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From the Amyloid Unit,*
Instituto de Biologia Molecular
e Celular, and the Instituto de Ciências Biomédicas Abel
Salazar,
Universidade do Porto, Porto,
Portugal; the Laboratoire d’Anatomie Pathologique et
Neuropathologique,
Faculté de
Médecine Paul Broca, Bordeaux, France; and the Médecine
Interne,§
Hôpital de Niort, Niort,
France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain,5
lysozyme,6
and
cystatin C.7 Since 1990 several amyloidogenic variants of apoAI, a 28-kd nonglycosylated protein that constitutes the major apolipoprotein of high-density lipoproteins (HDLs),8 have been reported. Most of the described mutations in apoAI are single amino acid substitutions resulting from point mutations in the gene.3,9-11 Two variants involve deletions from the gene in exon 4 that produce a variant protein with either a deletion and insertion of two amino acids12 or a deletion of three amino acids.13 In apoAI amyloidosis, amyloid fibrils are characterized by the deposition of N-terminal fragments of variable length of the mutated protein. No full-length apoAI has been detected so far in apoAI fibrils.
The majority of amyloidogenic apoAI variants carry an extra +1 charge with respect to normal apoAI and have their mutation in the N-terminal region. Gly26Arg,3 the first described variant, is associated with peripheral neuropathy, peptic ulcers, and nephrotic syndrome; Leu60Arg9 and Trp50Arg10 are also associated with renal involvement; and in the deletion variants,12,13 patients not only have renal but also cardiac amyloidosis. It was first hypothesized that the charge or electrostatic alteration might be one of the key features involved in the amyloidogenicity of apoAI variants.
The recently described substitution of proline for leucine at position 90,11 unlike other amyloidogenic apoAI variants, does not produce change in charge from neutral to positive, but is a neutral-to-neutral substitution. This mutation results in a unique clinical presentation of cutaneous amyloid deposition and restrictive cardiomyopathy.
Only very recently a mutation in apoAI was described that is in the C-terminus of the protein—Arg173Pro.14 Despite its C-terminal location, a clinical picture associated with cardiac, larynx, and cutaneous amyloidosis is observed and N-terminal fragments of the protein are found in the fibril deposits.
Here we report a new C-terminal variant of apoAI with a typical clinical picture of cardiac and larynx amyloidosis where co-localization of TTR and apoAI in the deposits occurs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proband is a French 41-year-old woman who was diagnosed in 1993 for dysphonia. Larynx amyloidosis, confirmed on a biopsy, was followed by a laryngoscopy. The left vocal chord presented a volumous polipus and the general aspect suggested a larynx papillomatose. Skin squamous lesions (yellow and maculopapular) were present at the face level, arms, and knees. Heart echography showed a myxoedematous aspect of the mitral and tricuspid valves, features that characterize the beginning of cardiac amyloidoses. Electromyography had signs of peripheral neuropathy with reduction of amplitude of the sensitive potentials. Apart from the symptoms described above, the kindred had no pathological antecedents and serum and urine protein electrophoresis did not detect monoclonal immunoglobulin.
A brother of the proband was deceased at 39 years of age with cardiac amyloidosis. In this case, larynx amyloidosis was also diagnosed after surgery to the vocal chords to overcome dysphonia. Electrocardiographic anomalies led to a myocardial biopsy that confirmed amyloidosis.
Two sisters of the proband have had regular cardiology consultations but amyloidosis has not been diagnosed. Both siblings refused blood/DNA testing. The mother of the proband is alive and healthy at 74 years of age; the father was deceased at 56 years of age with liver cirrhosis.
Histology
For light microscopy, tissues were fixed in 4% neutral buffered formalin at room temperature for 2 hours and embedded in paraffin. Paraffin embedded sections (20-µm thick) were used for histochemistry.
For histochemical demonstration of amyloid, paraffin sections were stained with Congo red and observed under a polarization microscope to detect the characteristic emerald green birefringence emitted from amyloid deposits.
