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Keywords:

  • Hermansky–Pudlak Syndrome;
  • BLOC-1;
  • HPS-8;
  • BLOC1S3

Summary

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgments
  9. References

Hermansky–Pudlak Syndrome (HPS) is a genetically heterogeneous disorder of lysosome-related organelle biogenesis and is characterized by oculocutaneous albinism and a bleeding diathesis. Over the past decade, we screened 250 patients with HPS-like symptoms for mutations in the genes responsible for HPS subtypes 1–6. We identified 38 individuals with no functional mutations, and therefore, we analyzed all eight genes encoding the biogenesis of lysosome-related organelles complex-1 (BLOC-1) proteins in these individuals. Here, we describe the identification of a novel nonsense mutation in BLOC1S3 (HPS-8) in a 6-yr-old Iranian boy. This mutation caused nonsense-mediated decay of BLOC1S3 mRNA and destabilized the BLOC-1 complex. Our patient’s melanocytes showed aberrant localization of TYRP1, with increased plasma membrane trafficking. These findings confirm a common cellular defect for HPS patients with defects in BLOC-1 subunits. We identified only two patients with BLOC-1 defects in our cohort, suggesting that other HPS genes remain to be identified.


Significance

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgments
  9. References

Hermansky–Pudlak Syndrome (HPS) is caused by mutations in genes whose protein products are involved in the biogenesis of lysosome-related organelles, such as melanosomes in melanocytes and delta granules in platelets. These proteins belong to four complexes, biogenesis of lysosome-related organelles complex (BLOC-1, BLOC-2, and BLOC-3) and adaptor protein complex-3 (AP-3). Mutations in genes that encode the BLOC-1 subunit proteins are rare, with only three mutations identified in three genes (DTNBP1, BLOC1S3, and PLDN). Here, we report a fourth individual with a BLOC-1 defect involving a novel mutation in BLOC1S3; this represents only the second identified HPS-8 patient.

Introduction

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgments
  9. References

Hermansky–Pudlak Syndrome (HPS; MIM# 203300) is a rare autosomal recessive condition characterized by reduced skin, hair and eye pigmentation and a bleeding diathesis. Occasionally, additional symptoms occur, including pulmonary fibrosis, granulomatous colitis, or neutropenia (Huizing et al., 2008). To date, nine human HPS subtypes (HPS1–9) and their associated genes have been identified (Anikster et al., 2001; Cullinane et al., 2011; Dell’angelica et al., 1999; Li et al., 2003; Morgan et al., 2006; Oh et al., 1996; Suzuki et al., 2002; Zhang et al., 2003). All of the HPS protein products are involved in the biogenesis of lysosome-related organelles such as melanosomes in melanocytes and delta granules in platelets (Huizing et al., 2008; Raposo et al., 2001; Wei, 2006), and all are components of one of four protein complexes: BLOC-1; BLOC-2; BLOC-3; or adaptor protein complex-3 (AP-3; Dell’angelica et al., 1999; Huizing et al., 2008; Wei, 2006).

The BLOC-1 complex contains eight subunits: BLOS1; BLOS2; BLOS3 (HPS-8); cappuccino; dysbindin (HPS-7); muted; pallidin (HPS-9); and snapin. HPS mouse models exist with mutations in five of the eight BLOC-1 subunits (Li et al., 2004). However, relatively little is known about the intracellular function of the BLOC-1 constituents, and only three human families with BLOC-1 defects are known (Cullinane et al., 2011; Li et al., 2003; Morgan et al., 2006). Specifically, HPS-7, HPS-8, and HPS-9 involve mutations in DTNBP1, BLOC1S3, and PLDN, respectively. Here, we describe the identification of another individual with a novel nonsense mutation in BLOC1S3, comprising only the second mutation leading to an HPS-8 subtype (Morgan et al., 2006).

