The intracellular quality control system down-regulates
the secretion of amyloidogenic apolipoprotein A-I
variants: A possible impact on the natural history of the
disease
Marta Marchesi, Cinzia Parolini, Caterina Valetti, Palma Mangione, Laura
Obici, Sofia Giorgetti, Sara Raimondi, Simona Donadei, Gina Gregorini,
Giampaolo Merlini, et al.
To cite this version:
Marta Marchesi, Cinzia Parolini, Caterina Valetti, Palma Mangione, Laura Obici, et al.. The
intracellular quality control system down-regulates the secretion of amyloidogenic apolipoprotein A-I variants: A possible impact on the natural history of the disease. BBA - Molecular
Basis of Disease, Elsevier, 2010, 1812 (1), pp.87. .
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The intracellular quality control system down-regulates the secretion of
amyloidogenic apolipoprotein A-I variants: A possible impact on the natural
history of the disease
Marta Marchesi, Cinzia Parolini, Caterina Valetti, Palma Mangione, Laura
Obici, Sofia Giorgetti, Sara Raimondi, Simona Donadei, Gina Gregorini,
Giampaolo Merlini, Monica Stoppini, Giulia Chiesa, Vittorio Bellotti
PII:
DOI:
Reference:
S0925-4439(10)00129-8
doi: 10.1016/j.bbadis.2010.07.002
BBADIS 63127
To appear in:
BBA - Molecular Basis of Disease
Received date:
Accepted date:
1 July 2010
2 July 2010
Please cite this article as: Marta Marchesi, Cinzia Parolini, Caterina Valetti, Palma Mangione, Laura Obici, Sofia Giorgetti, Sara Raimondi, Simona Donadei, Gina Gregorini,
Giampaolo Merlini, Monica Stoppini, Giulia Chiesa, Vittorio Bellotti, The intracellular
quality control system down-regulates the secretion of amyloidogenic apolipoprotein A-I
variants: A possible impact on the natural history of the disease, BBA - Molecular Basis
of Disease (2010), doi: 10.1016/j.bbadis.2010.07.002
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The intracellular quality control system down-regulates the
secretion of amyloidogenic apolipoprotein A-I variants: a possible
impact on the natural history of the disease
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Marta Marchesia, Cinzia Parolinia, Caterina Valettib, Palma Mangionec, Laura
Obicid, Sofia Giorgettic, Sara Raimondic, Simona Donadeid, Gina Gregorinie,
Department of Pharmacological Sciences, Università degli Studi di Milano, via Balzaretti
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a
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Giampaolo Merlinic,d, Monica Stoppinic, Giulia Chiesaa, Vittorio Bellottic,d,*
b
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9, 20133 Milan, Italy
MicroSCoBiO Research Center and IFOM Center of Cell Oncology and Ultrastructure,
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Dipartimento di Medicina Sperimentale, Università di Genova, via de Toni 14, 16132
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Genoa, Italy
Department of Biochemistry, University of Pavia, via Taramelli 3b, 27100 Pavia, Italy
d
Biotechnology Research Laboratories, IRCCS Fondazione Policlinico San Matteo, Viale
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Golgi 2, 27100 Pavia, Italy
e
Divisione di Nefrologia, Spedali Civili, Piazza Spedali Civili 1, 25123 Brescia, Italy
*Corresponding author. Fax: +39 0382 423108.
E-mail addess: [email protected] (Vittorio Bellotti).
Abbreviations: apoA-I, apolipoprotein A-I, HDL, high-density lipoproteins; IFLM, immunofluorescent light microscopy; ER, endoplasmic reticulum
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ABSTRACT
Hereditary systemic amyloidosis caused by apolipoprotein A-I variants is a dominantly
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inherited disease characterised by fibrillar deposits mainly localized in the kidneys, liver,
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testis and heart. We have previously shown that the apolipoprotein A-I variant circulates in
plasma at lower levels than the wild-type form (Mangione P et al. 2001, Obici L et al. 2004)
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thus raising the possibility that the amyloid deposits could sequester the circulating
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amyloidogenic chain or that the intracellular quality control can catch and capture the
misfolded amyloidogenic chain before the secretion. In this study we have measured
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plasma levels of the wild-type and the variant Leu75Pro apolipoprotein A-I in two young
heterozygous carriers in which tissue amyloid deposition was still absent. In both cases,
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the mutant was present at significantly lower levels than the wild-type form, thus indicating
that the low plasma concentration of the apolipoprotein A-I variant is not a consequence of
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the protein entrapment in the amyloid deposits. In order to explore the cell secretion of
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amyloidogenic apolipoprotein A-I variants, we have studied COS-7 cells expressing either
wild-type apolipoprotein A-I or two amyloidogenic mutants: Leu75Pro and Leu174Ser.
