|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3559-3566
Mutational Analysis in Murine Models for Myeloma-Associated Fanconi's
Syndrome or Cast Myeloma Nephropathy
By
C. Decourt,
A. Rocca,
F. Bridoux,
F. Vrtovsnik,
J.L. Preud'homme,
M. Cogné, and
G. Touchard
From the Laboratoire d'Immunologie, CNRS EP118, Centre Hospitalier
Universitaire de Limoges, Limoges; the Laboratoire d'Immunologie CNRS
ESA 6031, Université de Poitiers; Service de Néphrologie,
Centre Hospitalier Universitaire de Poitiers, INSERM U426,
Hôpital Bichat, Paris; and Institut Universitaire de France,
Limoges, France.
 |
ABSTRACT |
We have designed an in vivo model in which murine hybridoma cell
clones producing human Ig light chains (LC) are administred to mice.
Depending on which monoclonal LC is expressed, this model mimicks
either cast myeloma nephropathy or the pathological condition defined
as myeloma-associated Fanconi's syndrome (FS) with LC crystallization.
Morphological alterations of the kidney cells are thus obtained in
mice. All studied LC are closely related human monoclonal V I
proteins, which differ by a limited number of substitutions within the
variable region. In the case of an FS monoclonal LC, we show that
limited changes introduced through site-directed mutagenesis in the
variable domain may suppress formation of intracellular crystals within
tubular cells. We also show that multiple peculiarities of the variable
region are simultaneously needed to allow LC crystallization; this
property thus likely results from a unique LC tridimensional
conformation imposed by concomitant somatic mutations of a specific
germinally encoded framework.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
CAST NEPHROPATHY IS the most frequent
renal complication in myeloma patients with Bence-Jones
proteinuria.1 It is characterized by casts made up of
Tamm-Horsfall glycoprotein and monoclonal Ig light chains (LC)
obstructing distal tubule lumens.2 By contrast,
myeloma-associated Fanconi's syndrome (FS) is a rare entity; it is
characterized by alterations of proximal tubule functions and in most
patients is accompanied by crystalline inclusions in proximal tubular
cells, plasma cells, and macrophages. We recently studied and sequenced
at the cDNA level the monoclonal LC CHEB involved in
FS.3 Small protein-enriched gel filtration fractions from
patient's urine yielded crystals morphologically similar to those
found in patient's proximal tubular cells, with the same 60 Å periodicity on electron micrographs. N-terminal sequencing and mass
spectrometry studies showed that the crystals contained a 107 amino
acid fragment (with a C-terminal lysine) corresponding to the variable
(V) domain together with a low proportion of the entire chain. In
vitro trypsin, pepsin, or cathepsin B treatment of the native entire
LC yielded a homogeneous V domain fragment that, in contrast to
other monoclonal LC, was completely resistant to further
proteolytic digestion.4 The patient's chain also displayed an unusual self-reactivity by Western blotting. Genes encoding CHEB, as well as 2 other LCs from FS patients studied by our group, TRE and TRO,5 were found to
be highly homologous to the same germline VI gene O2/O12. All 3 patients (CHEB, TRO, and TRE) had numerous
intracellular crystals. LC sequences in another patient (DEL)
with myeloma associated FS, but no detectable crystals were homologous
to another closely related VI germline gene, O8/O18.5
We hypothesized that some specific and unusual V region sequences would
result in the physicochemical properties of LC in FS and be responsible
for their impaired trafficking and crystallization within proximal
tubular cells. The presence of polar residues at positions 30 and 94 in
the CHEB V region sequence was postulated to play a role in
crystallization.3,5 With the aim of evaluating the in vivo
potential nephrotoxicity of FS LCs, we developed an experimental model
in which transfected hybridoma cells expressing such LCs were
administred to mice. A similar strategy previously allowed us to
reproduce renal deposits featuring LC deposition disease in
mice.6 This model is based on the injection into mice of
syngeneic transfected tumor cells expressing either the CHEB FS
LC or closely related human I LC (269) as a control. CHEB
and 269 I chains, mutated at positions 30 or 94, were also expressed
in mice (Fig 1). This model allowed us to
show that limited sequence peculiarities of the variable region can
lead to the development of tubular lesions either typical of the FS with LC crystals or of cast myeloma nephropathy.
View larger version (24K):
[in this window]
[in a new window]
| Fig 1.
LCs sequences. Alignment of the primary sequences of
CHEB, 269, and mutated CHEBm30, CHEBm94, and
269m30 proteins. Dashes indicate identities with CHEB's
sequence. Bold residues indicate amino acids, which had been modified
by site-directed mutagenesis. Stars indicate absent residues. Amino
acids are numbered according to Kabat's
numbering.33
|
|
| |
MATERIALS AND METHODS |
Production of human LCs in vitro.