Results

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgments
  9. References

Clinical aspects of the HPS-8 patient

The HPS-8 patient is a boy born to Iranian first cousins. The boy had brown irides and nystagmus at birth with lighter skin pigment than either olive-complexioned parent (Figure 1A). His hair showed no abnormal pigment clumping under light microscopy (Figure 1B). Albinism and poor vision required corrective lenses at 3 months of age. Bilateral eye muscle surgery was performed for strabismus at 6 months. Upon admission to the NIH Clinical Center at age 6 yr, visual acuity was 20/200 OD and 20/125 OS with bilateral exotropia, strabismus, and fine horizontal nystagmus. Additional findings on ophthalmologic exams included iris transillumination, moderate pallor of the optic disks, absent foveal reflexes, and decreased pigment in the periphery of the retina (Figure 1C). Easy bruisability was evident on the boy’s legs, but had no early bleeding manifestations with circumcision nor tooth eruption, despite the absence of delta granules in his platelets (Figure 1D). However, the patient did suffer minor gum bleeds with four dental fillings and was treated with desmopressin (DDAVP) after the procedures. There was no history of epistaxis, gastrointestinal bleeds, surgery or trauma necessitating platelet transfusion. There were two episodes of asthma that required treatment in the emergency room, and allergy testing revealed reactions to pollen, dust mites, eggs, salmon, and tuna. A blood smear revealed no abnormal large granules in the neutrophils of the patient (Figure 1E) and there were no reports of serious infections. However, the patient had one episode of viral pneumonia. Dermal fibroblasts and melanocytes were cultured from a skin biopsy from this patient, and a packed pellet of cultured melanocytes was notably lighter than a melanocyte pellet from a control biopsy (Figure 1F).

image

Figure 1.  Clinical aspects of HPS-8 (bottom panels) compared with controls (top panels). (A) The patient has fair skin and hair compared with other family members. (B) The patient’s hair shaft shows significant pigmentation compared with that a dark haired/ethnically matched control, and no abnormal pigment clumping. (C) The patient has decreased retinal pigment in the periphery compared with control. (D) Whole-mount electron microscopy of the patient’s platelets revealed no delta granules, which are present in control platelets (arrows). (E) A blood smear revealed no abnormal large granules in the neutrophils. (F) Packed melanocytes show reduced pigment in the HPS-8 patient compared with a matched control.

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BLOC-1 subunit mutation screen

In the last decade, we have examined more than 250 patients with HPS-like features at the NIH Clinical Center, evaluating them at the clinical, molecular and cellular levels (Huizing et al., 2008). Most patients harbored mutations in genes encoding AP-3, BLOC-2, and BLOC-3 subunits. However, in 38 patients, we did not identify defects in any of the associated genes. As only a few mutations in BLOC-1 genes have been identified, we undertook a mutation screen of the BLOC-1 genes for this cohort of 38 unclassified HPS-like patients. This screen revealed a single patient with a new subtype of HPS (HPS-9), associated with homozygous nonsense mutations in PLDN encoding the BLOC-1 subunit pallidin (Cullinane et al., 2011). Furthermore, no other member of the cohort had mutations in PLDN.

Molecular analysis of the HPS-8 patient

Our mutation analysis revealed a novel BLOC1S3 mutation: c.131C>A, p.S44X (Figure 2A). This nonsense mutation was found in the homozygous state, as expected owing to the patient’s consanguineous background. Consistent with this, a SNP-chip microarray confirmed several regions of extended homozygosity throughout the genome, including homozygosity in the BLOC1S3 region on chromosome 19q13.3 (Figure 2B), consistent with the homozygous nature of the mutation.

image

Figure 2.  Molecular studies in the HPS-8 patient. (A) Sequencing chromatograms from control and patient genomic DNA. The patient is homozygous for c.131C>A in BLOC1S3 causing p.S44X at the protein level. (B) SNP-array data of chromosome 19 for the HPS-8 patient. The vertical red dotted line shows the position of BLOC1S3 and the red brackets show extended regions of homozygosity (top plot). On the B allele plot (upper chart), the middle dots represent heterozygous SNPs (AB), and the top and bottom borders represent homozygous SNPs (AA or BB). The log R ratio plot (lower chart) shows that there are normal SNP calls in both homozygous regions, indicating that no deletions or insertions are present.