Quantification of intracellular and extracellular apolipoprotein A-I alongside the intracytoplasmatic localization indicates that, unlike the wild-type protein, both variants are
retained within the cells and mainly accumulate in the endoplasmic reticulum. The low
plasma concentration of amyloidogenic apolipoprotein A-I may therefore be ascribed to the
activity of the intracellular quality control that represents a first line of defence against the
secretion of pathogenic variants.
Keywords: apolipoprotein A-I, amyloidogenic variants, quality control, protein folding
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1. Introduction
Systemic amyloidoses caused by peculiar variants of globular proteins such as lysozyme,
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transthyretin and apolipoprotein A-I (apoA-I) are late onset, autosomal dominant genetic
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diseases [1]. Each amyloidogenic globular protein causes a different clinical syndrome
since different tissues are involved. Moreover, different variants of the same protein can
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target different tissues and, in a few cases, heterogeneous phenotypes are reported even
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in the presence of identical mutations [2]. Additional heterogeneity is related to the onset
of the disease that can be highly variable even for the same mutation [3]. A key element in
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the formation of fibrils is represented by the concentration of the amyloidogenic precursor
both in the blood and the extracellular matrix at the site of amyloid deposition [4]. A
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seminal observation regarding the mechanisms responsible for the selectivity of
transthyretin deposition was reported by the Kelly's group. They hypothesized that tissue
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damage can be related to the local concentration of the protein, modulated by the variable
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efficiency of the quality control of protein folding and secretion at different sites of
synthesis [5]. No data have been so far reported about the possible role of the quality
control in modulating the plasma concentration of amyloidogenic apoA-I. Patients
heterozygous for apoA-I amyloidogenic mutations are frequently characterized by
hypoalphalipoproteinemia, with high density lipoproteins (HDL) being never below 50% of
the normal value [6]. Our research group was the first to observe that the wild-type chain is
far more abundant than the mutated chain in the HDL of patients heterozygous for either
Leu174Ser [7] and Leu75Pro variants [8]. Similar findings were reported for the Leu64Pro
mutation by the Solomon’s group [9]. Different mechanisms, acting independently or in
association one with the other, might be involved in the altered metabolism of the mutated
protein compared to its normal counterpart, either at the level of protein secretion during
the folding process, or after its secretion at the stage of apoA-I function and metabolism in
HDL. Clinical data and in vitro results of the present manuscript suggest that the low
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plasma concentrations of the apoA-I amyloidogenic variants largely depends on their
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impaired cellular secretion.
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2. Materials and methods
2.1 Comparative quantification of variant/wild-type chain in HDL.
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HDL were separated from plasma of two selected carriers of the Leu75Pro mutation by
sequential ultracentrifugation and apoA-I was purified as described by Calabresi et al. [10].
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Quantification of wild-type and Leu75Pro apoA-I was indirectly derived, as previously
described [7], from the measurement of specific peptides obtained from the digestion of
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apoA-I with V8 protease (Pierce). The digestion was carried out in bicarbonate buffer (50
mM at pH 8.0, 2mM EDTA) for 12 h at 37°C at an enzyme/protein ratio of 1:50 (w/w).
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Peptides obtained were then separated by reversed phase HPLC through a C18 column
(Vydac) at a flow rate of 1.5 mL/min. The mobile phases used were as follows: solvent A
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(0.05% trifluoroacetic acid (TFA) in water) and solvent B (0.05% TFA in acetonitrile).