The sequence of the FS chain CHEB encoded by a rearranged
VI-J1 gene was previously determined at the cDNA
level.3 The control I sequence 269 was cloned from human
polyclonal B cells and carried an in-frame VI-J1 coding region.
Sp2/0, a B-cell hybridoma lacking endogenous Ig, was transfected with a
modified pAKIV expression vector7 or with the pALI-Eµ
vecto,8 carrying the V sequence of either CHEB,
2 variants of CHEB (CHEBm30 [Ala 30  Ser],
CHEBm94 [Ile 94 Thr]), 269, or a variant of 269 (269m30 [Ser 30 Ala]). Cotransfection with a neo
gene was necessary, given the absence of resistance gene in pAKIV
and pALI Eµ. Geneticin-resistant cells were assayed for human LC
production by enzyme-linked immunosorbent assay (ELISA) and clones with
the highest secretion level of each I chain were selected: clones
S-CHEB (producing 35 µg/mL/24 h/106 cells of LC), S-269
(2 µg/mL/24 h/106 cells of 269 LC), S-CHEBm30 [Ala 30 Ser] (2.2 µg/mL/24 h/106 cells), S-CHEBm94
[Ile94 Thr] (35 µg/mL/24h h/106 cells), and
S-269m30 [Ser30 Ala] (1.4 µg/mL/24 h/106 cells).
Production of human LCs in vivo.
Selected clones, S-CHEB, S-269, S-CHEBm30, S-CHEBm94, and S-269m30 (4 × 106 cells) were injected
intraperitoneally into 8-week old BALB/c mice (Iffa Credo, L'Arbresle,
France). These mice were called CHEB, 269, CHEBm30,
CHEBm94, or 269m30 mice. After development of tumor, urine and
serum were collected and chain secretion was evaluated by ELISA.
Mice were killed about 5 weeks after injection (mean, 35.6 days;
standard deviation [SD], 4.75 days). At that time, the
average body weight of mice was 35.13 g (SD, 2.53 g).
Immunochemical studies.
For Western blots, culture supernatants, urine, and sera were analyzed
by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose sheets, and shown with antihuman chain alkaline-phosphatase-conjugated polyclonal
antiserum (Amersham, Buckinghamshire, UK).
Immunomorphologic analysis.
Light microscopic examination was performed on slides stained either
with hematoxilin/eosin (HES), Periodic Acid Schiff, light green
trichrome, silver methenamine, or toluidine blue. Frozen blocks of
liver, kidney, spleen, heart, and tumor obtained at death were cut in
4-µm-thick slices. Organs were studied for the presence of human chains by immunofluorescence with fluorescein-conjugated polyclonal
rabbit antisera against human or  chain (DAKO, Glostrup, Denmark) or with a combination of mouse monoclonal antibody against human chain and a rabbit antimouse Ig-rhodamine conjugate (Zymed, San Francisco, CA).
For immunoelectronmicroscopic studies, samples were fixed in 4%
glutaraldehyde. Staining with either biotinylated antihuman ,
antihuman sera, or biotinylated peptostreptococcus magnus protein L
(Zymed), shown by gold-tagged streptavidin (10-nm EM grade; Zymed), was
additionnally performed. Binding of rabbit anticathepsin D antiserum
(DAKO) was shown by 10-nm gold-conjugated goat antirabbit IgG (Sigma,
Aldrich Chimie, L'Isle d'Abeau Chesnes, France). Specificity was
controlled using normal rabbit serum instead of anticathepsin D
antibody. Sections were then stained with uranyl acetate and examined
in a Jeol 100 CX electron microscope (Akishima, Japan).
Nucleic acids studies and sequence analysis.
Total RNA (10 µg) from untransfected Sp2/0 cells, S-CHEB, and S-269
transfectomas from 269-4 and CHEB-9 mice tumors and from patient
CHEB's bone marrow were analyzed on 1% agarose, 0.7 mol/L formaldehyde gels, transferred onto nylon sheets (Amersham), and hybridized with a human C exon probe.
For sequencing, 2 µg of total RNA from 269-4 and CHEB-9 mice tumors
were used as templates for synthesis of complementary DNA (cDNA) by
reverse transcriptase (Boerhinger, Mannheim, Germany). cDNA were
amplified by polymerase chain reaction (PCR),9 cloned in
Sma I cut M13 mp19 vector, and sequenced by the
dideoxynucleotide method10 on an ABI 310 DNA sequencer
(Perkin-Elmer, Branchburg, NJ).
Site-directed mutagenesis.