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Both cultured fibroblasts and melanocytes from the patient showed a significant reduction in BLOC1S3 mRNA by quantitative real-time PCR (qRT-PCR), compared with control cells (Figure 3A; fibroblasts, 5.9 ± 1.2% of control; melanocytes, 4.1 ± 1.1% of control, P < 0.001, n = 3). BLOC1S3 (GenBank Accession NM_212550) is a two-exon gene, of which only exon 2 is protein coding (Figure 3B). As the qRT-PCR Taqman assay was located entirely within exon 2, genomic DNA could also potentially be amplified, even though all RNA samples were treated with DNase. Therefore, we designed PCR primers with one primer pair (F1-R) in the coding exon 2, and another pair with the forward primer (F2) in the non-coding exon 1 and the reverse in exon 2 (R) as before. Neither of these produced detectable BLOC1S3 amplified cDNA in either the patient’s fibroblasts or melanocytes, despite being present in control cells (Figure 3C). This suggests that nonsense-mediated decay (NMD) is occurring for this mutant transcript, even though the nonsense mutation does not occur more than 55 base pairs upstream of the last exon–exon boundary of the spliced transcript, which is the usual cutoff for NMD to occur (Maquat, 2004; Martina et al., 2003). Such atypical cases have been previously reported, however, and the term used for this event is boundary-independent NMD (Maquat, 2004; Martina et al., 2003). In any event, because no BLOC1S3 mRNA could be detected, very little protein, if any, is likely being synthesized.

image

Figure 3. BLOC1S3 mRNA and protein analysis of HPS-8 cells. (A) Quantitative real-time PCR results for BLOC1S3 mRNA expression in patient compared with control fibroblasts and melanocytes. Values shown are percentage expression of BLOC1S3 in patient cells compared with control cells, normalized by GAPDH (Error bars = ±1 SEM, n = 3, P < 0.001). (B) Schematic diagram depicting the exon structure of BLOC1S3, where only exon 2 is protein coding, and contains both the start (ATG; M) and stop (X) codons (gray box). Arrowheads show the position of the PCR primer used for (C) and arrow shows position of the HPS-8 patient’s mutation. (C) Standard PCR on cDNA of control and patient’s fibroblasts or melanocytes. Agarose gel images of PCR products show no detectable BLOC1S3 amplification in patient’s cDNA for either primer set (coding: F1-R; 5′UTR: F2-R); GAPDH was amplified. NTC, non-template control. (D) Immunoblots of fibroblast extracts from the HPS-8 patient and the previously reported HPS-9 patient for the BLOC-1 subunits cappuccino, dysbindin, pallidin and snapin. Cappuccino and dysbindin (and pallidin in the HPS-9 patient) are undetectable. Pallidin is reduced in the HPS-8 patient and snapin is also reduced in both BLOC-1 patients. (E) Immunoblots of lysates from HPS-8 and HPS-9 (BLOC-1), HPS-4 (BLOC-3) and HPS-5 (BLOC-2) patients using antibodies to the HPS4 and HPS5 proteins, showing independent assembly of the BLOC complexes. Loading was controlled by immunoblotting the same membrane for β-actin.

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Cellular characterization of the HPS-8 patient

As no commercial antibody is available for BLOS3, we could not perform immunoblotting to detect for the absence of this protein in the HPS-8 patient. However, we and others have shown in human and mouse cells that defects in one BLOC-1 subunit destabilize the entire complex at the protein level, resulting in the absence or significant down-regulation of other BLOC-1 subunits (Cullinane et al., 2011; Falcon-Perez et al., 2002; Moriyama and Bonifacino, 2002). Therefore, we subjected fibroblast lysates from control, HPS-8 and HPS-9 patients to immunoblotting with antibodies to the BLOC-1 subunits cappuccino, dysbindin (HPS-7), pallidin (HPS-9), and snapin (Figure 3D). This revealed the absence of pallidin in the HPS-9 patient, as expected, and a reduction in the HPS-8 patient (55.0% compared with control); furthermore, the cappuccino and dysbindin subunits were absent from both patients. Snapin was reduced in both BLOC-1 patients at 53.1 and 59.3% compared with control lysates for the HPS-8 and HPS-9 patients, respectively. Taken together, these data further suggest that when any one member of the BLOC-1 complex is mutated, the whole complex is unstable and prone to degradation. Consistent with previous data, the BLOC-1 patient lysates showed normal protein expression for HPS5 and HPS4, BLOC-2 and BLOC-3 subunits, respectively (Figure 3E). This confirms that the BLOC-2 and BLOC-3 complexes are normally expressed and form independently in BLOC-1-deficient patients.