Peptides were eluted by a gradient of 5%–100% solvent B over 60 min. The elution of
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peptides was monitored by UV absorbance at 220 nm and by diversion of 20% of the
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column flow into the mass spectrometer. Mass spectra were obtained on a Finnigan LCQ
ion trap mass spectrometer (Finnigan) with electrospray ionization (ESI). The ESI spectra
(positive ion mode) were collected from samples of 20pmoles/µL. The instrument was
calibrated by caffeine (m/z = 195), the peptide MRFA (m/z = 525), and ultramark (m/z =
1022, 1122, 1222, 1322, 1422, 1522, 1622).
2.2 In vitro mutagenesis of apoA-I cDNA.
A pRc/RSV plasmid containing the cDNA of the wild-type human apoA-I (kindly
provided by Dr. Alessandro Sidoli) was used as a template to create the two
amyloidogenic variants Leu174Ser and Leu75Pro. Mutations were carried out using an in
vitro QuikChange site-directed mutagenesis kit (Stratagene) and the appropriate
mutagenic primers. Leu174Ser ApoA-I vector was generated by using 5’-CTG CGC CAG
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CGC TCG GCC GCG CGC CTT GAG-3’ and 5’-CTC AAG GCG CGC GGC CGA GCG
CTG GCG CAG-3’ as forward and reverse primers, respectively. Similarly Leu75Pro apoA-
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I was created by using 5’-TGG GAT AAC CCG GAA AAG GAG ACA GAG-3’and 5’-CTC
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TGT CTC CTT TTC CGG GTT ATC CCA GAA CTC-3’ as forward and reverse primers.
The underlined codons represent the site of mutation. Mutagenesis was performed
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according to the manufacturer's protocol.
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2.3 Transient transfection of COS-7 cells.
COS-7 (monkey kidney) cells were plated on 6-well culture dishes the day prior to
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transfection at a density of 1.5x106 cells/plate in Dulbecco’s modified Eagle’s medium
(DMEM; Gibco Brl) supplemented with streptomycin-penicillin, L-glutamine (4mM), glucose
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(4500 mg/L), sodium carbonate (1500 mg/L) and 10% foetal bovine serum (FBS).
Subconfluent COS-7 cells were rinsed once with serum-free and antibiotic free medium
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(incomplete DMEM; Gibco Brl), and transfected with wild-type or mutated apoA-I cDNAs
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using the liposome transfection method (Lipofectamine 2000, Invitrogen). All transfections
were performed in triplicate. Transfection was carried out for 6 hours by adding 5µg/well
DNA and 2.5 µL/well of Lipofectamine 2000 to serum free transfection medium.
Transfected cells were washed twice with phosphate-buffered saline (PBS) to remove the
precipitate and than incubated with 2 mL DMEM containing 10% FBS for an additional 12
hours. Cell culture media were then replaced with serum-free media supplemented with
streptomycin-penicillin and harvested after 48 hours. Cells were then washed with PBS
and
lysed
with
25mM
Tris-phosphate,
diaminocyclohexane-N,N,N’,N’-tetraacetic
2mM
acid,
dithiothreitol
10%
glycerol,
(DTT),
1%
Intracellular extracts and supernatants were stored at -20°C until analysis.
2.4 Quantification of apoA-I expression and secretion.
2mM
Triton
1,2-
X-100).
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The culture media and the cell lysates normalized for the cellular protein content,
together with increasing concentrations of purified human apoA-I were loaded onto a 12%
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SDS-PAGE and proteins were then transferred to polyvinylidene difluoride membranes
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(HyBond-P, GE Healthcare). Membranes were incubated with a polyclonal rabbit antihuman apoA-I antibody (Dako, Denmark, 1:1000 dilution), and subsequently with a
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polyclonal swine-anti-rabbit horseradish peroxidase (Dako, Denmark, 1:10000 dilution). To
exclude any significant difference in affinity of the polyclonal antibodies for wild-type and
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mutated species, we have tested these antibodies against recombinant wild-type and
variants, Leu75Pro and Leu174Ser, both expressed and purified from CHO cell lines. Both
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the Western blot, performed after SDS-PAGE of denatured apoA-I, and dot blot, directly
the polyclonal antibodies.