Immunohistochemistry
For TTR and apoAI immunohistochemistry, sections were deparaffinated in xylol, 3 x 10 minutes and dehydrated in a descendent alcohol series (100%, 90%, 80%, and 70%, 10 minutes each). After a 30-minute treatment with 100% formic acid, endogenous peroxidase activity was inhibited with 0.3% hydrogen peroxide (H2O2) in methanol and sections were blocked in blocking solution containing 10% fetal calf serum (Life Technologies, Inc., Grand Island, NY) and 0.2% bovine serum albumin. Rabbit anti-human TTR (DAKO, Glostrup, Denmark) diluted 1:100, or goat anti-apoAI (Calbiochem, La Jolla, CA) diluted 1:100 in blocking solution were added for 3 hours at room temperature. Incubation with anti-rabbit (1:200), or anti-goat (1:200), respectively, Immunoglobulin G (IgG) coupled to horseradish peroxidase (The Binding Site, Birmingham, UK), diluted in blocking buffer, was performed for 1 hour at room temperature. Peroxidase activity was visualized with 3,3'[hyph]diaminobenzidine/H2O2 and the sections were counterstained with Gill’s hematoxylin. On parallel control sections, primary antibody was not used or primary antibodies pre-absorbed with excess antigen (300 µg TTR or apoAI/µl antibody, in a final volume of 100 µl) were used.
Fibril Extraction and Characterization of Amyloid Fibril Proteins
Fibrils from a 15-mg skin biopsy were extracted as described by Kaplan et al.15 Briefly, the tissue specimen was homogenized manually with a glass rod with 1 ml of ice-cold phosphate-buffered saline (PBS) and centrifuged for 10 minutes at 14,000 g. This procedure was repeated 3 times to remove soluble blood components. The resulting pellet was resuspended in 1 ml of 20% acetonitrile containing 0.1% trifluoroacetic acid. The mixture was incubated at room temperature for 1 hour with moderate mixing and centrifuged again. The incubation and centrifugation steps were repeated twice and the supernatants were pooled and lyophilized. The obtained fibril extracts were resuspended in 20 µl of water and used for immunochemical characterization of amyloid proteins. Coomassie-stained 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and anti-TTR and anti-apoAI immunoblotting were performed for the extracted fibrils. Standard apoAI was from Calbiochem. Recombinant TTR was produced and purified according to Almeida et al.16 For immunoblots, proteins were separated on 15% SDS-PAGE gels, transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany), and blocked with 5% skim milk. For TTR detection, rabbit anti-human TTR (DAKO) and anti-rabbit IgG horseradish peroxidase (The Binding Site) were used; for detection of apoAI, goat anti-human apoAI (Calbiochem) and anti-goat IgG horseradish peroxidase (The Binding Site) were used. Monoclonal antibodies against the C-terminus (4A12) and N-terminus (2G11) of apoAI17 were used to identify fragmentation of the protein. All antibodies were diluted 1:500 in PBS-0.05% Tween 20. Immunoblots were developed with chloronaphthol/H2O2.
Scanning of TTR Mutations
Single-strand conformation polymorphism for TTR exons was performed as described by Torres et al.18
MALDI-mass spectrometry analysis was carried out on TTR immunoprecipitated from whole sera with anti-human TTR (DAKO), separated by 15% SDS-PAGE, cut from the gel and digested with endoproteinase Lys-C,19 and peptides separated using a PerSeptive Voyager mass spectrometer in the linear mode.20
ApoAI DNA Sequence Analysis
Genomic DNA was isolated from leukocytes and/or cardiac biopsy by standard procedures.21 The primers used to amplify exons 3 and 4 of apoAI (that correspond to the mature form of the protein) were: for exon 3, E3S and E3A (5'-CCACCCTCAGGGA GCCAGGCTCGG-3' and 5'-TAGGTGAGGACTCGGCCAGTCTGG-3', respectively); exon 4 was amplified by two separate polymerase chain reactions, for the first part of exon 4, E4.1S and E4.1A primers were used (5'-CAGCCCTCAACCCTTCTGTCTCACC-3' and 5'-CAGATGCGTGCGCAGCGCGTCCACA-3', respectively); for the second part of exon 4 E4.2S and E4.2A primers were used (5'-AACGTTTATTCTGAGCACCGGGAAG-3' and 5'-AGCTGCAAGAGAAGCTGAGCCCACT-3', respectively). PCR conditions were 30 cycles of denaturation at 95°C, 1 minute; annealing at 65°C, 1 minute; and extension at 72°C, 1 minute. PCR products were electrophoresed on 1% agarose gels (Life Technologies, Inc.) and stained with ethidium bromide.