Early melanosomes mature into stage III melanosomes by acquiring melanogenic proteins, such as tyrosinase and TYRP1, which are crucial for melanin production and eventually produce the melanin-laden stage IV melanosomes required for pigmentation. We carried out immunofluorescence microscopy on control and patient melanocytes to test the effect of the unstable BLOC-1 complex on the ability to traffic the melanogenic proteins tyrosinase and TYRP1 (mutated in OCA-1 and OCA-3, respectively) to melanosomes (Huizing et al., 2008; Raposo et al., 2001). TYRP1 abnormally accumulated in the Golgi region in HPS-8 melanocytes, with occasional localization to non-Golgi-associated punctate perinuclear structures and the plasma membrane (Figure 4A). In contrast, tyrosinase localized normally to PMEL-17-labeled structures in the HPS-8 melanocytes (data not shown) and did not appear to significantly accumulate in the Golgi region (Figure 4B), suggesting that the mis-localization in BLOC1S3-deficient cells is cargo-specific. These findings are consistent with the situation in BLOC-1 deficient mouse melanocytes, in which immuno-electron microscopy showed normal PMEL17 and tyrosinase distribution, but abnormal TYRP1 accumulation in tubulovesicular structures and early vacuolar endosomes near the Golgi (Setty et al., 2007).

image

Figure 4.  Common defect of TYRP1 trafficking in BLOC-1 patients. (A) Confocal immunofluorescence images showing TYRP1 in the dendrites and tips of control melanocytes, while in the HPS-8 cells, the TYRP1 appears to be more perinuclear, in the plasma membrane, and colocalizes with the Golgi (inserts). (B) Tyrosinase localization appears normal in the HPS-8 patients’ melanocytes and is comparable with control cells. Golgi marked by TGN46, nuclei stained with DAPI and scale bar represents 20 μm. (C) Plasma membrane biotinylation assay shows increased TYRP1 protein on the membrane of the HPS-8 patients cells compared with control cells despite there being less TYRP1 in the whole-cell lysate from the patient. Blotting the same membrane for β-actin demonstrates purity of the membrane fraction, and equal loading of the whole-cell lysate. (D) TYRP1 internalization assay shows a decreased rate of endocytosis from the plasma membrane in the HPS-8 patient cells compared with that of control. Values shown are percentage of starting TYRP1 on the plasma membrane (error bars = ±1 SEM, n = 3).

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Previous studies of BLOC-1 mouse melanocytes showed enhanced flux of TYRP1 through the plasma membrane and decreased steady state TYRP1 levels because of lysosomal degradation of mis-trafficked TYRP1 (Setty et al., 2007). Our studies of HPS-8 melanocytes yielded similar results; steady state TYRP1 levels were decreased in the patient’s melanocytes by immunofluorescence microscopy (Figure 4A). Furthermore, by biotinylating the surface proteins before cell lysis, TYRP1 appeared significantly increased in the patient’s plasma membrane protein fraction (Figure 4C). Similar to our previous experiments in HPS-9 melanocytes, the TYRP1 on the plasma membrane showed a reduced rate of endocytosis compared with that of control cells (Figure 4D), possibly due to saturation of the endocytosis machinery by the increased TYRP1 on the membrane. From these results, we conclude that patients with BLOC-1 subunit defects have a similar cellular phenotype and that aberrant TYRP1 trafficking likely contributes to the hypopigmentation of BLOC-1 patients.