Site-directed mutagenesis was performed by PCR of the VJ segment cDNA:
the first 2 amplifications were performed separately on both flanks of
the mutation to be introduced, (1) on 1 side (PCR1) with a 5'
primer corresponding to the VI leader and a 3' primer
introducing the mutated base and (2) on the other side (PCR2), with a
5' primer introducing the desired mutation and a 3' primer
corresponding to J. Complementary primers introducing the mutations
were chosen so that both mutated fragments overlapped by
10 bp. The third round (PCR3) allowed mutated segments to anneal and
restored a complete mutated VJ region by using a 5' primer corresponding to the VI leader and a 3' primer corresponding to J. PCR1 and 2 consisted of 35 cycles of 94°C for 30 seconds, 53°C for 30 seconds, and 72°C for 30 seconds. PCR3 consisted of 3 cycles of 94°C for 30 seconds, 45°C for 30 seconds, and
72°C for 45 seconds for the hybridization of the 2 mutated
segments, followed by 30 cycles of 94°C for 30 seconds, 53°C
for 30 seconds, and 72°C for 30 seconds for the amplification of
the entire V domain.
The forward primer corresponding to the VI leader and used for all
I sequences was 5'- AAGTCGACATGGACATGAGGGTGCC-3'. The backward primers corresponding to the 3' end of the J of
CHEB and L269 were as follows: JCHEB: 5'-
TTCTCGAGACTTACGTTTGATTTCCACCTTGGT-3'; and J269:
5'- TTCTCGAGACTTACGTTTGATCTCCAGCTTGGT-3'.
Sequences of the other primers used in the directed mutagenesis were as
follows: (1) mutant CHEB m30 (Ala 30 Ser): PCR1 forward primer: VI; backward primer: 5'- TTAAAAAGGTGCT
AATGGTCTGA-3'; PCR2 forward primer: 5'- TCAGACCATTAG
CACCTTTTTAA-3'; backward primer: JCHEB; PCR3
forward primer: VI; backward primer: JCHEB; (2)
mutant CHEB m94 (Ile 94 Thr): PCR1 forward primer:
VI; backward primer: 5'-ACGTCCACGGGG TACTGTAACTC-3';
PCR2 forward primer: 5'-GAGTTACAGTAC CCCGTGGACGT-3';
backward primer: JCHEB; PCR3 forward primer VI;
backward primer: JCHEB; (3) mutant 269 m30 (Ser30
Ala): PCR1 forward primer: VI; backward primer: 5'-
TTAAATAGCTGGC AATGCTCTGA-3'; PCR2 forward primer: 5'-
TCAGAGCATTGC CAGCTATTTAA-3'; backward primer: J269;
PCR3 forward primer: VI; backward primer: J269.
ELISA.
96-well plates were coated with 100 µL of antihuman LC antibody
(Kallestad, Sanofi Diagnostic, Pasteur, France) diluted 1/1,000 in
phosphate-buffered saline (PBS) and then saturated with 200 µL of
PBS/0.1% bovine serum albumin. After incubation with samples, the
second antibody was an alkaline phosphatase-conjugated antihuman antibody (Sigma, L'Isle d'Abeau Chesnes, France). After addition of
the substrate, plates were kept at room temperature in the dark for 15 minutes; reaction was stopped with 50 µL NaOH 3N and plates were read
at 405 nm.
Tubule suspension and uptake studies.
Proximal tubules were isolated immediately after death: kidneys were
decapsulated and kept at 4°C in Hank's Balanced Salt Solution
(HBSS) supplemented with 10 mmol/L
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH
7.4, and 5 mmol/L D-glucose (HBSS-HEPES). The cortex was separated from
the medulla and dilacerated. Proximal tubules were isolated by
filtering on a Nylon mesh (106 µm) and washed twice with HBSS-HEPES.
Uptake of inorganic phosphate (Pi) was performed at 37°C in a
buffered solution with the following composition: 137 mmol/L NaCl, 5.4 mmol/L KCl, 1 mmol/L CaCl2, 1.2 mmol/L MgSO4,
and 15 mmol/L HEPES (pH 7.4). The sodium-free solution was made
iso-osmotic by replacing sodium chloride with N-methyl-D-glutamine.
