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. 2005 Mar 22;102(12):4342-7.
doi: 10.1073/pnas.0407287102. Epub 2005 Mar 7.

Disease-associated Mutations Cause Premature Oligomerization of Myelin Proteolipid Protein in the Endoplasmic Reticulum

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Free PMC article

Disease-associated Mutations Cause Premature Oligomerization of Myelin Proteolipid Protein in the Endoplasmic Reticulum

Eileithyia Swanton et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disease caused by mutations, deletions, or duplications of the proteolipid protein (PLP) gene. Mutant forms of PLP are retained in the endoplasmic reticulum (ER), and the resulting accumulation of mutant protein is thought to be a direct cause of oligodendrocyte cell death, which is the primary clinical feature of PMD. The molecular mechanisms underlying the toxicity of mutant PLP are however currently unknown. We report here that PMD-linked mutations of PLP are associated with the accelerated assembly of the protein into stable homooligomers that resemble mature, native PLP. Thus although WT PLP forms stable oligomers after an extended maturation period, most likely at the cell surface, mutant forms of PLP rapidly assemble into such oligomers at the ER. Using PLP mutants associated with diseases of varying severity, we show that the formation of stable oligomers correlates with the development of PMD. Based on these findings, we propose that the premature oligomerization of PLP in the ER of oligodendrocytes contributes to the pathology of PMD.

Figures

Fig. 1.
Fig. 1.
Structure and subcellular distribution of WT PLP. (A) Crude myelin extracts were analyzed by SDS/PAGE and Western blotting with a PLP-specific serum or the corresponding preimmune sera. (B) Diagram depicting the topology of PLP. Palmitoylation sites are shown by squiggles, disulfide bonds are shown by lines, and point mutations used in this study are shown by numbers 1–6. Mutations associated with mild PMD are in filled circles, and those associated with severe disease are in open circles. (C) COS-7 cells transiently transfected with WT PLPmh were stained with α-myc followed by a FITC-conjugated secondary antibody. (Scale bar: 20 μm.) (D) Lysates of cells transfected with WT PLPmh were analyzed by Western blotting with α-myc.
Fig. 2.
Fig. 2.
Oligomerization of WT PLPmh. (A) COS-7 cells transfected with WT PLPmh were solubilized with the indicated concentration of N-dodecylmaltoside (DDM) and analyzed by blue native gel electrophoresis and Western blotting with α-myc. (B) Cells stably expressing WT PLPmh were transfected with WT PLPha, solubilized, and immunoprecipitated with α-ha or control (ctl) antibody. Fifty percent of the lysate used for IP is present in lane 3. Lanes 4 and 5 show cells that were cross-linked with DPDPB before IP. Immunoprecipitated material was analyzed by Western blotting with α-myc. Fragments of the IP antibody heavy chain are visible at the top of the panel. (C) A stable cell line was induced to express WT PLPmh for the time indicated, and lysates were analyzed by SDS/PAGE and Western blotting with α-myc. Several different exposures were quantified by densitometry, and the dimer:monomer ratio was calculated.
Fig. 3.
Fig. 3.
Oligomerization of PLP mutants. (A) COS-7 cells transiently transfected with msd PLPmh were stained with α-myc followed by a FITC-conjugated secondary antibody. (Scale bar: 20 μm.) (B) Lysates of COS-7 cells transfected with msd PLPmh were analyzed by SDS/PAGE and Western blotting with α-myc. (C) COS-7 cells were transfected with WT or mutant forms of PLPmh and then analyzed as in B. (D) A stable cell line was induced to express msd PLPmh for the time indicated and then analyzed as in B. Note that approximately three times more cell lysate was used here than in Fig. 2C. (E) Lysates of COS-7 cells transfected with WT or msd PLPha, or with msd PLPha containing a glycosylation site in the first (msd CHO1) or second (msd CHO2) luminal loop, were analyzed by SDS/PAGE and Western blotting with α-ha. The position of glycosylated and nonglycosylated monomeric PLPha is shown by a curly bracket, and dimers are indicated by a square bracket. A dimer composed of glycosylated and nonglycosylated PLP is shown by an asterisk. (F) COS-7 cells were transfected as in C and then analyzed by SDS/PAGE and Western blotting with α-myc followed by [125I]protein A. PLPmh was quantified by autoradiography, and the amount of SDS-resistant dimer is expressed as a percentage of the total amount of dimer plus monomer. The chart shows the means of two to three independent experiments. The significance of the difference between mutants associated with mild and severe disease was determined by using the independent-samples t test. (G) COS-7 cells were transfected with TM1-3ha and stained with α-ha followed by a FITC-conjugated secondary antibody. (Scale bar: 20 μm.) (H) COS-7 cells were transfected with WT or msd PLPha, or with mutant 5 TM1-3ha and analyzed as in E.
Fig. 4.
Fig. 4.
Stable oligomers are not due to prolonged ER retention or aberrant disulfide bonds. (A and B) Stable cell lines were induced to express WT or msd PLPmh for 96 or 24 h, respectively. Lysates were separated on a Superdex 200 column, and fractions were analyzed by SDS/PAGE and Western blotting with α-myc. The amount of monomer and dimer was quantified by densitometry and is expressed as a percentage of the total amount of monomer/dimer. The chart shows the average of three to four independent experiments. The position of three soluble molecular weight markers is indicated. (C) Lysates of cells expressing msd PLPmh were separated as in B, and fractions 9 and 10 were analyzed by blue native gel electrophoresis and Western blotting with α-myc. (D) Lysates of cells expressing msd PLPmh were separated as in B, and fractions 4–7 and 8–11 were pooled and immunoprecipitated with α-myc or control (ctl) antibody. Immunoprecipitated material was analyzed by Western blotting with α-calnexin. Lanes 5 and 6 show one-thirtieth of the total lysate used for IP. (E) Stable cell lines were induced to express WT or msd PLPmh overnight in the presence of 5 μg/ml brefeldin A, and lysates were analyzed by SDS/PAGE and Western blotting with α-myc. (F) COS-7 cells were transfected with WT or msd PLPmh, or with WT or msd PLPmh in which the ER luminal cysteine residues were replaced with glycine (ΔCys). Lysates were analyzed as in A.

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