Discussion

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgments
  9. References

Here, we report the second case of HPS-8 to date, with albinism attributable to aberrant melanosome biogenesis and a bleeding diathesis attributable to absent platelet delta granules. Our patient had no clinical signs of additional HPS subtype-specific symptoms, including neutropenia, granulomatous colitis, or pulmonary fibrosis. As some of these symptoms may develop at a later age or may be mutation-dependent, it is crucial to follow clinical signs of this patient and to identify additional patients with BLOC-1 defects. Our molecular and cellular studies indicated that our patient exhibited a homozygous nonsense mutation in BLOC1S3, whose protein product, BLOS3, is a member of the BLOC-1 complex. The aberrantly expressed BLOS3 in the patient’s melanocytes destabilized the complex and caused mis-trafficking of TYRP1, which abnormally accumulated in the Golgi region and cell membrane; this severely reduced pigment production. Furthermore, the data presented here are consistent with findings in the HPS-9 patient, suggesting a common cellular defect and phenotype for all patients with mutations in the genes encoding BLOC-1 subunits. An explanation for this could be that the BLOC-1 complex becomes unstable and the proteins that constitute the complex become prone to degradation once one of the subunits is absent or mutated. The absence of the dysbindin and cappuccino subunits in our HPS-8 (BLOS3-deficient) and HPS-9 (pallidin-deficient) patients supports this idea. However, in contrast, the snapin subunit in both patients’ cells and pallidin in the HPS-8 patient’s cells are reduced but not completely absent. The residual expression of some BLOC-1 subunits in BLOC-1 patients’ cells may be due to subcomplexes forming within the BLOC-1 complex itself, as seen in the adaptor protein complexes (Boehm and Bonifacino, 2001). Alternatively, it may be due to the fact that members of the BLOC-1 complex, such as pallidin and snapin, have functions outside of the BLOC-1 complex. A structure of the BLOC-1 complex has recently been proposed based on direct protein interactions (Lee et al., 2012). However, this model does not explain the pattern of residual subunit expression in our BLOS3- or pallidin-deficient cells. We therefore favor the idea that certain subunits are spared from complete degradation because of their function outside of the BLOC-1 complex.

Of our entire cohort of over 250 patients with an HPS phenotype, we had assigned an HPS subtype (HPS-1 through HPS-6) by molecular methods to all but 38 individuals. Of these 38 unclassified HPS-like patients, we identified only two individuals with BLOC-1 defects; they had mutations in BLOC1S3 (HPS-8), reported here, and PLDN (HPS-9) reported previously (Cullinane et al., 2011). In addition to our BLOC-1 patients, there have been only two other families described to date that have mutations in a BLOC-1 subunit, that is, in DTNBP1 (HPS-7) and in BLOC1S3 (HPS-8; Li et al., 2003; Morgan et al., 2006). This may indicate that BLOC-1 defects are either extremely rare or embryonically lethal, or that we are not testing the correct patients for BLOC-1 defects. For example, we currently use the absence of delta granules in patients’ platelets as a sine qua non for diagnosing HPS, but it may be that a subset of patients with BLOC-1 defects have normal platelet delta granules. In addition, owing to the high expression of certain BLOC-1 subunits in the brain, and in some cases brain-specific transcripts, BLOC-1 patients might have a severe neurological phenotype in addition to classic HPS manifestations (Cullinane et al., 2011; Falcon-Perez et al., 2002). Nonetheless, we still have 36 patients with HPS-like symptoms that are not HPS1 through HPS-9, and this strongly suggests that there are other, yet to be identified, genes that when mutated cause HPS. Future elucidation of genetic defects in these HPS-like patients may shine more light on these issues of variable phenotypes.

Methods

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgments
  9. References

Patient

All 38 patients were enrolled in either clinical protocol NCT00001456 ‘Clinical and Basic Investigations Into Hermansky–Pudlak Syndrome’, or protocol NCT00369421, ‘Diagnosis and Treatment of Inborn Errors of Metabolism and Other Genetic Disorders’, approved by the NHGRI Institutional Review Board. All patients or their parents provided written informed consent. The HPS-8 patient was enrolled in protocol NCT00001456, and written informed consent was obtained from his parents.

Whole-mount electron microscopy of platelets

Patient or control blood was mixed with citric acid dextrose (CCD) in a ratio of nine parts blood to one part anticoagulant. Platelet-rich plasma (PRP) was prepared by centrifugation at 200 × g for 20 min at room temperature. Platelet counts and volume were determined using a Coulter HmX Hematology Analyzer (Beckman Coulter, Indianapolis, IN, USA). When necessary, platelet counts were adjusted to 300 000/ml. Small drops of citrate PRP were placed on formvar coated, carbon-stabilized grids (Electron Microscopy Sciences, Hatfield, PA, USA) and rinsed with drops of sterilized water, dried from the edges with filter paper, and air-dried to remove residual moisture. The grids were examined without fixation or staining in a Philips 301 electron microscope (FEI, Hillsboro, OR, USA).

