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1 Light Chain Deposition Disease
From the Department of Pathology,*
New York University
School of Medicine, New York, New York; the Facultad de
Química,
Universidad de la República Oriental del
Uruguay, Montevideo, Uruguay; and the Biosciences
Division,
Argonne National Laboratory,
Argonne, Illinois
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 light chains from patients with LCDD are
described and compared to seven other reported -LCDD proteins. The
N-terminal amino acid sequences of light chain GLA extracted from the
renal biopsy and light chain CHO from myocardial tissue were each
identical to the respective light chains isolated from the urines and
to the V-region amino acid sequences translated from the cloned cDNAs
obtained from bone marrow cells. The germline V-region
sequences, determined from the genomic DNA in both and in
MCM, a previously reported 1 LCDD light
chain, were identical and related to the L12a germline gene.
The expressed light chains in all three exhibit amino acid
substitutions that arise from somatic mutation and result in increased
hydrophobicity with the potential for protein destabilization and
disordered conformation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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than light
chain, have a patchy distribution within and among systemic organs, and
colocalize with amyloid P component, apolipoprotein-E (apo-E), and
glycosaminoglycans (GAGs).2,3
In contrast, the deposits in
LCDD are noncongophilic, have a granular rather than fibrillar
ultrastructure, and are more frequently than . In cases
completely examined at autopsy, deposits are uniformly distributed in
all the basement membranes of systemic organs4,5
and do
not associate with amyloid P component or apo-E.2
These
differences are of fundamental importance in determining the mechanisms
of protein deposition in tissues and the process of fibrillogenesis
possibly relevant to other common types of amyloidosis, such as
Alzheimer’s disease. However, the biophysical basis for their
differences is poorly understood. Knowledge of the primary structure of the protein deposits in LCDD is very limited; in only nine cases of LCDD has the complete light chain variable region (V-region) sequence been published.6-13 Thus, a comparison of the nonfibrillar and fibrillar forms of deposits in LCDD and AL that could identify differences in their primary structures and might relate to their dissimilar properties is hampered by the apparent scarcity of cases of LCDD to study and the unavailability of large amounts of tissues from postmortem examinations for biochemical analysis. This limitation is now partially overcome by microextraction methods to isolate and obtain the amino-terminal sequence of light chain deposits from milligram amounts of diagnostic biopsy tissues,14,15 as well as the application of molecular techniques to obtain the light chain V-region amino acid sequence translated from cloned cDNA of bone marrow cells. These methods, applied to more readily available biopsy tissues, allow the opportunity to build a primary structure data base for comparison with nonpathogenic Bence-Jones light chains as well as those in AL disease, with the goal of elucidating the mechanism(s) of tissue deposition and fibrillogenesis.
In a previous case of LCDD, we reported the biochemical data of a
V-region 1 (V-1) nonamyloidotic
immunoglobulin light chain, MCM, obtained by extraction of deposits
from myocardial tissue in which five unique amino acid substitutions
were identified.11
We now report two new 1
LCDD proteins: GLA and CHO. In both we determined the N-terminal amino
acid sequences of the light chains isolated from the tissues and
urines, the complete V-region light chain amino acid sequences deduced
from the cloned cDNAs, and the nucleotide germline sequences from
genomic DNA. We also determined the germline sequences from genomic DNA
of MCM. We conclude that the amino acid substitutions identified in GLA
and CHO, as well as in MCM, are due to somatic mutations and contribute
to protein instability, aggregation, and the deposition of the light
chains in the tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The diagnosis of LCDD was made in two patients who presented with
renal disease and whose renal biopsy tissues showed monotypic light
chain deposits.
A 61-year-old caucasian female (GLA) presented with increasing renal
functional impairment. Physical examination revealed no edema or other
abnormalities. The serum creatinine rose from 0.8 to 2.6 mg/dl over an
8-month period and to 3.7 mg/dl at the ninth month, the time of
admission and renal biopsy. The urine showed microhematuria and a urine
protein excretion of 0.16–0.37 g/24 hours. The serum cholesterol level
was 270 mg/dl, C3/C4 was 129/40 mg/dl (normal), and hematocrit was
32%. A diagnosis of LCDD was made by renal biopsy.
