An immunoblotting assay to facilitate the molecular diagnosis of Hermansky-Pudlak syndrome

Abstract

Hermansky-Pudlak syndrome (HPS) comprises a constellation of human autosomal recessive disorders characterized by albinism and platelet storage pool deficiency. At least eight types of HPS have been defined based on the identity of the mutated gene. These genes encode components of four ubiquitously expressed protein complexes, named Adaptor Protein (AP)-3 and Biogenesis of Lysosome-related Organelles Complex (BLOC)-1 through -3. In patients of Puerto Rican origin, the molecular diagnosis can be based on analysis of two founder mutations. On the other hand, identification of the HPS type in other patients relies on the sequencing of all candidate genes. In this work, we have developed a biochemical assay to minimize the number of candidate genes to be sequenced per patient. The assay consists of immunoblotting analysis of extracts prepared from skin fibroblasts, using antibodies to one subunit per protein complex. The assay allowed us to determine which complex was defective in each of a group of HPS patients with unknown genetic lesions, thus subsequent sequencing was limited to genes encoding the corresponding subunits. Because no mutations within the two genes encoding BLOC-3 subunits could be found in two patients displaying reduced BLOC-3 levels, the possible existence of additional subunits was considered. Through size-exclusion chromatography and sedimentation velocity analysis, the native molecular mass of BLOC-3 was estimated to be 140+/-30 kDa, a value most consistent with the idea that BLOC-3 is a HPS1HPS4 heterodimer (approximately 156 kDa) albeit not inconsistent with the putative existence of a relatively small third subunit.

Fig. 1

Specificity of antibodies selected for the assay. (A) Detergent extracts prepared from kidneys of mice from a wild-type strain (C57BL/6J) and the pallidin-null mutant strain pallid (B6.Cg-Pldn^pa/J), a whole-cell extract prepared from human HeLa cells, and bovine liver cytosol, were analyzed by immunoblotting using two monoclonal antibodies (mAbs; 2G5 and 2G6) raised against the pallidin subunit of human BLOC-1. Notice that moAb 2G6 recognized the pallidin protein from human, murine and bovine samples, whereas moAb 2G5 recognized only the human protein. (B and C) Detergent extracts prepared from human skin fibroblasts and Epstein-Barr virus (EBV)-transformed B-lymphocytes were analyzed by immunoblotting using antibodies to β-actin and α-tubulin as loading controls (B) and to the pallidin subunit of BLOC-1, the HPS6 subunit of BLOC-2, the HPS4 subunit of BLOC-3, and the μ3A subunit of AP-3 (C). The cell samples were obtained from Coriell Cell Repository (repository numbers indicated between parentheses) and derived from apparently normal individuals as well as from patients suffering from HPS due to mutations in genes encoding subunits of AP-3 (HPS-2 disease), BLOC-2 (HPS-6 disease) and BLOC-3 (HPS-1 disease). Asterisks denote bands deemed to represent non-specific cross-reactivity. Nazarian et al. Suggested size: 2 columns

Fig. 2

Immunoblotting analysis of cell samples derived from patients of unknown genetic lesion. Detergent extracts were prepared from the cultured skin fibroblasts of five patients with clinical diagnosis of HPS (45, 88, 94, 128 and 156), and two patients suffering from HPS-related disease (43* and 47*). As controls, detergent extracts prepared from an apparently normal individual and a patient suffering from HPS-4 (due to known mutations in the HPS4 subunit of BLOC-3) were analyzed in parallel. Nazarian et al. Suggested size: 1 column

Fig. 3

Selected portions of electrophoregrams obtained during sequencing of DNA samples from apparently normal individuals (A-C, left panels) and HPS patients 88 (A, right), 156 (B, right) and 128 (C, right). See the text for further details. Nazarian et al. Suggested size: 1 column

Fig. 4

Evolutionary conservation of amino acid residues within selected segments of the HPS5 and HPS1 proteins. (A) Alignment of residues 139-152 from the human HPS5 protein isoform a (GenBank accession no. NP_852608) and corresponding segments within the orthologs from the chimpanzee (Pan troglodytes; GenBank accession no. XP_508314), horse (Equus caballus; GenBank accession no. XP_001505011), pig (Sus scrofa; GenBank accession no. NP_001092073), cattle (Bos taurus; GenBank accession no. XP_869555), dog (Canis familiaris; GenBank accession no. XP_542523), mouse (Mus musculus; GenBank accession no. NP_001005247), chicken (Gallus gallus; GenBank accession no. XP_421011), frog (Xenopus tropicalis; GenBank accession no. NP_001090726), zebrafish (Danio rerio; GenBank accession no. AAI21197), fugu fish (Takifugu rubripes; GenBank accession no. DAA00973), tetraodon (Tetraodon nigroviridis; GenBank accession no. CAF95692), fruit fly (Drosophila melanogaster; GenBank accession no. NP_649810) and red flour beetle (Tribolium castaneum; GenBank accession no. XP_975447). The conserved residue that is mutated in one allele of HPS patient 88 is denoted with an arrow. (B) Alignment of residues 536-557 from the human HPS1 protein isoform a (GenBank accession no. NP_000186) and corresponding protein segments from the chimpanzee (GenBank accession no. XP_001165998), orangutan (Pongo pygmaeus; GenBank accession no. CAH92804), macaque (Macaca fascicularis; GenBank accession no. BAD51962), pig (GenBank accession no. NP_001092057), horse (GenBank accession no. XP_001500342), dog (GenBank accession no. XP_534985), cattle (GenBank accession no. ABH06318), rat (Rattus norvegicus; GenBank accession no. NP_414541), mouse (GenBank accession no. NP_062297), opossum (Monodelphis domestica; GenBank accession no. XP_001373344), platypus (Ornithorhynchus anatinus; GenBank accession no. XP_001508131), chicken (GenBank accession no. NP_001026751), frog (GenBank accession no. NP_001006903), zebrafish (GenBank accession no. NP_001032777), tetraodon (GenBank accession no. CAF89850), red flour beetle (GenBank accession no. XP_970712), fruit fly (GenBank accession no. NP_610997) and honey bee (Apis mellifera; GenBank accession no. XP_001121837). The residues mutated in one allele of HPS patient 128 are denoted with arrows. Nazarian et al. Suggested size: 1 column

Fig. 5

Estimation of the native molecular mass of human BLOC-3. (A) Size-exclusion chromatography. Freshly prepared cytosolic extracts from HeLa or pigmented melanoma MNT-1 cells were fractionated on a calibrated Superose 6 column (1 5 60 cm), and the resulting fractions analyzed by immunoblotting using the mAb against the HPS4 subunit of BLOC-3. The column’s exclusion volume (V₀), and the elution positions of standard proteins (Stokes radii in Ångstroms) that were analyzed on the same column just before fractionation of each cytosolic sample, are indicated on the top. (B) Sedimentation velocity analysis. Freshly prepared cytosol from HeLa or MNT-1 cells was fractionated by ultracentrifugation for 16.5 and 13 h, respectively, on a 5-20% (w/v) sucrose gradient at 39,000 rpm in a SW41 rotor (∼261,000 5 g), at 4°C. Fractions were collected from the bottom of the tube and analyzed by immunoblotting using mAb against HPS4. Fraction 1 corresponds to the top of the gradient. The positions of standard proteins (sedimentation coefficients in Svedbergs) that were analyzed in parallel with each cytosolic extract are indicated on the top. Nazarian et al. Suggested size: 1.5 column