Tissue culture

Primary patient and control fibroblasts and melanocytes were cultured from a forearm skin biopsy. Fibroblasts were grown in high-glucose (4.5 g/l) DMEM supplemented with 10% fetal calf serum (FCS; Gemini Bio-Products, West Sacramento, CA, USA), 2 mm l-glutamine, MEM non-essential amino acid solution and penicillin–streptomycin. Melanocytes were cultured in Ham’s F10 (Invitrogen, Carlsbad, CA, USA), supplemented with 5% FCS, 5 μg/l basic fibroblast growth factor (Sigma, St. Louis, MO, USA), 10 μg/l endothelin (Sigma), 7.5 mg/l 3-isobutyl-1-methylxanthine (Sigma), 30 μg/l choleratoxin (Sigma), 3.3 μg/l phorbol 12-myristate 13-acetate (Sigma), 10 ml pen/strep/glutamine (Invitrogen), and 1 ml fungizone (Invitrogen).

gDNA analysis: sequencing and SNP-array

For BLOC-1 subunits’ gDNA sequencing, primers were designed to cover all coding exons and flanking intronic regions of BLOC1S1 (NT_029419.12), BLOC1S2 (NT_030059.13), BLOC1S3 (NT_011109.16), CNO (NT_006051.18), DTNBP1 (NT_007592.15), MUTED (NT_007592.15), PLDN (NT_010194.17), and SNAPIN (NT_004487.19); primer sequences available on request. Direct sequencing was carried out using the di-deoxy termination method (ABI BigDye Terminator v3.1) on an ABI 3130xl DNA sequencer (Applied Biosystems, Austin, TX, USA). Results were analyzed using sequencher v4.9 software (Gene Codes Corporation, Ann Arbor, MI, USA). The HPS-8 patient’s BLOC1S3 mutation was verified bi-directionally based on accession number NM_212550.3. For SNP genotyping, genomic DNA was run on a Human 1M-Duo DNA Analysis BeadChip and the data analyzed using the genomestudio software (both Illumina, San Diego, CA, USA).

RNA analysis: extraction, cDNA, and qRT-PCR

Total RNA was isolated from control and patient fibroblasts and melanocytes using RNA-Easy Mini-Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. RNA was treated with a DNase kit (DNA-free) to remove all remaining DNA according to the manufacturer’s protocol (Applied Biosystems). RNA concentrations and purity were measured on the Nanodrop ND-1000 apparatus (Nanodrop Technologies, Wilmington, DE, USA). First-strand cDNA was synthesized using a high-capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer’s guidelines. For q-RT-PCR, Taqman gene expression master mix reagent and Assays-On-Demand (Applied Biosystems) were obtained for BLOC1S3 (Assay ID Hs03028695_s1) and a control gene, GAPDH (Assay ID Hs99999905_m1). Q-RT-PCR was performed using 100 ng cDNA, on an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) using the comparative CT method (ΔΔCT); this method measures relative gene expression (Livak and Schmittgen, 2001). The cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, and 40 cycles at 95°C for 15 s, and 60°C for 60 s. For PCR on cDNA, a primer pair (F1-R) was designed entirely within the coding exon of BLOC1S3 and a second pair (F2-R) with the forward primer in the BLOC1S3 non-coding exon 1 and the same reverse primer as before. PCR was performed on cDNA for both primer pairs and a GAPDH primer pair as a control. Products were run on 2% agarose gels.

Antibodies

Mouse monoclonal antibodies against the following proteins were acquired as follows: β-actin (Clone AC-15; Sigma-Aldrich, St Louis, MO, USA), TYRP1 (used for immunofluorescence and internalization assay; clone TA99, ATCC, Manassas, VA, USA), tyrosinase (clone T311) and cappuccino (Clone S-5; both Santa Cruz Biotechnology, Santa Cruz, CA, USA). Rabbit polyclonal antibodies against the following proteins were acquired as follows: TYRP1 (used for immunoblotting) and HPS4 (both Santa Cruz Biotechnology); HPS5 and snapin (both ProteinTech, Chicago, IL, USA). The goat polyclonal antibody against dysbindin was purchased from Santa Cruz Biotechnology, and a sheep polyclonal antibody against TGN46 was obtained from AbD Serotec (Raleigh, NC, USA). The mouse monoclonal antibody against pallidin was a kind gift from Dr. E. Dell’Angelica (UCLA School of Medicine, Los Angeles, CA, USA).

