Hermansky-Pudlak syndrome type 7 (HPS-7) results from mutant dysbindin, a member of the biogenesis of lysosome-related organelles complex 1 (BLOC-1)

Wei Li, Qing Zhang, Naoki Oiso, Edward K Novak, Rashi Gautam, Edward P O'Brien, Caroline L Tinsley, Derek J Blake, Richard A Spritz, Neal G Copeland, Nancy A Jenkins, Dominick Amato, Bruce A Roe, Marta Starcevic, Esteban C Dell'Angelica, Rosemary W Elliott, Vishnu Mishra, Stephen F Kingsmore, Richard E Paylor, Richard T Swank, Wei Li, Qing Zhang, Naoki Oiso, Edward K Novak, Rashi Gautam, Edward P O'Brien, Caroline L Tinsley, Derek J Blake, Richard A Spritz, Neal G Copeland, Nancy A Jenkins, Dominick Amato, Bruce A Roe, Marta Starcevic, Esteban C Dell'Angelica, Rosemary W Elliott, Vishnu Mishra, Stephen F Kingsmore, Richard E Paylor, Richard T Swank

Abstract

Hermansky-Pudlak syndrome (HPS; MIM 203300) is a genetically heterogeneous disorder characterized by oculocutaneous albinism, prolonged bleeding and pulmonary fibrosis due to abnormal vesicle trafficking to lysosomes and related organelles, such as melanosomes and platelet dense granules. In mice, at least 16 loci are associated with HPS, including sandy (sdy; ref. 7). Here we show that the sdy mutant mouse expresses no dysbindin protein owing to a deletion in the gene Dtnbp1 (encoding dysbindin) and that mutation of the human ortholog DTNBP1 causes a novel form of HPS called HPS-7. Dysbindin is a ubiquitously expressed protein that binds to alpha- and beta-dystrobrevins, components of the dystrophin-associated protein complex (DPC) in both muscle and nonmuscle cells. We also show that dysbindin is a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1; refs. 9-11), which regulates trafficking to lysosome-related organelles and includes the proteins pallidin, muted and cappuccino, which are associated with HPS in mice. These findings show that BLOC-1 is important in producing the HPS phenotype in humans, indicate that dysbindin has a role in the biogenesis of lysosome-related organelles and identify unexpected interactions between components of DPC and BLOC-1.