Single-strand DNA sequencing was performed using as template one tenth of the above PCR product after 20 minutes of incubation at 37°C with 10 units of exonuclease I (Amersham Pharmacia Biotech, Buckinghamshire, UK) and 2 U of shrimp alkaline phosphatase (Amersham Pharmacia Biotech). Internal primers of each of the amplified PCR products were used for sequencing purposes with T7 sequencing kit (Amersham Pharmacia Biotech) following the manufacturer’s instructions. Samples were electrophoresed on 8% polyacrylamide gels at 2000 V for 3 hours, dried, and exposed overnight for autoradiography.
Restriction Fragment Length Polymorphism (RFLP) Analysis
A region encompassing the mutation was amplified by PCR using the same conditions as above and the following primers: R4-S 5'-ATGTGGACGCGCTGCGCACG-3' and R4-A 5'-GACCTTGAAGCTCTCCAGCA C-3'. The resulting 255-bp PCR amplified DNA fragments were digested with NlaIII at 37°C for 3 hours, electrophoresed on 4% Nusieve GTG agarose gels (FMC, Rockland, ME), and stained with ethidium bromide. Fifty genomic DNA samples from the same geographical region of the proband were also tested by RFLP analysis. The one-kilobase DNA ladder was from MBI Fermentas (Vilnius, Lithuania).
Isoelectric Focusing and Immunoblotting
Delipidated plasma22 was subjected to isoelectric focusing at 2000 V for 5 hours in 7.5% polyacrylamide/4.5 mol/L urea using Pharmalyte, pH 4 to 6.5 (Pharmacia, Kalamazoo, MI). The separated proteins were transferred to a 0.45-µm nitrocellulose membrane (Schleicher and Schull) in 0.1% acetic acid and blocked with 5% skim milk. Immunostaining for apoAI was performed using as first antibody goat polyclonal anti-human apoAI (Calbiochem) and as secondary antibody peroxidase labeled anti-goat immunoglobulins (The Binding Site); immunoblots were developed with chloronaphthol/H2O2.
Lipoprotein Analysis
Total cholesterol and triglyceride levels were measured using standard enzymatic procedures (Boehringer Mannheim, Mannhein, Germany and Sentinel, Birmingham, UK respectively). HDL cholesterol levels were measured after precipitation of apoB-containing lipoproteins using phosphotungstic acid and low-density lipoproteins cholesterol levels were measured using polyvinylsulphate both following the manufacturer’s instructions (Boehringer Mannheim). ApoAI levels were measured by radial immunodiffusion assay (The Binding Site).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of cardiac amyloidosis and neuropathy led us to
perform initially immunostaining of amyloid deposits with anti-TTR in
myocardiac and laryngeal biopsies of the proband and her brother. Both
biopsies were positive for TTR. However, a recent report by Hamidi Asl
et al11
describing a variant apoAI (apoAI Pro90Leu)
related to cardiac and larynx amyloidosis led us to perform apoAI
immunohistochemistry of serial sections of the larynx and skin biopsies
of the proband. Anti-apoAI immunohistochemistry (Figure 1A)
showed massive deposition of apoAI.
The proband’s larynx biopsy was confirmed to contain extensive amyloid
deposits showing typical green birefringence when stained with Congo
red (Figure 1B)
. Anti-TTR immunohistochemistry both with a polyclonal
antibody (Figure 1C)
and a monoclonal antibody (not shown) showed the
presence of TTR in the amyloid deposits, with co-localization with
sites of apoAI fibril deposition. Specificity of TTR staining was
demonstrated by using the primary antibody preabsorbed with antigen
(Figure 1D)
. A skin biopsy from the proband was available and
immunohistochemistry again revealed that apoAI and TTR were present and
co-localized in the fibrils (data not shown).