Tubule suspensions were washed twice with the uptake solution and
incubated in the presence of
K2H32PO4 (0.5 µCi/mL) and 100 µmol/L KH2PO4 at 37°C. Uptake was stopped by washing the suspension twice with ice-cold solution 137 mmol/L NaCl,
15 mmol/L HEPES, pH 7.4. Cells were then solubilized in 0.5% Triton
X-100 and aliquots were counted by liquid scintillation. Protein
concentrations were determined by Bradford's method.11
| |
RESULTS |
A high secretion rate of human chain was observed in vitro after
transfection of Sp2/0 hybridoma cells with cDNA expression vectors
encoding either the FS I chain CHEB, the control I chain 269, the 2 mutated CHEB chains (CHEBm30 and
CHEBm94), or the mutated 269m30 chain. The mutated CHEB
chains did not differ from the original CHEB chain in their
apparent molecular mass in PAGE-SDS or in their ability to polymerize
as dimers and tetramers (Fig 2A and B). In
vivo, human chains were produced in mice injected with these
transfected clones (Table
1), but not in control mice injected with untransfected Sp2/0 cells (C mice; not shown). In C mice,
kidneys were normal. In 7 of 10 CHEB mice, light microscopy examination of kidney samples showed marked proximal tubular cell lesions with clear cytoplasmic atrophy and numerous intracellular crystals; toluidine blue typically stained multiple rhomboid crystals within a large number of proximal tubular cells
(Fig 3A). Rarely (2 of 10 mice) were
crystals found in the tubular lumens, but no myeloma casts were found.
By immunofluorescence, these intracellular crystals strongly stained
with an antihuman chain (Fig 3B). Electron microscopic analysis of
proximal tubular cells of CHEB mice showed that inclusions in
proximal tubular cells were surrounded by smooth membranes and were
located predominantly in the apical pole of cells (Fig 3C) and also
confirmed the crystalline nature of the hexagonal inclusions with a
regular striation of 60 Å periodicity (Fig 3D). By
immunoelectronmicroscopy, these crystals were stained with antihuman
chain, anticathepsin D sera, and protein L (which specifically
binds V domains). The results observed with CHEB mice were
very similar to those observed in patient CHEB, either by
immunofluorescence labeling pattern with the antihuman chain (Fig 4A) or by electronmicroscopic
examination of intracellular crystals (Fig 4B and C). Transfected
hybridoma cells themselves producing the CHEB chain did not
contain any intracytoplasmic crystalline inclusions (data not shown).
Similar analysis on kidney biopsies from CHEBm30 and
CHEBm94 mice did not show crystals in tubular cells
(Fig 5A and B), but only cytoplasmic
droplets indicating internalization of the chain by the normal process
of proximal tubular reabsorption (9 of 9 and 7 of 7, respectively).
View larger version (69K):
[in this window]
[in a new window]
| Fig 2.
Western blot pattern for CHEB LC and its mutants.
Western blot analysis of LCs produced in vitro by transfected Sp2/0
cells. (A) Nonreduced conditions. Lane 1, CHEB chain; lane 2, CHEBm30; lane 3, CHEBm94. (B) Reduced conditions. Lane
1, CHEB chain; lane 2, CHEBm30; lane 3, CHEBm94. Molecular weight markers are indicated in kilodaltons.
The difference in migration of molecular weight markers between (A) and
(B) reflects nonreducing versus reducing conditions.
|
|
View larger version (137K):
[in this window]
[in a new window]
| Fig 3.
Mouse CHEB kidney. (A) Light microscopy,
toluidine blue staining (original magnification × 500): strong
staining of crystalline inclusions in the cytoplasm of proximal tubular
cells. (B) Immunofluorescence microscopy, anti- human conjugate
(original magnification × 500): these inclusions are strongly stained
with a pattern similar to that found in patients' CHEB tubular
proximal cells. Electron microscopy: (C) original magnification × 1,200; (D) original magnification × 25,000: these crystalline
inclusions are osmiophilic and similar to that found in patient CHEB
with the same 60 Å periodic striation.
|
|
View larger version (207K):
[in this window]
[in a new window]
| Fig 4.
Patient CHEB, kidney biopsy. (A)
Immunofluorescence microscopy, anti- conjugate (original
magnification × 500), heavy staining of cytoplasmic inclusions in
proximal tubular cells. (B) Electron microscopy (original magnification × 2,600): numerous crystalline and osmiophilic inclusions in the
cytoplasm of proximal tubular cells. (C) Electron microscopy (original
magnification × 16,000): details of intracytoplasmic crystalline
inclusions.
|
|
View larger version (84K):
[in this window]
[in a new window]
| Fig 5.
Mutant CHEB mice and 269 mice kidneys. (A) Mouse
CHEBm30 no. 1, kidney, electron microscopy (original
magnification × 6,000). (B) Mouse CHEBm94 no. 1, kidney,
electron microscopy (original magnification × 2,500). (C) Mouse 269 no. 10, kidney, electron microscopy (original magnification × 4,000):
moderate or heavy increase of lysosomal component in proximal tubular
cells with variable apical vacuolation secondary to reabsorption of
mutated CHEB or 269 LCs. Crystalline inclusions are absent in the
lysosomal compartment.
|
|
Finally, in I 269 control mice, the proximal tubular cells contained
droplets (8 of 10) and were sometimes atrophic, but no crystal
inclusions were found (Fig 5C). Surprisingly, large tubular myeloma
casts were found in 5 of 10 269 mice (Fig
6A), which strongly stained with antihuman conjugate (Fig 6B and C). The same pattern was observed in mice expressing the mutant 269m30
of the control 269 chain, with myeloma casts in 4 of 8 mice and no
crystals (Table 1). Interestingly, CHEBm30 chains, sharing
both Ser30 and Ile94 residues with 269 LC, also led to the formation of
myeloma casts in 2 injected mice (no. 1 and 9 in Table 1).