Conflict of interest statement

Competing Interests Statement: The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1
High-resolution genetic and physical maps of the sdy gene region of mouse chromosome 13. (a) High-resolution genetic map of the sdy gene region. Numbered D13Mit markers are listed above (without the prefix). The centromere is represented by a solid circle. (b) High-resolution physical map based on the BAC contig and Celera database. Two known genes in the sdy critical region are in bold with arrows indicating directions of transcription and relative sizes. A BAC contig (with RPCI-23 library designations) spans the sdy genetic interval (∼570 kb). Arrows, proportional in length to the BAC sizes and oriented with the SP6 end at the arrowhead and the T7 end opposite, represent BACs.
Figure 2
Figure 2
Dtnbp1 mRNA is smaller and dysbindin is undetectable in tissues of sdy mutant mice. (a) Genomic PCR. We amplified genomic DNA from control DBA/2J, sdy/+, sdy/sdy and various inbred strains by a duplex PCR method targeting exon 7 of Dtnbp1 and designed to produce PCR products of 472 bp from wild-type DNA and 274 bp from sdy/sdy DNA. sdy arose in the DBA/2J strain. (b) Northern-blot analysis. We hybridized total RNA (20 μg) from kidney, brain and heart of wild-type (DBA/2J), heterozygous (sdy/+) and homozygous (sdy/sdy) mice with Dtnbp1 (upper panels) and Gapd (lower panels) cDNA probes. Dtnbp1 mRNA in sdy tissues was 1.5 kb in size compared with 1.65 kb in control DBA/2J; mRNA of heterozygous sdy/+ mice contains both ∼1.5-kb and 1.65-kb Dtnbp1 mRNAs. (c) Western-blot analysis. We resolved kidney extracts of control DBA/2J, sdy/+ and sdy/sdy mutant mice together with transgenic progeny containing (+) or lacking (−) BAC54F9, which contains the entire Dtnbp1 genomic region, in denaturing gels, blotted gels and incubated them with polyclonal antibody to dysbindin 3111A (top). The blots were reprobed with antibody to Rab4 as a loading control (bottom). Immunoblot analyses of brain, heart, liver and skeletal muscle (data not shown) also showed lack of expression of dysbindin in sdy/sdy mutants.
Figure 3
Figure 3
Destabilization of the dysbindin, pallidin and muted proteins in extracts of three HPS mutants (sdy, pa and mu). We separated kidney extracts from 16 mouse HPS or HPS-related mutants and 4 control strains (DBA/2J, mu/+, C57BL/6J and C3H/HeJ) on denaturing gels and probed them with antibodies to dysbindin, muted and pallidin as indicated at left. Dystrobrevins were similarly analyzed in the brain as expression of α-dystrobrevin is weak in kidney. Inbred strain DBA/2J served as the control for sdy/sdy, mu/+ for mu/mu, C3H/HeJ for sut/sut, ash/ash and ru6J/ru6J and C57BL/6J for the remaining mutants. Antibodies to Rab4 and α-tubulin served as loading controls.
Figure 4
Figure 4
Association of dysbindin with muted, pallidin and β-dystrobrevin. (a) Yeast two-hybrid interactions. We transformed yeast strain AH109 with constructs expressing the entire coding regions of the indicated proteins fused to binding domains (left) or activation domains (top) and spotted cotransformants on plates containing high-stringency and low-stringency media. All combinations grew vigorously on low-stringency plates, indicating that no construct was lethal to the cells. Cotransformants pGBKT7-53 and pGADT7-T are positive controls for interacting proteins; pGBKT7-LAM and pGADT7-T are negative controls. AD (N) and BD (N) represent ‘empty’ activation and binding domain constructs, respectively. sdy/sdy dysbindin is the sdy mutant form of dysbindin with the 52-amino-acid deletion. We were unable to test for interactions among wild-type dysbindin, sdy/sdy dysbindin and muted because they autoactivated in the binding domain. sdy/sdy dysbindin did not interact with dystrobrevin but did interact with pallidin. (b) Coimmunoprecipitation shows direct association of dysbindin with components of BLOC-1. We cotransfected COS-7 cells with either dysbindin or sdy/sdy dysbindin (the sdy mutant form of dysbindin with the 52-amino-acid deletion) and Myc-pallidin, Myc-muted, β-dystrobrevin or Myc-pearl. Proteins were immunoprecipitated with antibody to dysbindin or an unrelated polyclonal antibody (control IgG) and detected with an antibody to Myc. Muted and pallidin were detected when immunoprecipitated with dysbindin but not with an unrelated IgG, indicating that both muted and pallidin form a complex with dysbindin. The cell lysate is shown for comparison. In control experiments, β-dystrobrevin coimmunoprecipitated with dysbindin as described previously but pearl did not. Pallidin and muted immunoprecipitated with sdy/sdy dysbindin, but to a lesser extent. β-dystrobrevin, wild-type dysbindin and pearl did not coimmunoprecipitate with sdy/sdy dysbindin. The asterisk indicates the IgG heavy chain that is detected by the secondary antibody.
Figure 5
Figure 5
Dysbindin and pallidin are components of a common protein complex in mouse liver. (a) Size exclusion chromatography. We fractionated mouse liver cytosol on a calibrated Superose 6 column and analyzed the resulting fractions by immunoblotting with antibodies to dysbindin, pallidin, α1-dystrobrevin, β-dystrobrevin and the dystrophin isoform Dp71 (ref. 12). The exclusion volume (V0) and the elution positions of standard proteins (Stokes radii given in Angstroms) are indicated at the top. (b) Sedimentation velocity analysis. We fractionated mouse liver cytosol by ultracentrifugation on a 5–20% (w/v) sucrose gradient and analyzed the resulting fractions by immunoblotting as in a. Fractions 1 and 28 correspond to the top and bottom ends of the gradient, respectively. The positions of standard proteins (sedimentation coefficients given in Svedberg units) are indicated at the top. Taken together, the size-exclusion chromatography and sedimentation velocity analyses indicate a native molecular mass of ∼230 kDa (221–247 kDa) and a frictional ratio of ∼2.4 for mouse BLOC-1. Both numbers are in close agreement with those calculated for bovine BLOC-1 (ref. 11), indicating that mouse BLOC-1 is similarly a large asymmetric protein complex.
Figure 6
Figure 6
Retinal melanosomes are deficient and abnormal in sdy mice. Retinal sections of DBA/2J (a) and sdy/sdy (b) mice. In each case, the hyphenated partial line at the left indicates the interface between the retinal pigment epithelium (above) and the choroid (below). Scale bars = 2 μm.

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