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performed on native apoA-I, demonstrated that the mutation does not affect the affinity of
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Immunoblots were developed with an enhanced chemiluminescence system (ECL Plus,
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GE Healthcare) and apoA-I was visualized on autoradiography film (Hyperfilm, GE
Healthcare). Images were imported into Multi Gauge (FujiFilm) and apoA-I concentration
in the samples was calculated based on the standard apoA-I curve. Non parametric
Kruskal-Wallis test was used to estimate the significance of the differences between
intracellular and extracellular levels of each variant versus wild-type.
Transfection efficiency was evaluated on cell lysates by real-time PCR analysis. DNA was
purified from aliquots of cell lysates per each well, by phenol/chloroform extraction and
wild-type, Leu174Ser or Leu75Pro apoA-I genes were quantified on an Applied
Biosystems 7900 sequence detector using SYBR® Green I and the following specific
primers:
sense,
ATCGAGTGAAGGACCTGGC
and
antisense,
AGCTTGCTGAAGGTGGAGGT. The primers amplify a 154-bp product. Real-time
detection was performed in a volume of 25 μL containing 100 nmol/L of each primer and
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iTaq SYBR Green Supermix with ROX 2X as recommended by the manufacturer (BioRad). Conditions were 95°C for 10 minutes, followed by 40 cycles of 30 seconds at 95°C,
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30 seconds at 55°C and 30 seconds at 72°C. A final melting curve guaranteed the
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authenticity of the target product. The housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used for normalization, with the following specific primers:
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sense, CTTCACCACCATGGAGAAGGC and antisense, AGTTGTCATGGATGACCTTGG
manufacturer's recommendations.
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2.5 Immunofluorescence microscopy.
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[11]. All of the procedures and calculation of the results were carried out according to
COS-7 cells, plated on 10-mm2 coverslips 24 h before transfection, were transfected
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with the cDNAs of wild-type, Leu174Ser or Leu75Pro apoA-I basically as reported in the
transient transfection paragraph, and analyzed 48 h later. In the experiments, the
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expression of wild-type and apoA-I mutants was carried out simultaneously in order to
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analyze the expression under identical transfection conditions. 15-18 hours before fixation,
cell culture media were replaced with serum free media supplemented with streptomycinpenicillin. Cells were fixed with 4% paraformaldehyde (PFA) in PBS pH 7.4 and
permeabilized with 0.1% saponin (Sigma). Primary and secondary antibodies were
incubated for 20 min with 0.1% saponin in PBS. The following antibodies were used: a
rabbit polyclonal anti-human apoA-I (DAKO), a monoclonal anti-GM-130, identifying the
Golgi structure (BD Transduction Labs), a monoclonal anti-PDI, a marker of the
endoplasmic reticulum (Abcam) and a monoclonal anti-LAMP2, a lysosomal protein (clone
H4B4 kindly provided by the Developmental Studies Hybridoma Bank University of Iowa,
Iowa City, IA, USA) [12]. Secondary antibodies were immunoadsorbed FITC-conjugated
donkey anti-mouse IgG and TRITC-conjugated donkey anti-rabbit IgG and purchased from
Jackson ImmunoResearch Laboratories. Labelled cover-slips were mounted using Mowiol
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reagent (Calbiochem) and were inspected on an Olympus inverted fluorescence
microscope (model IX70). Images were recorded with a digital CCD camera (model
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C4742-95, Hamamatsu), using the IPLab spectrum V3.2 software package (Scanalytics,
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Inc.) and processed with Adobe Photoshop 7.0 (Adobe Systems, Inc.).
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3. Results
3.1 Clinical case
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A 28-year-old male and a 44-year-old woman were positive for the Leu75Pro mutation
at pre-symptomatic genetic test performed for family history of apoA-I amyloidosis. They
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were subsequently investigated to exclude early signs of the disease. Serum creatinine,
urinalysis, transaminases, alkaline phosphatase, N-Terminal pro-Brain Natriuretic Peptide,
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gonadotropins and testosterone levels, in the male patient, were all within the reference
range. Total cholesterol, HDL-cholesterol and apoA-I plasma concentrations were 103
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mg/dL (u.r.l. < 200), 35 mg/dL (normal levels ≥ 45) and 105 mg/dL (reference range
110-205) in the young man and 140 mg/dL, 43 mg/dL (normal levels ≥ 55) and 113 mg/dL
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(reference range 125-215) in the woman, respectively. Abdominal ultrasound showed no
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abnormalities. An abdominal fat aspirate was negative for amyloid deposits in both
subjects. After written informed consent, a 15 mL plasma sample was obtained for
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purification of total apoA-I from their circulating HDL.