Protein extraction and immunoblotting

Cells were grown to confluency in 75-cm2 flasks, washed twice with ice-cold PBS and scraped into 250 μl of cell lysis buffer containing 50 mm Tris–HCl (at pH7.5), 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 mm EDTA, 1 mm EGTA, 0.27 m sucrose, 1% Triton X-100 and Complete, Mini Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN, USA). Cell lysates were centrifuged (20,000 × g, for 15 min at 4°C); supernatants were removed for immunoblotting. Twenty micrograms of total protein, as determined by the Dc Protein assay (Bio-Rad, Hercules, CA, USA), was loaded onto 4–12% Tris-Glycine gels. For HRP detection, proteins were blotted onto PVDF membranes, or nitrocellulose membranes for Li-Cor detection, using the iBlot transfer system (Invitrogen). After blotting, membranes were probed with the appropriate antibodies and loading was controlled by blotting the same membranes with β-actin. HRP-conjugated secondary anti-mouse or anti-rabbit antibodies (Amersham Biosciences, Piscataway, NJ, USA) and IRDye 800CW conjugated secondary anti-mouse or anti-rabbit antibodies (Li-Cor Biosciences, Lincoln, NE, USA) were used. The antigen–antibody complexes were visualized with an Enhanced Chemiluminescence kit (Amersham Biosciences) or detected using the Li-Cor Odyssey Infrared imaging system. All blots were detected using HRP except for HPS5 and β-actin when appropriate, where Li-Cor detection was used.

Plasma membrane protein biotinylation

For membrane protein biotinylation, control and patient melanocytes were incubated with 500 μl of 0.25 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Thermo Scientific, Waltham, MA, USA) for 30 min at 4°C. This reaction was quenched by incubating with 1 m NH4Cl for 5 min. Protein was then extracted using the protocol above. The biotin-labeled proteins were separated from the total lysates using streptavidin Dynabeads (Invitrogen) according to the manufacturer’s instructions. The protein samples were loaded directly onto SDS-PAGE gels for immunoblotting analysis.

Immunofluorescence microscopy

Cells were grown in 4-well chamber slides and fixed using 4% paraformaldehyde and permeabilized using 0.1% Triton X-100. Alexafluor 488 and 555 secondary antibody conjugates were purchased from Invitrogen, and nuclei were counterstained with DAPI (Vector Laboratories, Burlingame, CA, USA). Cells were imaged with a Zeiss 510 META confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY, USA) with the pinhole set to 1 Airy unit. A series of optical sections were collected from the xy plane and merged into maximum projection images.

TYRP1 internalization assay

This assay was carried out essentially as previously described (Setty et al., 2007). Briefly, control and HPS-8 patient melanocytes were trypsinized and washed twice with Ham’s F10 medium containing 10% FBS and 25 mm HEPES. For each time point, 2 × 105 cells were used and incubated with a monoclonal antibody against TYRP1 on ice for 30 min to allow antibody binding to plasma membrane TYRP1. Cells were then washed twice with medium and transferred to 37°C for different amounts of time (0, 5, 10, 15, 30, and 60 min), after which they were incubated on ice with Alexafluor 488 conjugated secondary antibodies for 30 min. The cells were then washed twice with medium and resuspended in FACS buffer (5% FBS, I mm EDTA in 1× PBS). The samples were sorted on a FACSCalibur (Becton Dickenson, Franklin Lakes, NJ, USA) flow cytometer and green fluorescence quantified and analyzed using flowjo software (Tree Star, Ashland, OR, USA).

Acknowledgments

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgments
  9. References

We appreciate the excellent technical assistance of Roxanne Fischer and Carla Ciccone. We thank Dr. Thomas Markello for assistance with SNP arrays and Dr. E. Dell’Angelica for supplying the pallidin antibody. This study was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA. All authors declare no conflicts of interest.

References

  1. Top of page
  2. Summary
  3. Significance
  4. Introduction
  5. Results
  6. Discussion
  7. Methods
  8. Acknowledgments
  9. References