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, lane 1). One of the major
protein components seen by Coomassie staining has an apparent molecular
weight equivalent to mature apoAI (Figure 2
, lane 2). Presence of both
full-length and fragments of apoAI (Figure 2
, lane 3) was confirmed by
anti-apoAI immunoblot. Full-length apoAI was identified by monoclonal
antibodies against both the N and C terminus of the protein (data not
shown). However, fragments of the protein were only identified by the
monoclonal antibody against the N-terminus of apoAI (data not shown)
thus showing C-terminal fragmentation of apoAI. A TTR fragment of ~11
kd, and high molecular weight products staining for TTR (Figure 2
, lane
4) were seen by anti-TTR immunoblot, further confirming the presence of
both TTR and apoAI in the amyloid deposits.
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The presence of both TTR and apoAI in the amyloid deposits of this
kindred suggested that both proteins could possibly be mutated.
However, scanning of TTR mutations by single-strand conformation
polymorphism analysis did not find evidence for any TTR mutation.
MALDI-mass spectrometry analysis of immunoprecipitated TTR revealed
only peaks of normal TTR (data not shown) further showing that the
protein is not mutated. For the apoAI gene, amplification and
sequencing of exon 4 revealed that the proband (Figure 3A)
and her brother (not shown) were
heterozygous for a single-base substitution in this exon changing the
codon for residue 178 of the mature protein from CTT (Leu) to CAT
(His). The remainder of the sequence was normal. The thymidine for
adenine transition corresponding to the second base of codon 178 of
apoAI results in the creation of a restriction site for
NlaIII. RFLP analysis of the 255-bp product from exon 4 by
digestion with NlaIII originated bands corresponding to the
expected 183-bp and 72-bp digestion products plus the undigested 255-bp
band indicating heterozygosity at this position (Figure 3B)
. To find
out if the mutation represented a polymorphism, 50 DNAs from the same
geographical region of the proband were analyzed by RFLP and were found
negative for the Leu178His mutation (data not shown).
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Delipidated plasma from the proband analyzed by isoelectric
focusing and immunoblotted with anti-apoAI, showed both normal apoAI
forms and an abnormal additional band with a pI corresponding to one
extra positive charge (Figure 4)
. This is
in agreement with the predicted substitution of a neutral amino acid
(Leu) to a positively charged amino acid (His). Total cholesterol, LDL-
and HDL-associated cholesterol, and triglyceride levels were within the
normal range (188 mg/dL, 132 mg/dL, 35 mg/dL, and 104 mg/dL,
respectively). ApoAI levels, 161 mg/dL, were also normal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reasons why mutant forms of apoAI lead to amyloid fibril formation
have not been identified. Unlike other amyloid precursor proteins such
as TTR and immunoglobulin light chain, which are rich in ß structure,
apoAI has little, if any, ß structure. ApoAI has been predicted to
contain a number of 22-residue tandem repeats23,24
that
form amphipathic -helices divided into 2- to 11-residue subhelices
interconnected by ß-turns at Pro or Gly residues, and a number of
flexible loops that allow adaptation of the conformation of apoAI to
varied lipoprotein surfaces and lipid environments. Although several
groups have performed extensive secondary-structure analysis of human
apoAI,25,26
no three-dimensional structure for the
full-length protein has been reported to date. However, the crystal
structure of a truncated form of apoAI (residues 44 to 243) has been
reported27
and shows essentially helices. A model for
association of a four-helix bundle has been proposed. This sort of
structure would be unlikely to rearrange to the ß structure necessary
for fibril formation. Several hypothesis have been raised to explain
the amyloidogenicity of apoAI:
First, as all of the first described amyloidogenic variants for apoAI had a +1 alteration in charge, it was first hypothesized that the charge or electrostatic alteration, also observed in the mutant here described, might be one of the key features involved in the amyloidogenicity of apoAI variants. However, the report of the neutral-to-neutral substitution Leu90Pro,11 argues against this hypothesis.