3.2. Quantification of the amyloidogenic apoA-I polypeptide in HDL
In the absence of an efficient chromatographic method suitable for the separation of the
mutant from the wild-type apoA-I, the problem of measuring both apoA-I species in
heterozygous carriers of amyloidogenic mutations was by-passed through the analysis of
the V8 protease digest derived from the full-length protein [7]. The concentration of the
mutated polypeptide in the young pre-symptomatic carrier of the Leu75Pro mutation was
even lower than expected on the basis of the results obtained from previously described
patients [8] and represented less than 10% of the total apoA-I chain. As shown in Fig. 1,
the mutated chain in the young male was detectable in mixture in the HPLC
chromatograms. Similar results were obtained using HDL from the second carrier
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analyzed (data not shown).
3.3. Expression and secretion of the amyloidogenic variants in mammalian cell models
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The efficiency of secretion of wild-type apoA-I and Leu75Pro or Leu174Ser variants was
examined in transiently transfected COS-7 cells.
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Transfection efficiency was measured in each experiment and no significant differences
were found between wild-type apoA-I and mutants (P>0.05). The average amount of
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Leu174Ser cDNA was 91.7±9.0% and that of Leu75Pro was 107.0±12.3% vs cDNA of
wild-type apoA-I.
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ApoA-I levels in both cell lysates and culture media were visualized by western blotting
analysis (Fig. 2a). ApoA-I was quantified in all samples using a calibration curve
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constructed with known concentrations of apoA-I (Fig. 2a) and a linear plot in the
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concentration range between 0.054 and 1 ng/µL was obtained (data not shown). The
results of three separate experiments are reported in Fig. 2b where intra- and extra-
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cellular distribution of wild-type or mutant apoA-I are compared. These results clearly
indicate that the relative amounts of the apoA-I secreted in the medium and the intracytoplasmatic apoA-I are completely different when the cells synthesize the wild-type
rather than the amyloidogenic variants. Both the Leu174Ser and Leu75Pro variants are
largely retained within the cells whereas wild-type apoA-I is mostly secreted in the
medium. Quantification of total (intra- and extra-cellular) wild-type and mutant apoA-I
indicates that Leu174Ser and Leu75Pro are present at lower amounts than the wild-type
form. Particularly, the total Leu174Ser was estimated 63 ± 4.9 ng, the total Leu75Pro was
74 ± 13 ng, whereas wild-type apoA-I was 120 ± 30 ng.
3.4. Sub-cellular localization of the amyloidogenic variants in mammalian cell models
The intracellular distribution of wild-type and apoA-I mutants in COS-7 cells was
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investigated by immunofluorescence microscopy. Cells transfected with wild-type apoA-I
showed an intracellular distribution of the protein characterized by a strong localization in
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the Golgi and faint presence in the endoplasmic reticulum (ER); almost 87% of the cells
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exhibit this intracellular distribution, as shown in Fig. 3 a-d. On the contrary, apoA-I
mutants displayed an opposite localization, with a prevalent accumulation in the ER and a
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scarce presence in the Golgi, as shown in panel e-f for Leu75Pro, and in panel g-h for the
Leu174Ser mutant. Particularly, the phenotype related to the Leu174Ser mutant was more
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extreme as 34% of the cells showed an exclusively ER pattern suggesting a block of
secretion. Interestingly, both apoA-I mutants showed around 30% of the expressing cells
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with vacuolar and cytosolic pattern (Fig. 4a) indicating cytosolic protein accumulation. This
finding suggests that the quality control degradative pathway has reached saturation as a
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consequence of reduced secretion of both the mutated apoA-I species. Those vacuolar
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structures were further characterized, showing that they did not display co-localization
neither with the ER marker PDI, nor with the golgian GM130 protein (not shown), or with
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the lysosomal protein Lamp2 (Fig. 4b-d) that suggests a similar nature to the cytoplasmic
droplets [13].