View larger version (124K):
[in this window]
[in a new window]
| Fig 6.
Mice 269 kidneys. (A) Mouse 269 no. 3, kidney, light
microscopy, HES staining (original magnification × 200): glomeruli without abnormalities, tubular casts are large with
fracture lines, tubular lumen are enlarged. (B) Mouse 269 no. 8, kidney, immunofluorescence microscopy, antihuman conjugate
(original magnification × 500): numerous reabsorptive granules in the
cytoplasm of proximal tubular cells with a pattern different from that
noted in Fig 3 upper right quadrant. (C) Immunofluorescence microscopy,
antihuman LC conjugate: strong staining of a cast in 1 tubular
lumen. Note fracture lines in cast. Tubular basement membranes are
unstained. Staining for human LC and mouse Igs were negative. (D)
Electron microscopy (original magnification × 10,000): osmiophilic
myeloma cast without crystalline substructure.
|
|
Identity of LC produced in mice with the expected translation products
of the expression vectors was checked at the mRNA and protein levels.
Northern blotting of total RNAs from the transfected clones grown in
vitro or isolated from tumors showed mRNAs of the size expected
from the splice sites used in the vectors (data not shown). No human
mRNA was detected in C mice tumors. No staining was found in
proximal tubular cells of C mice with antihuman conjugate. Staining
with the antihuman conjugate was negative in all mice. Western
blots of urines of CHEB and 269 mice showed normal sized chains (data not shown). The absence of any mutation in transfected
genes during tumoral growth in mice was checked by sequencing cDNAs
from representative CHEB and I mice.
Preliminary uptake studies on renal tubule suspensions showed a
significant decrease in the uptake of Pi in CHEB mice when compared with normal BALB/c control mice and BALB/c mice injected with
untransfected Sp2/0 cells (P < .05). A similar reduction in
the Pi uptake was also observed in CHEBm94 mice and in 269 mice, when compared with control mice injected with Sp2/0 and uninjected BALB/c mice (P < .05). Because there was no
significant difference in the reduction of Pi uptake between
CHEB mice and CHEBm94 mice or 269 mice (P = .10), the alteration of proximal tubule Pi uptake could not be ascribed
to the presence or the absence of intracytoplasmic crystals
within proximal tubular cells (Table 2).
| |
DISCUSSION |
We have established a murine model of human FS with I LC
crystallization in proximal tubular cells (CHEB mice expressing a human FS I chain) and by chance, a murine model of myeloma cast
nephropathy without crystals in mice secreting a randomly-selected I
chain. Long ago, it had been postulated that peculiarities of certain
LC V domains may promote their agregation into fibrils in
AL-amyloidosis,12-22 their deposition along basement
membranes in nonamyloid LC deposition disease,6,23-29 or
their crystallization in the cytoplasm of proximal tubular cells in
FS.3,5 We speculated that an in vivo model of FS, allowing
site-directed mutagenesis on the sequence of a nephrotoxic human I
chain, would be ideally suited for addressing the issue of which
particular residues are responsible for the abnormal behavior of the protein.
Indeed, when mice were injected with tumours secreting the CHEB
FS I chain, pathologic alterations of the kidneys typical of FS with
intracellular crystals were readily induced and physiological alterations of phosphate tubular reabsorption also appeared. By contrast, mice injected with tumors secreting a control I chain (269) yielded no crystals, but did develop intratubular formations with
the typical aspect of myeloma casts. Although this control I chain
was chosen randomly among I chain sequences from polyclonal B cells,
its ability to induce cast nephropathy when expressed in high amounts
was not unexpected, given the high frequency of this nephropathy in LC
myeloma patients.