Second, until very recently, all of the described amyloidogenic
variants of apoAI presented the N-terminus of the protein (amino acids
1 to 93) incorporated into amyloid fibril deposits. The deposition of
the amyloidogenic variants in the form of N-terminal fragments was
thought to be due to a local distortion caused by the N-terminal
mutation that would change lipid-apoAI interactions and/or expose sites
for proteolytic cleavage. It was speculated that during the catabolic
processing of the protein, the N-terminal portion could assume a ß
structure after being proteolytically released from the -helical
carboxyl portion of the apoAI molecule. The Arg173Pro
mutation,14
in the C-terminus of the protein argues
against the importance of the N-terminus of apoAI for amyloid formation
and this is further reinforced by our description of a new C-terminal
amyloidogenic apoAI variant. In the recently described apoAI Leu174Ser
mutant,28
amyloid fibrils are constituted by the
93-residue N-terminal polypeptide. In this case, visualization of the
mutation in the three-dimensional structure of lipid-free apoA-I,
composed of four identical polypeptide chains, indicated that position
174 of one chain is located near position 93 of an adjacent chain. A
new model for apoAI amyloid formation was therefore
suggested28
in which the amino acid replacement in
position 174 was permissive for a proteolytic split at the C-terminal
of Val93, leading to the deposition of N-terminal fragments of the
protein.
Third, decreased plasma HDL cholesterol levels in carriers of the Arg173Pro mutation14 suggested an increased rate of catabolism as has been shown for the amyloidogenic Gly26Arg mutation.29 It was speculated, that the normal metabolism/catabolism may be altered, leading to significant changes in lipoprotein composition and subclasses, producing the amyloid deposition. In the Leu178His mutation, all of the cholesterol-related parameters were within the normal range, the same happening to the levels of circulating apoAI. This finding argues against an altered metabolism/catabolism for apoAI as an important feature in apoAI fibril formation.
Therefore, additional factors should be responsible for apoAI amyloidosis as well as for the determination of location and clinical effects of amyloid deposition. It is interesting to note that the clinical presentation of Leu178His, with cardiac amyloidosis and deposition of amyloid in the skin is very similar to what has been described for the deletion variants and for the Pro90Leu and Arg173Pro variants.
Our finding of wild-type TTR fragments in the apoAI Leu178His amyloid fibrils raises several questions; namely whether nonmutated wild-type TTR interferes with deposition of apoAI. It is worth to mention that so far, the only described form of wild-type TTR deposition is senile systemic amyloidosis, a noninherited condition that affects 25% of people older than 80 years of age.30 In this case, nonmutated full-length and fragments of wild-type TTR form deposits in the heart which are probably related to the high content of ß structure of the protein. It is also interesting to note that mutated TTR preferentially deposits in the peripheral nerve and heart which are also sites of apoAI amyloid deposition.
Similarly to what has been described to all of the other apoAI amyloidogenic variants,3,9-14 the fragments seen on the fibrils correspond to C-terminal fragmentation of the variant protein. Further studies are necessary to analyze TTR and apoAI composition in apoAI Leu178His amyloidosis, namely to determine the sites of cleavage. However, in contrast to all reports of apoAI amyloidosis that exist so far, in Leu178His the fibrils contain full-length apoAI. Whether this corresponds to wild-type and/or mutated protein has to be investigated in the future.
We have previously determined that, in most individuals, a small percentage of TTR (1 to 2%) circulates in plasma bound to HDL and that the interaction of the protein with the lipoprotein vesicle occurs through binding to apoAI.31 The physiological meaning of this observation remains to be explained, but the present report further suggests that, indeed, TTR and apoAI interaction might be relevant not only in physiological conditions but also in amyloidosis.
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Acknowledgements |
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Footnotes |
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Supported by grants BIA/459/94 and SAU/1290/95 from PRAXIS XXI, Portugal. M. M. Sousa is a recipient of a scholarship from the Gulbenkian PhD Program in Biology and Medicine.
Accepted for publication February 9, 2000.
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