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4. Discussion
A general assumption regarding the mechanisms of hereditary amyloidosis is that, despite
their propensity to misfold, the pathogenic variants escape the quality control system, and
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are secreted as much as the wild-type counterpart [14]. For certain amyloidogenic proteins
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such as fibrinogen [15] and several transthyretin variants [16] this assumption is
experimentally confirmed by the presence, in plasma, of an equimolar balanced
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concentration of both variant and wild-type chains. These data would suggest that the
intracellular quality control, which is accustomed to remove not properly folded proteins,
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cannot detect the intrinsic propensity to misfold and aggregate caused by these mutations.
However, in the case of peculiar transthyretin species like the Asp18Gly and Ala25Thr
variants, the effect of these mutations on protein conformation is apparently recognized by
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the quality control system and these peculiar variants are significantly retained in the ER of
certain cellular models [5] and forced to be degraded [17]. The natural clinical history of
Asp18Gly carriers is characterized by the presence of amyloid in the leptomeninges
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without any deposits in other tissues. Such a discrepancy was apparently dictated by a low
efficiency of the quality control of choroid cells in recognizing and degrading this
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amyloidogenic variant whereas a more effective quality control system of liver cells is
capable of recognizing and blocking the secretion of the pathogenic variant. The down-
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regulation of secretion of the amyloid protein precursor and the consequent reduction of its
concentration in the body fluids can therefore represent a first line of defence of the
organism against the disease. Any clinical and biological evidence suggesting the capacity
of the cell to recognize the abnormal conformation of amyloidogenic protein before the
secretion should be carefully studied because it could shed light on specific features of the
disease and can represent a highly sensitive therapeutic target. Regarding the hereditary
amyloidosis associated to mutations in the apoA-I gene, we were the first to discover a
disequilibrium between wild-type and the amyloidogenic variant in circulating HDL of
symptomatic patients carrying the mutation Leu174Ser or Leu75Pro (7-8) and displaying
histological evidence of amyloid deposits. That observation was then confirmed in other
subjects with different mutations, such as the Leu64Pro [9] and the Gly26Arg [18]. The
population expressing Leu75Pro apoA-I is the largest so far discovered for this type of
amyloidosis. More than 200 subjects heterozygous for this mutation have been identified
and they represent a highly heterogeneous variety of clinical cases that are extremely
informative on the natural history of the disease. In particular, we have confirmed the
disequilibrium between the wild-type and the mutated chain, observed in symptomatic
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patients, also in two young carriers of the mutation that were pre-symptomatic and free of
any biochemical and histological evidences of the disease. These data are clearly against
the hypothesis that the low plasma concentration of the Leu75Pro variant can be simply
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ascribed to the absorption of the amyloidogenic chain on the amyloid fibrils and on the
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contrary strengthen the idea that a reduced cellular secretion might substantially contribute
to the chain imbalance in HDL. The latter hypothesis has been explored by studying the
expression and the secretion of two amyloidogenic variants, Leu174Ser and Leu75Pro, in
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a mammalian cell line, COS-7 cells. These non-hepatic, non-lipoprotein-producing cells do
not express endogenous apoA-I, constitutively. COS-7 cells have been previously used for
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monitoring the intracellular trafficking and the expression of variants of human apoA-I [19]
since they can produce and secrete apoA-I upon transient transfection even if with a lower
efficiency compared with hepatic cells. Our results show that the total amount of the
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secreted amyloidogenic variants was lower than that of wild-type apoA-I. No differences in
transfection efficiency were detected among apoA-I types, therefore an intracellular
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degradation of the two amyloidogenic variants could explain the observed results.
A remarkable difference in the ratio between intracellular and secreted apoA-I was found
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in COS-7 cells expressing the wild-type or the amyloidogenic variants. The amyloidogenic
Leu174Ser and Leu75Pro apoA-I resulted entrapped in the ER, whereas the wild-type was
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mainly localized in the Golgi. Furthermore, both the cytoplasm outside the ER and the
Golgi contained apoA-I aggregates adopting a doughnut like shape that remind the apoA-I
aggregates localized in the cells expressing other variants of apoA-I associated with
hypoalphalipoproteinemia rather than amyloidosis [20]. The cells appeared unable to
dispose of the misfolded and aggregated apoA-I that was sequestered in the ER and well
separated from the lysosomal system, which, on the contrary, is known to efficiently
interact with amyloidogenic proteins and contribute to their catabolism as reported in other
models of intracellular accumulation of amyloidogenic proteins [21]. Altogether our results
suggest that circulating levels of apoA-I variants are significantly affected by an
impairment in their secretion. It should be noted that an impaired secretion would not be a
unique feature of amyloidogenic apoA-I variants, since it has been also demonstrated in
primary hepatocytes for non-amyloidogenic apoA-I mutations [22-23]. In addition to an
impaired secretion, data reported by Rader et al [24], regarding the in vivo metabolism of
the Arg26Gly apoA-I, clearly demonstrated a reduced half-life of the variant in comparison
to the wild-type, thus suggesting that the low plasma level of the variant might be also due
to a faster uptake of the protein in a metabolically active target tissue. Overall, how even
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very low plasma concentrations of the variants may cause the disease at target tissues
remains an intriguing and open issue. It is possible that the persistently low concentration
of the precursor may be at the basis of both the slow progression of the disease and the
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relatively benign phenotype of this condition, thus representing a protective mechanism.
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Acknowledgements
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We are very grateful to Dr. Alessandro Sidoli for providing the pRC/RSV plasmid with the
cDNA of wild-type human apoA-I. This work was supported by MIUR (PRIN 2007XY59ZJ,
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2007AE8FX2 and 20083ERXWS); EURAMY project that has received research funding
from the European Community’s Sixth Framework Program; Fondazione Cariplo (Milano,
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Italy; Nobel Project, Transcriptomics and Proteomics Approaches to Diseases of High
Sociomedical Impact: A Technology-Integrated Network; and project 2007.5151); and
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(526D/63).
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Ricerca Finalizzata Malattie Rare, Ministero della Salute, Istituto Superiore di Sanità
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Legend to Figures
Fig. 1. (a) Reversed-phase chromatography of V8 protease digestion of apoA-I purified
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from HDL of a young carrier of the Leu75Pro mutation without clinical evidence of amyloid
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deposits. The peptide indicated as 71–78 (sequence: FWDNLEKE) presents a missed
cleavage site. 71-78* (sequence: FWDNPEKE) is the peptide containing Pro75. (b) ESI-
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Ion Trap Mass spectra of peptide 71–76 (eluted at 21.01 minutes), peptide 71–78 (eluted
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at 19.15 minutes) and peptide 71–78* carrying mutation (eluted at 14.55 minutes) shown
in panel A.
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Fig. 2. ApoA-I expression in COS-7 cell line. Wild-type apoA-I levels in COS-7 cytoplasm
and medium were compared with Leu174Ser and Leu75Pro variants respectively.
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were analysed by Western blotting as described in the Material and Methods section in
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three separate experiments. Results of one experiment, for each variant, are reported in
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panel a. Average values (mean ±SD) for the three experiments on each mutant are given
in panel b, as percentage of the total amount of expressed protein. Statistical significance
for pair wise comparison (variant versus wild-type) is represented by * for P< 0.05.
Fig. 3. ApoA-I expression in COS-7 cells. Exogenous apoA-I was expressed for 48h.
Panel a shows wild-type apoA-I distribution in the Golgi compartment as displayed by the
co-localization with the Golgi matrix protein GM130 (cis-Golgi marker) in panel b. No
evident co-localization is present between wild-type apoA-I in panel c, and the protein
disulphide isomerase (PDI), ER marker in panel d. Panels e-h show mutant apoA-I
distribution in comparison with cis-Golgi staining, for Leu75Pro in panel e-f, and
Leu174Ser in panels g-h, respectively.
Fig. 4. ApoA-I expression and vacuolar structures. Panel a shows vacuolar and cytosolic
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staining for apoA-I Leu174Ser mutant. No evident co-localization is detected between
apoA-I positive vacuoles and the lysosomal protein Lamp-2, as shown in panels c, d and
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in the overlay, panel b, in wild-type cells.
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Fig. 3
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Research highlights
Amyloid formation in vivo is highly dependent by the concentration of the amyloidogenic
precursors > In carriers of amyloidogenic ApoA-I, concentration of the pathogenic chain is
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lower than the wild type > The intracellular QC detects and down regulates the secretion of
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the misfolded ApoA-I >This process represents a first line of defence against the disease