We then decided to express variants of these 2 I chains in the same
model. LCs are of the type in more than 90% of myeloma-associated FS with intracellular crystals, with a strong overrepresentation of the
VI subgroup.5 Although up to 56% of myeloma cases
produce chains of the I subgroup, FS stands as a rare
complication of myeloma30,31; it thus appears likely that
specific somatic mutations of I genes are involved in nephrotoxicity
and that subgroup-specific germline sequences are not sufficient to
confer toxicity. This is in clear contrast with the situation of 6
LCs in AL-amyloidosis, in which all known myeloma patients with free 6 monoclonal LCs had amyloidosis.13 In FS, it is likely
that a germinally encoded framework is needed, but that somatically created mutations finally confer a nephrotoxic potential to LC; indeed,
among published FS LC sequences, 3 were derived from the same germline
gene O2/O12 and were associated with LC crystallization, the fourth one
derived from the closely related O8/O18 gene did not yield any crystal.
Comparison of these sequences focused our attention on specific
substitutions at position 30, where the unusual presence of nonpolar
residues is shared by the 3 FS LC CHEB, TRE, and
TRO5 featuring crystallization. In LC CHEB, Ala 30 is encoded by the codon GCC and may either result from allelic
variation or from a block mutation of the original AGC codon of the
published O2/O12 germline gene. In FS patient DEL5 without crystallization, VI position 30 was occupied by serine, the
most frequent amino acid at this position.
The importance of Ala30 in protein CHEB is most clearly seen in
mice expressing CHEBm30 LC with an AlaSer substitution
introduced through site-directed mutagenesis, because these mice had no
intracellular crystals. Indeed, 2 of these mice presented tubular cast
nephropathy, thus showing that a single amino acid substitution can
change the crystal-forming property into the ability to yield partially organized agregates. Another unusual nonpolar residue of protein CHEB, Ile 94, was targeted in the same way: the CHEBm94 variant I chain did not yield crystals either. Taken together, our data directly demonstrate that sequence peculiarities of V domains are
implicated in the process of LC crystallization that occurs in most
cases of myeloma-associated FS and show that slightly different V
domain sequences may confer distinct pathogenetic properties on LC,
leading either to FS or cast myeloma. Although FS I chains likely
originate from a limited set of germline genes, we show that, in the
case of protein CHEB, concomitant alterations of such a
germline sequence are needed at least at 2 different positions (30 and
94) for the process of crystallization to occur. Interestingly,
simultaneous occurrence of these very same substitutions in the context
of a closely related VI framework is not sufficient to induce
crystallization in I variant 269m30. Because protein crystal
formation probably requires nucleation-dependent
initiation,32 unusual amino acids at position 30 may
increase the stability of monomer or dimer interactions, as suggested
for amyloidogenic LC21 and could thus initiate the
formation of crystal-forming units.32 However, the rare
ability of chains to form crystals also clearly involves the whole
tridimensional structure of the V domain and a precise combination of
germinally and somatically encoded residues.
In vivo studies and clearance measurements could not be performed.
Consequently, proximal tubule function was evaluated ex vivo by uptake
studies on freshly isolated proximal tubule suspensions. Phosphate
proximal tubule uptake was significantly decreased in proximal tubule
cells from CHEB mice, thus suggesting that transfection of LC induced 1 cardinal feature of the proximal tubule dysfunction characteristic of FS. In our model, the mechanisms of the alteration of
phosphate proximal tubule uptake remain to be elucidated. Notably, tubular necrosis was not observed and inhibition of phosphate uptake
could theoretically result either from inhibition of the Na/Pi brush
border membrane cotransport or from decreased availability of the
transporter. Interestingly, CHEBm94 mice and I mice did not
have any intracellular crystal formation in proximal tubular cells, but
exhibited a significant reduction of the phosphate uptake. These
results suggest that the ability of CHEB LC to inhibit
tubular phosphate uptake may be independent from its property to
crystallize within proximal tubule cells. Indeed, clinical reports have
well established the absence of crystal formation in some patients with
multiple myeloma-associated FS.5
| |
FOOTNOTES |
Submitted March 29, 1999; accepted July 16, 1999.
C.D. and A.R. contributed equally to this work.
Supported by grants from Fondation contre la Leucémie (Grant No.
91001123), Ligue contre le Cancer (Comité Régional de la
Vienne), Conseil Régional du Limousin, Association pour la Recherche sur la Cancer (Grant No. 9121), INSERM 4R001B, and PHRC AOM
96058. C.D. is the recipient of a fellowship from Association pour la
Recherche sur le Cancer.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to M. Cogné, MD,
Laboratoire d'Immunologie, CNRS EP118, Faculté de
Médecine, 2 rue du Dr Marcland, F87025 Limoges, France; e-mail:
cogne{at}unilim.fr.
| |
REFERENCES |
1.
Pirani C, Silva F, D'Agati V, Chander P, Striker LMM:
Renal lesions in plasma cell dyscrasias: Ultrastructural observations.
Am J Kidney Dis
10:208, 1987[Medline]
[Order article via Infotrieve]
2.
