceptor peptide is also shown in the N-terminus at proline 4. <i>B,</i> a sequence alignment of known PDZ-containing ABCC family members is shown. The Type I consensus is shown above the alignment as is represented as: A, acidic; P, polar, X, any; and H, hydrophobic amino acids. The PXE-associate G1501S site is highlighted in red. <i>C,</i> representative western blots of the wildtype and Δ6-COOH ABCC6 proteins are shown after expression in HEK293 cells. The core and complexly glycosylated species are indicated on the left by B and C, respectively. Two exposures are shown for the Δ6-COOH to illustrate the formation of both band B and C at low levels in the mutant protein. <i>D,</i> endoglycosidase assays confirm the glycosylation state and differential electrophoretic migration of the ABCC6 proteins. The differential digestion of the band C protein by EndoH and PNGaseF demonstrates complex glycosylation, consistent with trafficking through the Golgi. The N15D substitution blocks N-linked glycosylation and is a reference for the unglycosylated wildtype and Δ6-COOH proteins. <i>E</i>, representative western blots from cell surface biotinylation experiments are shown. Cell surface expression is shown for cells mock transfected (CNTL) or transfected with wildtype or Δ6-COOH ABCC6, <i>Cell Surface</i>. Whole cell lysates are shown, <i>Total</i>, from samples prior to streptavidin pull-down. The control samples are taken from non-adjacent wells on a single gel/film. <i>F,</i> immunofluorescence images of the wildtype and Δ6-COOH proteins are shown. The ABCC6 proteins are shown in green, phalloidin is shown in red, and DAPI is shown in blue. Colocalization of the ABCC6 protein with phalloidin is consistent with ABCC6 trafficking to the cell surface in the wildtype protein and is decreased by the Δ6-COOH mutant. <i>G,</i> immunofluorescence images of the wildtype and Δ6-COOH ABCC6 proteins are shown after expression in polarized MDCK cells. The wildtype ABCC6 protein localizes to the basolateral membrane in polarized MDCK cells, <i>left.</i> The Δ6-COOH protein shows significant intracellular staining and a loss of basolateral targeting in MDCK cells, <i>right</i>. Both X–Y, <i>top</i>, and X–Z, <i>bottom</i>, images are shown. For <i>G</i>, ABCC6 is stained in <i>green</i> and ZO1 is shown in <i>red</i>. Western blots are representative of samples from at least three independent experiments.</p