Immunoelectrophoresis revealed no monoclonal protein in the serum and a
mixture of albumin and monoclonal Bence-Jones light chain,
estimated at 30 mg/dl, in 100-fold concentrated urine. A bone marrow
biopsy showed features of a B-lymphocyte neoplasm. Despite chemotherapy
with Melphalan and Prednisone, irreversible renal failure developed,
requiring maintenance hemodialysis.
The second patient, a 64-year-old Asian female (CHO), was admitted to
the hospital for complaints of fatigue, anorexia, back pain, and acute
renal failure that developed after abdominal computed tomography (CT)
with radio contrast. She was known to have normal renal function, with
a serum creatinine of 0.9 mg/dl, 2 years before. After CT studies the
creatinine was 5.7 mg/dl and rose to 9.1 mg/dl 4 days later at the time
of admission. The past medical history was unremarkable except for
hepatitis B surface antigenemia for many years. Her blood pressure was
normal and there was no edema. The significant laboratory findings
included urine protein excretion, 1.0 g/24 hour; microhematuria; blood
urea nitrogen, 100 mg/dl; serum Ca, 10.4 mg/dl; Phos, 6.7 mg/dl. A
diagnosis of LCDD was made by renal biopsy. The initial serum and
urine immunoelectrophoresis showed no monoclonal Ig, but subsequently
Bence-Jones light chain was identified in concentrated urine, and
lytic lesions were detected in the skull. Bone marrow examination
revealed multiple myeloma. Renal failure worsened and expiration
occurred 2 months after admission. A postmortem examination was
performed.
Pathological and Immunohistological Studies
Paraffin sections of formalin-fixed tissues were stained with
hematoxylin and eosin, periodic acid silver methenamine, and Congo red.
Standard immunofluorescence microscopy examination of frozen sections
was performed on renal biopsy tissues from both patients and on
systemic tissues obtained at necropsy from CHO. Sections were incubated
with a panel of fluorescein-labeled rabbit polyclonal anti-human Ig
chain-specific antibodies (
, µ, 
, , and ), C3, C1q,
fibrin and unlabeled rabbit polyclonals anti-amyloid P component (Dako,
Carpenteria, CA), and anti-apo-E (Chemicon, Temecula, CA), followed by
fluorescein-conjugated swine anti-rabbit Ig (Dako). Immunoperoxidase
examinations of formalin-fixed, paraffin-embedded bone marrow biopsy
specimens from both patients incubated with polyclonal anti- and
anti- antibodies (Dako) were performed as described.16
Electron microscopic studies were carried out on ultrathin sections of glutaraldehyde-fixed epon-embedded renal biopsy tissues and on frozen cardiac tissue stored at -70°C.
Light Chain Isolation from Tissue and Urine Specimens
The GLA residual 1-mm3 frozen renal biopsy tissue was washed three times in 500 µl of 50 mmol/L phosphate/150 mmol/L NaCl (PBS) (pH 7.2) and centrifuged at 2500 rpm. The tissue pellet was placed in dissociating buffer (50 mmol/L Tris, pH 6.8, 2% sodium dodecyl sulfate, 5% glycerol, 0.1% bromophenol blue), incubated at (80°C) for 2 hours with continuous agitation and centrifuged at 2500 rpm for 5 minutes. The supernatant was boiled for 5 minutes after the addition of dithiothreitol (DTT) to a final concentration of 100 mmol/L.
Light chain deposits were extracted from CHO myocardial tissue stored at -70°C as previously described.11 Briefly, tissue (5–8 g) was repeatedly homogenized in 10 mmol/L phosphate/2.7 mmol/L KCl/137 mmol/L NaCl, pH 7.4, with a cocktail of protease inhibitors (Complete, plus 1 µmol/L pepstatin and 1 µmol/L leupeptin, all from Boehringer Mannheim, Indianapolis, IN), and 1 mmol/L EDTA, followed by centrifugation. The pellet was extracted with 6 mol/L guanidine-HCl in 50 mmol/L Tris-HCl, pH 10.2, containing 170 mmol/L DTT under constant stirring for 48 hours at room temperature. The extracted material was centrifuged at 100,000 x g for 1 hour at 4°C, and the crude extract was dialyzed at 4°C in membrane tubing with a cutoff of 2 kd against distilled water and lyophilized.
The GLA and CHO 24-hour urine specimens were dialyzed against PBS in
tubing with a molecular mass cutoff of 2 kd and concentrated 100-fold.
A sample diluted 1:3 in PBS was filtered through a 0.45-µm disk
(Millipore, Bedford, MA) and run three times through a 1-ml KappaLock
sepharose column (Zymed Lab, San Francisco, CA) according to the
manufacturer’s instructions. The column was washed with 10 column
volumes of PBS before the bound chains were eluted with 2 column
volumes of 500 mmol/L propionic acid (Fisher Scientific, Pittsburgh,
PA). All of the eluates were lyophilized.
Gel Electrophoresis and Western Blotting Analysis
The protein extracts from the renal biopsy and myocardial tissues
and the purified Bence-Jones protein from the urines were analyzed by
fractionation in 12.5% and/or 15% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
electrotransferred onto polyvinyldifluoride membranes in
3-cyclo-hexylamino-1-propanesulfonic acid (Sigma Chemical Co., St.
Louis, MO) buffer, pH 11, containing 10% methanol. The membranes were
washed in distilled deionized water. Strips removed for immunoblotting
were blocked for 1 hour in PBS–0.05% Tween 20 containing 5% nonfat
dry milk and 1% bovine serum albumin, incubated with polyclonal rabbit
anti-human antibody 1:500 (Dako) for 11/2 hours at
room temperature, washed three times in PBS–0.1% Tween 20, followed
by incubation with horseradish peroxidase-conjugated donkey anti-rabbit
Ig (1:2500; Amersham, Arlington Heights, IL) for 2 hours. The reaction
was developed with 4-chloro-1-naphthol peroxidase substrate system
(Kirkegaard and Perry Laboratories, Gaithersburg, MD) or by fluorograms
prepared with an ECL Western blotting kit (Amersham) according to the
manufacturer’s specifications. For sequence purposes, membranes were
stained with 0.1% Coomassie blue R 250 in 40% methanol–1% acetic
acid (high-performance liquid chromatography (HPLC) grade), destained
with 50% methanol (HPLC grade), and extensively washed in deionized
water, and the bands were excised and sequenced. Automated Edman
degradation analysis was carried out on a 477A microsequencer, and the
resulting phenylthiohydantoin derivatives were identified using the
on-line 120 A PTH analyzer (Applied Biosystems, Foster City, CA).
cDNA Cloning
The amino-terminal sequence of the light chain deposits extracted
from the renal biopsy and the myocardial tissue, as well as the amino
acid sequences obtained from the light chains isolated from the urines
of both subjects, indicated that both light chains belonged to the
V-1 subgroup.17
Accordingly,
oligonucleotide forward and reverse primers were synthesized as
described.9
Total RNA isolated from the GLA frozen
residual diagnostic bone marrow aspirate and CHO frozen vertebral bone
marrow fragments obtained at autopsy were used in reverse
transcriptase-polymerase chain reaction (RT-PCR) experiments to isolate
and clone the cDNAs. Briefly, bone marrow cells were washed with 10
mmol/L phosphate-buffered saline, pH 7.4, and 150 mmol/L NaCl, and
total RNA was isolated by the guanidine isothiocyanate method, using
Trizol LS (Gibco BRL, Gaithersburg, MD). Reverse transcription of RNA
was performed with a first-strand cDNA synthesis kit (Boehringer
Mannheim), using the avian myeloblastosis virus reverse transcriptase
with the downstream primer. PCR amplification of the first-strand cDNA
produced by reverse transcription was performed by introducing the
upstream amplimer in 1x PCR buffer. Each PCR cycle, consisting of a
denaturation step (94°C/1 minute), an annealing step (42°C/1
minute), and an elongation step (72°C/2 minute), was repeated 30
times. For control purposes RNA samples were pretreated with RNase A
for 30 min at 37°C and subjected to RT-PCR. In these controls as well
as in RT-PCR of RNA extracted from other diagnostic bone marrows
without plasma cell dyscrasia, no specific amplification was observed.
PCR products were fractionated on 5% polyacrylamide gels and
visualized under UV light. The resulting PCR products were subcloned
into the pCR2.1 vector (TA cloning kit; Invitrogen, Carlsbad, CA), and
the isolated recombinant plasmids were sequenced by the dideoxy chain
termination method in both directions.
Germline V-Region Sequences
Genomic DNA was isolated from lymphocytes in fresh peripheral blood (GLA) and from stored frozen cardiac tissues (CHO, MCM) obtained at necropsy. Specific PCRs were set up using forward and reverse primers: 5'-GTCTTCCYAYAATATGATC-3' and 5'-AGGACCACT-CTCAGCTGATA-3', respectively, for gene L12. Five hundred nanograms of each DNA were added to 25 pg of each primer, 200 µmol/L dNTP, and 1.5 units of Taq DNA polymerase in a final volume of 50 µl of appropriate buffer (Pharmacia, Uppsala, Sweden), denatured at 94°C for 5 minutes, and amplified through 30 cycles, including 30-second steps at 94°C, 55°C, and 72°C, followed by elongation at 72°C for 10 minutes. To rule out possible errors of Taq polymerase, at least two independent PCR reactions were performed on DNA extracted from each specimen. Products were ligated to pCR2.1 vector, and a minimum of 10 clones from each case were sequenced in both directions.
Structural Analysis
The spatial locations of the amino acid substitutions of the GLA
and CHO light chains were computationally mutated on the backbone
structure of REI, a 1-soluble nonpathogenic Bence-Jones
light chain of known three-dimensional structure,18,19
with the computer program Insight II (Biosym, San Diego, CA).
Isoelectric Points of -Light Chains
The theoretic isoelectric points of the V-region Ig light chains were determined as previously described.20 In this calculation cysteine residues involved in disulfide bond function were assumed to be nonionizable.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the GLA renal biopsy tissue there were 29 glomeruli, four of
which were obliterated by sclerosis. All of the remaining glomeruli
exhibited normal cellularity, and there were no mesangial nodules. A
few had thickened wrinkled basement membranes but most had minimal
abnormalities. There was widespread interstitial edema and fibrosis,
focal lymphocytic infiltration, and focal tubular atrophy. No prominent
casts with multinucleated giant cells were seen. Congo red stain for
amyloid was negative. Immunofluorescence microscopy demonstrated bright
staining of all glomerular, tubular, and vascular basement membranes
for (Figure 1A)
, but not light
chains; , µ, or heavy chains; C3; C1q; fibrin; amyloid P
component; or apoE (not shown). Clustered granular deposits in the
glomerular and tubular basement membranes (Figure 1B)
, typical of LCDD,
were found ultrastructurally. A bone marrow aspirate and biopsy
exhibited increased lymphocytes, dispersed as well as in small
aggregates, some with plasmacytoid differentiation; cell marker
analysis revealed predominantly B cells expressing surface
immunoglobulin of the isotype with a : ratio of 30. A
diagnosis of lymphoplasmacytoid lymphoma was rendered.
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but not light
chain nor , µ, or heavy chains; C3; C1q; fibrin; amyloid P
component; or apoE. Clustered, granular electron-dense deposits in
glomerular and tubular basement membranes were ultrastructurally
typical of LCDD. The bone marrow biopsy displayed nodules and sheets of
-bearing plasma cells and only a few scattered -staining cells,
diagnostic of myeloma. At autopsy the remarkable findings were a firm
enlarged 400-g heart with left ventricular hypertrophy and a liver with
nodular cirrhosis. There were no vertebral lytic lesions. Light
microscopic examination confirmed the renal and bone marrow biopsy
findings. The myocardium exhibited mild interstitial widening by loose
eosinophilic material, but no evidence of inflammation or muscle
damage. Congo red stains of all systemic organs showed no amyloid
deposits. Immunofluorescence microscopy demonstrated deposits of
(Figure 1C)
, but not light chain (Figure 1D)
, around cardiac
myocytes, in fibrous septae and vascular walls, and diffusely in the
basement membranes of all systemic organs. Western Blotting, Amino Acid Sequencing, and cDNA Cloning
The electrotransferred material extracted from the tissues and the
urines of both subjects is shown in Figure 2
. The GLA extract from the kidney biopsy
tissue (lane 1) yielded intact protein and fragments reactive with
anti- (lane 2). The sequences obtained from all bands were
homologous to the intact N-terminus of the 1 light chain
subgroup (Figure 3A)
; the 13-kd protein
yielded the sequence from residues 1–8 (16 kd, 1–15; 17 and 18 kd,
1–10; and the 28-kd protein the sequence from positions 1–6). The
urine eluate from the KappaLock column was mainly comprised of a single
protein of 28 kd (Figure 2
, lane 3) that was reactive with anti-
(Figure 2
, lane 4) and revealed the sequence from positions 1 to 35.
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,
lane 5), also consisting of intact protein and fragments, demonstrated
immunoreactivity with anti- (Figure 2
, lane 6); the sequence,
homologous with the N-terminus of the 1 light chain
subgroup (Figure 3B)
, was obtained from bands at 6.5 kd yielding
residues 1–10 (8 kd, 1–16; and 28 kd, 1–39). A 7-kd protein gave the
sequence from residues 51–70. Light chain from the urine eluted from
the KappaLock column, composed of a 28-kd protein (Figure 2
, lane 7),
was immunoreactive with anti- (Figure 2
, lane 8) and rendered the
sequence from residues 1–37.
Several independent cDNA clones of GLA, isolated from
reverse-transcribed RNA obtained from bone marrow cells, were sequenced
and found to be identical. The same was true for cDNA clones of
CHO. The deduced amino acid sequence (Figure 3, A and B)
was in full
agreement with the N-terminal amino acid sequence retrieved from the
tissues and the urines. When the cDNA sequences were compared with the
V-1 germline sequences, the best homology was found with
the L12a gene.21
Germline V-Region Sequences
The cloned and sequenced nucleotides of the full-length germline
V- genes from genomic DNA, obtained from GLA and CHO, as well as
MCM, were all identical to the L12a germline subset of the
1 light chain. In Figure 4
, a comparison of the germline sequence
and the translated sequences from the cDNAs of GLA and CHO, together
with the previously extracted and sequenced tissue deposits of
MCM,11
demonstrates that the amino acid substitutions are
the result of somatic mutation in the proliferating clones instead of
inherited susceptible variant genes. As shown, there are a total of
nine mutations in GLA, seven in CHO and 13 in MCM. The mutations occur
throughout the domain: in GLA two are in FR1, one is in CDR1, three are
in FR2, and one each in FR3 and CDR3; in CHO one is in FR1, one in
CDR2, two in FR3, and three are in CDR3; in MCM the mutations are three
in CDR1, two in FR2, one in CDR2, three in FR3, and four in CDR3. The
spatial locations of the substitutions in GLA and CHO are depicted in
Figure 5, A and B
.
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Phe
substitution at position 14 breaks the hydrogen bonding pattern in the
beta turn of FR1 and results in exposure of the hydrophobic Phe to the
solvent. The mutation IlePhe at position 21 in GLA interferes with
packing by replacing a moderate-sized hydrophobic side chain with a
much larger hydrophobic Phe. At position 66 in MCM a highly conserved
small Gly is replaced by the bulky side chain of Glu that may disrupt
packing and the turn in FR3. The SerPhe mutation at position 67 in
CHO interferes with the turn in the FR3 by inserting a hydrophobic Phe
into the solvent. In MCM, a SerLys at position 77 may destabilize
the turn in FR3; the replacement of Asp with Ile at residue 82 in MCM
is severely destabilizing by removing a highly conserved salt bridge
with Arg61 and replacement of a strongly hydrophilic
residue with a hydrophobic side chain. The replacement of ThrIle at
position 85 in GLA results in a hydrophobic side chain exposed to
solvent. At position 95, the SerLeu in CHO and SerPro in GLA and
MCM increase hydrophobicity and potentially destabilize the turn in
CDR3. Taken together, GLA, CHO, and MCM all exhibit destabilizing
mutations that increase hydrophobicity.
In Figure 6
, constructed models depicting
the hydrophobic residues in solvent-exposed CDR-presenting surfaces of
the V-region dimers of GLA, CHO, and MCM are compared to that of
REI, a nonpathogenic 1 light chain;18,19
all three of the LCDD proteins show an increased proportion of
hydrophobic side chains exposed to the solvent that could contribute to
protein destabilization. Trp at position 32 in the CDR1 is a
genetically encoded feature of 1a (gene, L12a) light chain, and it
is not found in 1b (gene O18-O8) protein REI.
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The calculated pI values were 6.42 (5.6–7.5) and 9.22 for GLA and CHO, respectively, and 8.21 for MCM.11
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-LCDD proteins, GLA and CHO, obtained by N-terminal
sequence analysis of light chain deposits extracted from the tissues,
light chains isolated from the urines, and the V-region amino acid
sequences deduced from the cloned and sequenced cDNAs, and
MCM,11
are compared to the germline sequence determined
from genomic DNA in each. The findings indicate that the amino acid
substitutions in GLA, CHO, and MCM are the result of somatic
mutations in the proliferating clones rather than from inherent variant
alleles.
Although there are well over 150 published cases of nonamyloidotic
LCDD, the primary structure of the involved light chains has been
determined in only 11 cases. Nine, including GLA and CHO (Table 1)
, are light chain, and two are
light chain.12
One (MCM) was obtained by sequence analysis
of light chain deposits extracted from tissue,11
two (SCI,
BURN) by sequence analysis of the monoclonal light chain from the
urine,7,13
and the others (ISE, REV, BLU, FRA, BOU, and
RAC) were deduced from cloned cDNAs.6,8-10,12
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-LCDD proteins mentioned thus far (Table 1)
are
V-region light chains that belong to three different light chain
subgroups. Four of them (CHO, GLA, MCM, ISE) belong to the
1 subgroup, three (BLU, BURN, FRA) to IV,
and two (REV, SCI) belong to III. The two -LCDD
proteins (BOU, RAC) belong to the II subgroup. Among the
nine -LCDD proteins summarized in Table 1
, all have amino acid
substitutions that occur in all domains, but more frequently in the
CDRs than in the FRs (51 versus 37), possibly reflecting
immunoglobulin antibody specificity that is antigen driven.
Collectively there are six in the FR1, 21 in the CDR1, eight in the
FR2, 12 in the CDR2, 23 in the FR3, and 18 in the CDR3.
All -LCDD proteins exhibit one and as many as three mutations that
can be expected to be structurally and functionally significant in
affecting protein destabilization; in each at least one mutation is in
a turn/loop exposed to solvent. All LCDD proteins characterized to
date have come from the three major germline genes that encode a Trp
residue at solvent accessible positions, ie, L12a, Trp32;
L2–L16, Trp94; B3, Trp50. Trp, together with
the other hydrophobic residues at the surface sufficient to exclude the
light chain from the solvent, could cause aggregation and precipitation
in tissues. Hydrophobic residues (Figure 6)
are increased in relation
to REI, and such substitutions in the FR regions (Table 1)
could
distort the ß structure, producing amorphous rather than fibrillar
deposits.
The diffuse organ distribution of nonamyloidotic deposits in MCM and
CHO and in other cases of LCDD4,5
is in contrast to the
more patchy distribution of deposits in AL disease. The uniform light
chain deposits in systemic basement membranes of MCM suggested a unique
affinity for some component of the basement membrane, possibly due to
an immunological or electrostatic interaction. The calculated pI values
of the V-region amino acid sequences in MCM (8.21), as well as pI
values calculated from published sequences of ISE and BLU (8.20, 8.19,
respectively), were high.11
This observation raised the
possibility that the affinity might be due to charge interactions
between cationic light chains and anionic GAGs in basement
membranes,22
a mechanism of immune complex deposition
demonstrated in vivo in a murine model.23
The
calculated isoelectric point of the complete V-region in CHO is also
high (9.22). In GLA the pI is lower (5.5–7.5). The high pIs of the
light chains in LCDD are in contrast to the acidic pIs of amyloidogenic
Bence-Jones proteins (mean, 4.8 ± 1.1),24
suggesting
a different mechanism for tissue deposition. Furthermore, if one
compares the pI values determined by the N-terminal 50 amino acids
encoded by the germline genes, one finds acidic pI values for 1b
(6.4) and 2 peptides (3.6–4.5). In contrast, all of the other genes
encode basic 50-mers, with 10.4, 9.9, and 9.8 deduced for 1a, 3a,
and 4, respectively, the three gene families for which LCDD
representatives have been identified to date.
Why some Bence-Jones proteins are soluble and nonpathogenic, whereas others form either fibrillar or nonfibrillar granular aggregates in AL or LCDD, or even coexisting fibrillar and nonfibrillar deposits,25 remains elusive. In AL, although the primary protein structure likely plays an important role in fibril formation, amyloidogenic motifs have not been identified in the primary sequence data of more than 60 light chains extracted from amyloid tissue deposits. Moreover, the identity of the complete primary structure of both the deposited amyloid fibril protein and the soluble precursor Bence-Jones protein DIA26 and the amino-terminal identity of extracted light chains from coexisting fibrillar deposits of AL and granular deposits of LCDD25 argue in favor of extrinsic factors and other molecules that might play a part in the processing of susceptible light chains. For example, the detection of amyloid P component and apo-E in amyloid, but not in nonamyloid light chain deposits,2 suggests a function, as yet unclear, for these molecules. On the other hand, spontaneous in vitro fibril formation of amyloid ß peptides27 and other proteins not known to cause amyloidosis28,29 relates to a physical-chemical environment favoring intermolecular interactions.
It is now apparent that a number of different diseases, including AL
and LCDD, involve aberrant protein folding.30,31
Based
upon the accumulated data, LCDD and AL are considered to be
conformational disorders. The evidence suggests that somatic mutational
effects may be responsible for destabilization of the normal soluble
globular light chain structure.32
The increased
hydrophobicity of GLA, CHO, and MCM relative to REI shown in Figure 6
and similar observations reported by others33,34
could
promote the formation of aggregation-prone, partially unfolded
intermediates, thereby reducing solubility and favoring deposition in
tissues. In vitro studies have shown that the Bence-Jones
light chains in LCDD and AL were more unstable than a nonpathogenic
Bence-Jones protein, and AL light chain was more unstable than the LCDD
light chain.35
In the context of current concepts of
protein folding,36,37
the interesting question posed is
the relationship between LCDD and AL, and whether LCDD represents a
partially unfolded or misfolded intermediate species stabilized in a
transition state that favors off-pathway aggregation and precipitation
in tissues, or alternatively is a preamyloid form of disease with
restriction of maturation on pathway to fibril formation. The latter
possibility is supported by both in vivo and in
vitro ultrastructural observations showing a transition between
granular and fibrillar structures in tissue deposits in a patient with
coexistent LCDD and AL,38
and the progressive formation in
solution of Congophilic fibrils within granular
aggregates.29
As new data develop on protein folding and
protein-protein interactions, it is likely that new insights into the
different disease expressions exhibited in AL and LCDD and,
importantly, the mechanisms of fibrillogenesis will be achieved.
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Footnotes |
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Supported in part by a grant from the National Institutes of Health (AR 02594, Merit), by the U.S. Department of Energy, Office of Health and Environmental Research (contract W-31-109-ENG), and by the U.S. Public Health Service (grant DKY 3757).
R. V. and F. G. contributed equally to this work.
P. A.’s current address: Hôpital Necker, 161 rue de Sèvres, 75743 Paris CEDEX 15, France.
Accepted for publication August 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain of the V-IV subgroup in the kidney and plasma cells in light chain deposition disease. J Clin Invest 1991, 87:2186-2190
Sci, the Bence Jones protein isolated from a patient with light chain deposition disease. Biochim Biophys Acta 1991, 1097:177-182[Medline]
chain in myeloma with light chain deposition disease. Clin Exp Immunol 1992, 87:122-126[Medline]
1 subgroup (ISE) in light chain deposition disease. Clin Exp Immunol 1993, 91:506-509[Medline]
III immunoglobulin light chain in myeloma with light chain deposition disease. Clin Exp Immunol 1996, 106:357-361[Medline]
immunoglobulin light chains in myelomas with nonamyloid (Randall-type) light chain deposition disease. Am J Pathol 1998, 153:313-318 Light Chain Subgroup I. Sequences of Proteins of Immunological Interest. 1991, :pp 103-112 National Institutes of Health, Bethesda, MD,
genes and their hypermutation. Eur J Immunol 1993, 23:3248-3271[Medline]
light chain deposits. A human disease to study alterations of protein conformation. Clin Exp Immunol 1997, 110:472-478[Medline]
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