Sanders PW, Booker BB, Bishop JB, Cheung HC:
Mechanisms of intranephronal proteinaceous cast formation by low molecular weight proteins.
J Clin Invest
85:570, 1990
3.
Aucouturier P, Bauwens M, Khamlichi AA, Denoroy L, Spinelli S, Touchard G, Preud'homme JL, Cogné M:
Monoclonal Ig L chain and L chain V domain fragment crystallisation in myeloma-associated Fanconi's syndrome.
J Immunol
150:3561, 1993[Abstract]
4.
Leboulleux M, Lelongt B, Mougenot B, Touchard G, Makdassi R, Rocca A, Noël LH, Ronco PM, Aucouturier P:
Protease resistance and binding of Ig light chains in myeloma-associated tubulopathies.
Kidney Int
48:72, 1995[Medline]
[Order article via Infotrieve]
5.
Rocca A, Khamlichi AA, Touchard G, Mougenot B, Ronco P, Denoroy L, Déret S, Preud'homme JL, Aucouturier P, Cogné M:
Light chain V region gene usage restriction and peculiarities in myeloma-associated Fanconi's syndrome.
J Immunol
155:3245, 1995[Abstract]
6.
Khamlichi AA, Rocca A, Touchard G, Aucouturier P, Preud'homme JL, Cogné M:
Role of light chain variable region in myeloma with light chain deposition disease: Evidence from an experimental model.
Blood
86:3655, 1995[Abstract/Free Full Text]
7.
Khamlichi AA, Rocca A, Cogné M:
Effect of intron sequences on expression levels of Ig cDNAs.
Gene
150:387, 1994[Medline]
[Order article via Infotrieve]
8.
Chauveau C, Decourt C, Cogné M:
Insertion of the IgH locus 3' regulatory palindrom in expression vectors warrants sure and efficient expression in stable B cell transfectants.
Gene
222:279, 1998[Medline]
[Order article via Infotrieve]
9.
Saïki RK, Scharf S, Fallona F:
Enzymatic amplification of  -globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.
Science
230:1350, 1985[Abstract/Free Full Text]
10.
Sanger F, Nicklen S, Coulson AR:
DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci USA
74:5463, 1987
11.
Bradford MM:
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:248, 1976[Medline]
[Order article via Infotrieve]
12.
Sletten K, Natvig JB, Husby G, Juul J:
The complete amino acid sequence of a prototype immunoglobulin-lambda light-chain-type amyloid-fibril protein AR.
Biochem J
195:561, 1981[Medline]
[Order article via Infotrieve]
13.
Solomon A, Frangione B, Francklin EC:
Bence Jones proteins and light chains of immunoglobulins. Preferential association of the VVI subgroup of human light chains with amyloidosis AL(1).
J Clin Invest
70:453, 1982
14.
Eulitz M, Linke R:
Amyloid fibrils derived from V-region together with C-region fragments from a lambda II-immunoglobulin light chain (HAR).
Biol Chem Hoppe-Seyler
366:907, 1985[Medline]
[Order article via Infotrieve]
15.
Toft KG, Sletten K, Husby G:
The amino-acid sequence of the variable region of a carbohydrate-containing amyloid fibril protein EPS (immunoglobulin light chain, type lambda).
Biol Chem Hoppe-Seyler
366:617, 1985[Medline]
[Order article via Infotrieve]
16.
Tveteraas T, Sletten K, Westermark P:
The amino acid sequence of a carbohydrate-containing immunoglobulin-light-chain-type amyloid-fibril protein.
Biochem J
232:183, 1985[Medline]
[Order article via Infotrieve]
17.
Dwulet FE, Strako K, Benson MD:
Amino acid sequence of a lambda VI primary (AL) amyloid protein (WLT).
Scand J Immunol
22:653, 1985[Medline]
[Order article via Infotrieve]
18.
Dwulet FE, O'Connor TP, Benson MD:
Polymorphism in a kappa I primary (AL) amyloid protein (BAN).
Mol Immunol
23:73, 1986[Medline]
[Order article via Infotrieve]
19.
Holm E, Sletten K, Husby G:
Structural studies of a carbohydrate-containing immunoglobulin lambda-light-chain amyloid-fibril protein (AL) of variable subgroup III.
Biochem J
239:545, 1986[Medline]
[Order article via Infotrieve]
20.
Benson MD, Dwulet FE, Madura D, Wheeler G:
Amyloidosis related to a lambda IV immunoglobulin light chain protein.
Scand J Immunol
29:175, 1989[Medline]
[Order article via Infotrieve]
21.
Liepnieks JJ, Dwulet FE, Benson MD:
Amino acid sequence of a kappa I primary (AL) amyloid protein (AND).
Mol Immunol
27:481, 1990[Medline]
[Order article via Infotrieve]
22.
Aucouturier P, Khamlichi AA, Preud'homme JL, Bauwens M, Touchard G, Cogné M:
Complementary DNA sequence of human amyloidogenic immunoglobulin light-chain precursors.
Biochem J
285:149, 1992
23.
Preud'homme JL, Aucouturier P, Touchard G, Khamlichi AA, Rocca A, Denoroy L, Cogné M:
Monoclonal immunoglobulin deposition disease: A review of immunoglobulin chain alterations.
Int J Immunopharmacol
16:425, 1994[Medline]
[Order article via Infotrieve]
24.
Cogné M, Preud'homme JL, Bauwens M, Touchard G, Aucouturier P:
Structure of a monoclonal kappa chain of the VIV subgroup in the kidney and plasma cells in light chain deposition disease.
J Clin Invest
87:2186, 1991
25.
Bellotti V, Stoppini M, Merlini G, Zapponi MC, Meloni ML, Banfi G, Ferri G:
Amino acid sequence of Sci, the Bence Jones protein isolated from a patient with light chain deposition disease.
Biochem Biophys Acta
1097:177, 1991[Medline]
[Order article via Infotrieve]
26.
Khamlichi AA, Aucouturier P, Silvain C, Bauwens M, Touchard G, Preud'homme JL, Nau F, Cogné M:
Primary structure of a monoclonal chain in myeloma with light chain deposition disease.
Clin Exp Immunol
87:122, 1992[Medline]
[Order article via Infotrieve]
27.
Rocca A, Khamlichi AA, Aucouturier P, Noël L, Denoroy L, Preud'homme JL, Cogné M:
Primary structure of a variable region of the VI subgroup (ISE) in light chain deposition disease.
Clin Exp Immunol
91:506, 1993[Medline]
[Order article via Infotrieve]
28.
Decourt C, Cogné M, Rocca A:
Structural peculiarities of a truncated VIII immunoglobulin light chain in myeloma with light chain deposition disease.
Clin Exp Immunol
106:357, 1996[Medline]
[Order article via Infotrieve]
29.
Decourt C, Touchard G, Preud'homme JL, Vidal R, Beaufils H, Diemert MC, Cogné M:
Complete primary sequences of two immunoglobulin light chains in myelomas with non-amyloid (Randall type) light chain deposition disease.
Am J Pathol
153:313, 1998[Abstract/Free Full Text]
30.
Solomon A, Weiss DT:
A perspective of plasma cell dyscrasias: Clinical implications of monoclonal light chains in renal disease, in
Minetti L,
d'Amico G,
Ponticelli C
(eds):
The Kidney in Plasma Cell Dyscrasias. Dordrecht, The Netherlands, Kluwen, 1988, p 3
31.
Defronzo RA, Thier SO:
Inherited disorders of renal tubule function, in
Brenner BM,
Rector FC
(eds):
The Kidney. Philadelphia, PA, Saunders, 1986, p 1287
32.
Jarret TJ, Lansbury PT Jr:
Seeding "one-dimensional crystallization" of amyloid: A pathogenic mechanism in Alzheimer's disease and scrapie?
Cell
73:1055, 1993[Medline]
[Order article via Infotrieve]
33.
Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C:
in US Department of Health and Human Services, Public Health Service, National Institutes of Health: Sequences of proteins of immunological interest (ed 5). Bethesda, MD, NIH Publications, 1991
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. Sirac, F. Bridoux, C. Carrion, O. Devuyst, B. Fernandez, J.-M. Goujon, C. E. Hamel, J.-C. Aldigier, G. Touchard, and M. Cogne
Role of the monoclonal {kappa} chain V domain and reversibility of renal damage in a transgenic model of acquired Fanconi syndrome
Blood,
July 15, 2006;
108(2):
536 - 543.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Decourt, H. R. Galea, C. Sirac, and M. Cogne
Immunologic basis for the rare occurrence of true nonsecretory plasma cell dyscrasias
J. Leukoc. Biol.,
September 1, 2004;
76(3):
528 - 536.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Lebeau, E. Zeindl-Eberhart, E.-C. Muller, J. Muller-Hocker, P. R. Jungblut, B. Emmerich, and U. Lohrs
Generalized crystal-storing histiocytosis associated with monoclonal gammopathy: molecular analysis of a disorder with rapid clinical course and review of the literature
Blood,
August 13, 2002;
100(5):
1817 - 1827.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-U. Rengers, G. Touchard, C. Decourt, S. Deret, H. Michel, and M. Cogne
Heavy and light chain primary structures control IgG3 nephritogenicity in an experimental model for cryocrystalglobulinemia
Blood,
June 1, 2000;
95(11):
3467 - 3472.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION