From the Department of Internal Medicine, Malmö
General Hospital, Lund University, S-214 01 Malmö, Sweden, the
¶ Institute for Biochemistry, Medical Faculty, University of
Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany, the
M. E. Müller Institute for Biomechanics, University of
Bern, CH-3010 Bern, Switzerland, the ** Division of Orthopedic Surgery,
University of Wisconsin, Madison, Wisconsin 53792, the
Department of Cell and Molecular Biology,
Lund University, S-221 00 Lund, Sweden, and the
§§ Department of Protein Chemistry, Max Planck
Institute for Biochemistry, D-82152 Martinsried, Germany
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ABSTRACT |
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A large protein was purified from bovine kidney,
using selective extraction with EDTA to solubilize proteins anchored by
divalent cation-dependent interactions. An antiserum raised
against the purified protein labeled the apical cell surface of the
epithelial cells in proximal tubules and the luminal surface of small
intestine. Ten peptide sequences, derived from the protein, all matched
the recently published sequences for rat (Moestrup, S. K.,
Kozyraki, R., Kristiansen, M., Kaysen, J. H., Holm Rasmussen, H.,
Brault, D., Pontillon, F., Goda, F. O., Christensen, E. I.,
Hammond, T. G., and Verroust, P. J. (1998) J. Biol. Chem. 273, 5235-5242) and human cubilin, a receptor for
intrinsic factor-vitamin B12 complexes, identifying the
protein as bovine cubilin. In electron microscopy, a three-armed
structure was seen, indicating an oligomerization of three identical
subunits. This model was supported by the Mr values of about 1,500,000 for the intact protein and 440,000 for its
subunits obtained by analytical ultracentrifugation. In a search for a
potential assembly domain, we identified a region of heptad repeats in
the N-terminal part of the cubilin sequence. Computer-assisted analysis
supported the presence of a coiled-coil The receptor for intrinsic factor-vitamin B12
complexes, cubilin, is expressed in intestinal epithelium and, more
abundantly, in kidney tubular epithelium and yolk sac (1). In the
kidney, it is found at the base of the brush border at the apical
surface of the epithelial cells of proximal tubuli, as well as in
endocytic vesicles in the apical portion of the cell (2). It
codistributes with the transcobalamin-vitamin B12 receptor
megalin, and recently an interaction between cubilin and megalin was
demonstrated (3). Megalin may mediate the intracellular trafficking of
cubilin, which serves the function of facilitating the endocytic uptake of intrinsic factor-vitamin B12 complexes (3). After
degradation of intrinsic factor in the lysosomes, vitamin
B12 is secreted to plasma in a complex with transcobalamin
(4).
The complete amino acid sequences were recently determined for rat (3)
and human (5) cubilin. The two sequences show 69% identity and predict
a peripheral membrane protein of Mr 396,953 in
rat and 396,280 in human with a signal peptide, a stretch of about 110 amino acids without homology to known proteins, followed by 8 EGF1 repeats and 27 CUB
domains. Whereas the EGF domain, patterned on epidermal growth factor,
has been recognized in many extracellular proteins (6), the CUB domain
was more recently defined (7) on the basis of a module found in the C1r
and C1s components of complement.
In early studies, the intrinsic factor-vitamin B12 receptor was
isolated from porcine (8-10) and canine (11) ileal mucosa. When
extracted by Triton X-100, the receptor was detected in several complexes with masses ranging from 800,000 to 12,000,000 Da, depending on source and method of analysis. Upon SDS-PAGE, polypeptides ranging
from 40,000 to 180,000 Da were recovered. More recently, the receptor
has been purified from rat kidney as a protein of Mr 230,000 (1) and 460,000 (cubilin) (2) by
SDS-PAGE, which showed a tendency to form higher aggregates. The clones
containing the cDNA coding for the 396,953-Da rat cubilin were
identified by use of specific antibodies raised against purified
cubilin and confirmed by the demonstration of matching sequences in
peptides derived from this receptor protein (3). The same antibodies detect a band of apparent Mr 460,000 also in
intestinal mucosa, albeit some lower Mr
immunoreactive material could also be seen in immunoblots from this
source (3). It may be that the lower values for
Mr obtained in the early studies of the
intrinsic factor-vitamin B12 receptor from ileal mucosa were due to
degradation of the receptor when isolated from this source, which is
particularly rich in proteases.
During studies of extracellular matrix proteins from bovine kidney
(12), we found an abundant, large, oligomeric protein, which could be
selectively extracted with chelating agents and which was localized to
kidney tubules. We have now, by determining amino acid sequences from
peptides spanning a large portion of the protein, shown it to be the
bovine homologue of cubilin. The access to highly purified protein,
obtained under mild conditions, allowed us to use electron microscopy
and analytical ultracentrifugation to show the organization of cubilin
into a noncovalently associated trimer in which the subunits are
connected by a coiled-coil Purification of Cubilin--
Fresh bovine kidneys were cut in
1-3-g pieces (approx. 400 g/batch) and homogenized in 20 volumes
(ml/g) of cold Tris-buffered saline (TBS) (0.15 M NaCl, 50 mM Tris-HCl, pH 7.5), using a Polytron homogenizer at full
speed, and centrifuged (10,000 rpm for 20 min at 4 °C) in a Beckman
JA 10 rotor. Protease inhibitors (5 mM
N-ethylmaleimide, 5 mM phenylmethylsulfonyl
fluoride) were added to all extraction buffers. All extraction and
purification steps were performed at 4 °C. The pellet was
resuspended by brief homogenization in 20 volumes of TBS and
centrifuged. This process was repeated two or three times. The pellets
were resuspended and further extracted by stirring for 2-15 h with 5 volumes of TBS containing 10 mM EDTA. After centrifugation,
the EDTA extraction was repeated.
The combined EDTA-extracts were diluted 2:1 with distilled water before
addition of DEAE-Sepharose Fast Flow (Amersham Pharmacia Biotech) (100 ml of gel/200 g of tissue), equilibrated in 0.1 M NaCl, 10 mM EDTA, 40 mM Tris-HCl, pH 7.5. After
end-over-end rotation overnight, the ion-exchanger was allowed to
sediment, and the supernatant, containing the cubilin, was collected.
The pH was adjusted to 8 by the addition of NaOH, and a stronger
anion-exchanger, Q-Sepharose Fast Flow (Amersham Pharmacia Biotech)
(100 ml of gel/200 g of tissue; equilibrated in 0.1 M NaCl,
10 mM EDTA, 40 mM Tris-HCl, pH 8.0) was added.
After end-over-end rotation overnight, the supernatant was decanted,
and the gel was poured into a column (2.6 × 40 cm) and washed
(20-60 ml/h) with equilibration buffer (500-1000 ml). Bound material
was eluted with 0.5 M NaCl in the same buffer and
centrifuged at 100,000 g for 1 h. The supernatant was
applied (80-100 ml per run at 80 ml/h) to a column (5 × 100 cm)
of Sepharose CL 4B (Amersham Pharmacia Biotech) equilibrated in TBS-10
mM EDTA, containing 0.5 mM
N-ethylmaleimide and 0.5 mM phenylmethylsulfonyl
fluoride. Fractions containing cubilin were pooled, diluted 2:1 with
distilled water, and passed through a column of heparin-Sepharose
(1.5 × 15 cm) that was equilibrated in 0.1 M NaCl, 10 mM EDTA, 10 mM Tris, pH 7.5. Cubilin, which does not bind heparin, was concentrated on a column (1.5 × 10 cm)
of Q-Sepharose Fast Flow that was equilibrated in 0.1 M
NaCl, 10 mM EDTA, 10 mM Tris, pH 8.0. The
flow-through material from the heparin column was adjusted to pH 8.0 before application (at approximately 20 ml/h) to the Q-Sepharose. After
washing, each column was separately eluted with 0.5 M NaCl,
5 mM EDTA, 50 mM Tris, pH 7.5. For final
purification, an additional ion exchange chromatography was performed.
The cubilin pool was dialyzed into 0.1 M NaCl, 10 mM EDTA, 40 mM Tris, pH 7.5, and loaded onto a column (1.5 × 15 cm) of DEAE-Sepharose Fast Flow. Cubilin eluted in the flow-through and was concentrated by binding to Q-Sepharose Fast
Flow as described above.
Electrophoresis--
SDS-PAGE was performed in gradient gels
using the Laemmli (13) buffer system. Samples were applied reduced or
not reduced with 2% Immunohistochemistry--
A specific antiserum against cubilin
was raised in rabbit. Cubilin bands were cut out from a SDS-PAGE gel,
on which purified cubilin had been loaded, and used as antigen. Rat
kidney (2-mm-thick slices) was fixed either twice for 12 h in
fresh 0.5% (w/v) paraformaldehyde in PBS (0.15 M NaCl, 8 mM sodium phosphate, pH 7.4) and overnight in 2% uranyl
acetate for immunofluorescence staining, or twice for 12 h in
fresh 4% (w/v) paraformaldehyde in PBS for immunoperoxidase staining.
Rat small intestine was cleared of feces by rinsing in PBS and fixed
twice for 12 h in freshly prepared 4% (w/v) paraformaldehyde in
PBS. The fixed tissue was extensively washed in PBS, soaked in 1 M sucrose overnight, equilibrated in 2.3 M
sucrose, and frozen on dry ice in Tissue TekTM (Miles). Rat
kidney cryosections (6 µm) were cut at Trypsin Digestion--
A cubilin sample was dialyzed against
0.15 M NaCl, 20 mM Tris-HCl, 1 mM,
pH 7.5, and either CaCl2 or EDTA was added to a final concentration of 1 mM. Trypsin (bovine pancreas,
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated,
Sigma) was added to a ratio of 1:200 (weight of enzyme/weight of
substrate). The sample was incubated for 24 h at room temperature,
and digestion was terminated by precipitation with ethanol (9 volumes).
Cyanogen Bromide Cleavage--
A modification of the method of
Gross and Witkop (16) was used. 300-400 µg of highly purified protein
was dissolved in 100 µl of 6 M guanidine HCl, 0.1 M Tris-HCl, pH 8.0-8.5. 10 µl of N-terminal Sequence Analysis of Peptides--
Tryptic peptides
were separated by SDS-PAGE on a 3-15% gel and electroblotted to
a ProblottTM membrane (Applied Biosystems). The
membrane were briefly stained with Coomassie Brilliant Blue and
destained in 40% (v/v) high performance liquid chromatography-grade
methanol. Bands were cut out, dried, and frozen until use. The
CNBr-peptides were separated according to size on a Superdex Peptide
HR10/30 column (Amersham Pharmacia Biotech) in 0.1% trifluoroacetic
acid containing 25% acetonitrile at a flow rate of 0.3 ml/min.
Selected pools were further purified by reversed phase high performance
liquid chromatography on a 3 × 250-mm column filled with Vydac
C18 phase with a gradient of 3-42% acetonitrile in 0.1%
trifluoroacetic acid in 160 min at a flow rate of 0.25 ml/min. Fragment
CB1 was further cleaved with lysyl endopeptidase (WAKO, Richmond, VA)
at an enzyme to substrate ratio of 1:100 in 0.1 M ammonium
hydrogen carbonate containing 4 M urea for 16 h at
23 °C. The resulting peptides were separated by reversed phase high
performance liquid chromatography as above. Peptides were sequenced
using an Applied Biosystems 470A sequenator.
Electron Microscopy--
The rotary shadowing technique was
adapted from Shotton et al. (17) and used as described
previously (18). Protein samples were dissolved in 1 M
ammonium acetate (10-50 µg/ml) and, after addition of an equal
volume of glycerol, sprayed onto freshly cleaved mica disks. Negative
staining followed a previously published procedure (18).
Preparation of Subunits--
Purified cubilin was dialyzed
against 6 M guanidine HCl, 10 mM Tris-HCl, pH
7.5. Dithiothreitol was added to a final concentration of 10 mM, and the reaction vials were flushed with nitrogen and incubated at 60 °C for 4 h. Free thiol groups were blocked with an excess of iodoacetamide (final concentration, 30 mM) for
20 min, and the samples were dialyzed against 6 M guanidine
HCl, 10 mM Tris-HCl, pH 7.5.
Analytical Ultracentrifugation--
A Beckman model XL-A
analytical ultracentrifuge was used with the absorption optics at 275 nm. The sedimentation velocity runs were carried out at 52,000 rpm and
20 °C with a 12-mm double-sector Kel-F cell. The sedimentation
equilibrium runs were done at 20 °C using a 12-mm double sectors
charcoal-filled Epon cell. The two sectors had been filled (0.1 ml)
with sample or dialyzed reference solvent. Speeds of 4400-8000 rpm
were used depending on the Mr of the sample.
Mr was calculated with a program similar to the EQASSOC program (19), using a floating baseline equivalent to that
described by Chernyak et al. (20). A partial specific volume of 0.73 cm3/g was assumed in all calculations.
Solvent viscosity and density has been corrected to standard
H2O values according to Kawahara and Tanford (21).
Isolation and Characterization of Cubilin from Bovine
Kidney--
During studies of basement membrane proteins (22, 23), we
found that extraction of tissues with chelating agents, such as EDTA,
selectively solubilizes a limited set of proteins, which appear to be
anchored by divalent cation-dependent interactions. When we
applied this approach to bovine kidney with the purpose of isolating
kidney-specific laminin isoforms (12), we noticed the presence of
substantial amounts of a protein with an apparent subunit mass of
400,000 Da on SDS-PAGE (Fig. 1,
lane 1). Unlike laminin, this protein did not bind to
DEAE-Sepharose at 0.1 M NaCl and pH 7.5, but it could be
collected on the stronger anion-exchanger Q-Sepharose, after adjustment
of the pH to 8.0. The protein eluted from the Q-Sepharose was further
purified by gel filtration on Sepharose CL 4B, where it appeared in an
early peak together with residual amounts of laminin. The laminin
contamination could be removed by passing the material over
heparin-Sepharose as laminin binds to heparin with moderate strength
(24), whereas the novel protein is not bound. Repetition of some of
these chromatographic steps increased the purity further (Fig. 1,
lane 3). When SDS-PAGE was performed without reduction and
avoiding boiling in SDS, variable amounts of larger complexes were seen
(Fig. 1, lane 6), indicating that the native protein may
have an oligomeric structure. This observation was supported by
analytical ultracentrifugation in the absence of SDS (see Table
I and below). Tryptic digestion of
purified cubilin yielded distinct fragments, which were larger when the
digestion was performed in the presence of calcium (Fig. 1, lanes
4 and 5).
Immunohistochemistry--
An antiserum against the novel protein
was raised in rabbit and used in indirect immunofluorescence and
immunoperoxidase microscopy on sections of rat kidney (Fig.
2). The antigen had been purified as
described above and subjected to SDS-PAGE, and the narrowly cut-out
Mr 400,000 band was used for immunization. A
very selective staining of a subpopulation of tubuli in the kidney
cortex was observed (Fig. 2b), and higher resolution
immunohistochemistry, performed using a peroxidase-labeled second
antibody, showed in transverse sections a continuous staining along the
apical surface adjacent to the brush border in proximal tubules (Fig.
2c). In addition to this staining along the apical surface,
dots of staining were seen that might represent membrane-bound
vesicles. Low magnification immunofluorescence of rat small intestine
showed a continuous staining of the luminal surface, and at higher
magnification of cross-sections, staining of the brush border was seen
(Fig. 3).
Determination of Peptide Sequences--
To determine the identity
of the novel protein, it was cleaved with trypsin, and the fragments
were separated by SDS-PAGE. Two peptides of apparent
Mr 140,000 and 90,000 (Fig. 1, lane
4; Fig. 4, T140 and
T90) proved to be accessible to N-terminal sequencing. Further peptides could be obtained by cyanogen bromide and lysyl endopeptidase cleavage and yielded eight additional sequences (Fig. 4,
CB1-4 and CB-K1-4). A search of the translated
EMBL/GenBankTM data bases showed that each sequence was
identical or homologous with rat (3) and human (5) cubilin. The ten
peptides were derived from both EGF repeats and CUB domains and spanned
a large portion of the cubilin sequence.
Electron Microscopy--
Glycerol spraying/rotary shadowing
electron microscopy of cubilin showed relatively homogenous but often
collapsed particles of a star-shaped structure (Fig.
5). Analysis of well spread particles revealed three similarly sized arms (about 75 nm in length) extending from a central point (Fig. 5B). In negative staining (Fig.
5C), each arm was resolved into a number of globular or
elongated domains in a tandem array. The convoluted appearance of the
arms, as well as the frequent observation of collapsed particles,
indicates that these domains are connected by highly flexible
regions.
Analytical Ultracentrifugation--
Molecular mass and
sedimentation coefficient were determined for intact cubilin under
native conditions as well as in 6 M guanidine HCl and for
cubilin subunits, obtained after reduction and alkylation in 6 M guanidine HCl (Table I). In both solvents, a molecular
mass for cubilin of about 1,500,000 Da was obtained, whereas the
sedimentation coefficient decreased from 19.9 to 13.0 S upon exposure
to 6 M guanidine HCl, suggesting unfolding of the
polypeptide chains. The cubilin subunit obtained after reduction and
alkylation had a molecular mass of 440,000 Da. The relationship between
the molecular mass of the intact protein and of the subunit most
closely fits a trimer.
We have isolated cubilin, the intrinsic factor-vitamin
B12 receptor, from bovine kidney as a large oligomeric
protein. The selective solubilization by EDTA yielded cubilin in a
native trimeric form that was characterized by analytical
ultracentrifugation and electron microscopy. The data allow us to
derive a model for cubilin structure and domain organization.
Antibodies against cubilin revealed its presence at the apical surface
in the proximal tubules of kidney and at the luminal surface of the
small intestine. As the sequence of cubilin (3, 5) does not predict a
transmembrane domain, cubilin seems to be peripherally bound to the
plasma membrane or a receptor in a divalent
cation-dependent manner. Similarly, Moestrup et
al. (3) showed that release of cubilin from rabbit renal membranes is facilitated by EDTA and that cubilin binding to megalin is abrogated
by EDTA. We were able to show that the presence of calcium stabilizes
cubilin against cleavage by trypsin (Fig. 1, lanes 4 and
5) and a variety of proteases (results not shown),
indicating that calcium binding induces a conformational change in the
molecule. This calcium-dependent conformation may favor the
interaction of cubilin with the plasma membrane or its receptor. EGF
repeats 2, 4, 5, and 8 are of the calcium binding type (3), but it remains to be shown whether these domains are directly involved in interactions.
The intrinsic factor-vitamin B12 receptor was recently
isolated as a monomeric Mr 460,000 protein from
Triton X-100 solubilized kidney cortex membranes (2). From a
combination of electron microscopy, analytical ultracentrifugation, and
SDS-PAGE we propose that cubilin isolated without detergents is a
trimer of identical Mr 440,000 subunits. This
would yield a molecular mass of 1,320,000 Da for the trimer, whereas
the estimated mass from ultracentrifugation was about 1,500,000 Da. The
discrepancy could be due to the presence of aggregates, leading to an
overestimation from the ultracentrifugation study. The trimer appears
to be more stable to denaturation with the chaotropic agent guanidine
HCl than with the detergent SDS. The higher sensitivity to detergents
hints at an importance of hydrophobic interactions for the formation of
the oligomer (see below). The use of detergents in previous
purification protocols for cubilin may explain why the trimeric
structure was not revealed.
Each subunit of cubilin has a length of about 75 nm and is made up of a
tandem array of globular domains connected by segments that give the
overall structure a high degree of flexibility. Cubilin is made of a
N-terminal stretch of 110 amino acids, followed by 8 EGF-like domains
and 27 CUB domains. Assuming lengths of 4.4 nm for the N-terminal
domain (see below) and 20 nm for eight EGF domains, the CUB domains
should extend over 50.6 nm, or 1.9 nm per CUB domain. CUB domains form
a compact ellipsoid The observation of the trimeric structure led to a search for an
assembly domain. Indeed, scrutiny of the rat (3) and human (5) cubilin
sequences revealed a N-terminal region with a repeating pattern of
hydrophobic residues. Alignment with the heptad repeat indicated the
presence of four heptads between amino acids 103 and 132 preceding the
first EGF-like domain (Fig. 6). Positions a and d of the heptads are occupied by residues Ile, Leu, Val, and Phe,
which come into close contact in an -helix between amino acids
103 and 132 of the human cubilin sequence and predicted the formation
of a triple coiled-coil. We therefore conclude that cubilin forms a
noncovalent trimer of identical subunits connected by an N-terminal
coiled-coil
-helix.
INTRODUCTION
Top
Abstract
Introduction
References
-helix.
MATERIALS AND METHODS
-mercaptoethanol. Proteins were stained with
Coomassie Brilliant Blue R250.
27 °C, adsorbed to
gelatin-coated slides, air-dried, and immunolabeled with the antibody
(diluted 1:100 or 1:50) using either the fluorescence (14) or
peroxidase (15) protocol with 10% human serum in TBS for blocking.
Similarly prepared cryosections of rat small intestine were
immunolabeled with the antibodies diluted 1:100 using Texas Red-conjugated secondary antibodies (Dako) and 1% BSA in TBS for blocking.
-mercaptoethanol was
added, and the sample was incubated for 2 h at 50 °C to
reduce disulfide bonds. Thiol groups were alkylated by addition of 20 µl of vinylpyridine and incubation for 2 h in the dark at room
temperature. The protein was extensively dialyzed against 5% acetic
acid, lyophilized, and subsequently dissolved in 100 µl of formic
acid. One crystal of cyanogen bromide was added, and the sample was
incubated for 24 h in the dark at room temperature. After
drying the sample with a stream of nitrogen, the cleavage was checked
by SDS-PAGE.
RESULTS
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Fig. 1.
SDS-PAGE of bovine kidney cubilin at
different stages of purification and after tryptic digestion.
Electrophoresis was performed under reducing conditions of samples of
the combined EDTA-extracts (lane 1), of the flow-through
from the heparin-Sepharose column after chromatography on DEAE- and
Q-Sepharose (lane 2), and of the fully purified protein
after repeated chromatography on DEAE- and Q-Sepharose (lane
3). Tryptic digestion of the purified cubilin was performed in the
presence of 1 mM CaCl2 (lane 4) or 1 mM EDTA (lane 5), and the fragments obtained
were separated by SDS-PAGE. Samples identical to those in lane
2 were also electrophoresed without prior reduction before
(lane 6) and after (lane 7) boiling in the SDS
sample buffer. The gels were gradients of 3-10% polyacrylamide and
stained with Coomassie Brilliant Blue. The arrowheads mark
cubilin monomers, the arrow marks oligomers, and
asterisks mark the Mr 140,000 and
90,000 tryptic fragments used for N-terminal sequencing. The molecular
masses of protein standards are given in kDa. L and
L
designate the migration positions of the
1
(Mr 400,000),
1, and
1
(Mr 200,000) chains from Engelbreth-Holm-Swarm
tumor laminin.
Hydrodynamic parameters of intact cubilin and its subunit obtained
after reduction and alkylation
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Fig. 2.
Immunohistochemical demonstration of cubilin
in kidney proximal tubules. Overviews of rat kidney are shown in
phase contrast (a) and after indirect immunofluorescence
staining for cubilin (b). In c, immunoperoxidase
staining along the apical surface of proximal tubules is shown, and
d shows a similar control section, at which the specific
antibody had been exchanged for a nonspecific IgG. Note the distinct
localization of the protein in proximal tubules (b) and the
circular staining of the apical aspect of the tubular epithelial cells
(c). The brush border remains largely unstained. The
bars correspond to 150 µm (a and b)
and 10 µm (c and d).
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Fig. 3.
Immunohistochemical localization of cubilin
in small intestine. An overview of a longitudinal section
through rat small intestine is shown in phase contrast (top
panel) and after indirect immunofluorescence for cubilin
(middle panel). In a cross-section at the level of the
crypts, a circular staining surrounding the lumen was seen when differential interference contrast and fluorescence images
were overlaid (bottom panel). The bars correspond
to 200 µm (top and middle panels) and 30 µm
(bottom panel).
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Fig. 4.
Alignment of peptide sequences with rat and
human cubilin. The sequences of two tryptic (T90 and T140) and
eight cyanogen-bromide-derived (CB) and lysyl
endopeptidase-derived (K) peptides were aligned with the
corresponding sequences from rat and human cubilin. Lowercase
letters designate residues for which the identification was not
unambiguous.
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Fig. 5.
Electron microscopy of cubilin after glycerol
spraying/rotary shadowing (A and B)
and negative staining (C). A shows a
representative overview, whereas B and C show
selected well spread particles of cubilin. A structure with three arms
is seen in most well resolved particles. With negative stain, each arm
is resolved into a tandem array of globular or elongated domains
connected by more flexible regions.
DISCUSSION
-sandwich structure (25). Seminal plasma PSP-I
and PSP-II are each composed of one CUB domain and form heterodimers
(26). The N- and C-terminal ends of PSP-I and PSP-II are located at the
same side of a 5-stranded
-sheet and are buried in the interface. It
is unlikely that such an interaction also occurs between tandem CUB
domains, as the arrangement does not allow the formation of rows of
consecutive domains. The electron microscopic pictures of cubilin show
globules arranged in a zig-zag manner in the outer segments of the
arms, demonstrating that the relative orientation of CUB domains in cubilin is variable and not rod-like.
-helical coiled-coil and
stabilize it by hydrophobic interactions. Further coiled-coil stabilization may occur through intrahelical ionic interactions between
oppositely charged side chains of the type i
i + 3 and i
i + 4 (27, 28).
Cubilin residues at positions b, c, e, f, and g are mainly hydrophilic,
and two intrachain ionic interactions are possible (Fig. 6). These
findings were confirmed when the sequence was analyzed by different
algorithms for predicting
-helical coiled-coil structures (Fig.
7). All programs agree in their
assignment of heptad positions (abcdefg) to each residue. A pair of
charged residues, Lys-115-Glu-120, is located in heptad positions
g-e', compatible with the formation of an interchain ionic
interaction. Such ionic interchain interactions have been shown to
determine the packing and the oligomerization state of the
-helices
(29, 30).
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Fig. 6.
Sequence of cubilins arranged in heptad
repeats. The amino acid sequences of the N-terminal domain from
rat (3) and human (5) cubilins are aligned. The letters a-g
indicate positions in the heptad coiled-coil repeats. Hydrophobic
residues in positions a and d are shown in
black squares. Putative ionic interactions between charged
residues either within a peptide chain (spacing i i + 3 or i
i + 4) or between
adjacent chains in a coiled-coil conformation (g-e') are
indicated by brackets. Numbers refer to the position in the
complete cubilin precursors. Note that only the C-terminal
sequences show good agreement with the heptad consensus (A),
whereas frequent interruptions can be found in the N-terminal part
(B). Cys-133 and Cys-135 are the first amino acids in
EGF-like domains.
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Fig. 7.
Coiled-coil probability of the N-terminal
domain of human cubilin. The sequence of the amino acids 50-135
was analyzed for its coiled-coil forming potential using the programs
Coils ( ) (34), applying the MTIDK matrix and using a 2.5 weight on
residues in heptad positions a and d; Paircoil (- - - -) (35);
and MultiCoil (31), which differentiates for the possibilities of
forming a three-stranded (- - - - -) or two-stranded
(- - - - - -) conformation. In each case, a window size of 28 residues was used. No significant difference was observed when the rat
cubilin sequence was analyzed (not shown).
The MultiCoil program (31) allows a discrimination between dimer and
trimer coiled-coil formation and favors a trimer assembly for cubilin
(Fig. 7). Assuming a length of 0.15 nm/residue, a triple coiled-coil of
29 amino acids would yield a rod-like structure of about 4.4 nm (32).
The N-terminal sequence of these heptads is preceded by a proline- and
glycine-rich stretch that should interrupt formation of an -helical
coiled-coil. Within amino acids 53-102, heptad repeats can be
detected, but frequent interruption of the pattern of hydrophobic
residues at positions a and d makes the formation of a second
coiled-coil region questionable. This coiled-coil also could not be
detected by the algorithms.
It remains to be studied how the formation of the cubilin trimer
affects its function during endocytosis, but a multivalency might
strengthen the interactions with large ligands such as megalin. Already, the 27 CUB domains contained in each subunit provide multiple
interaction sites for proteins or membrane phospholipids, but possibly
a protein containing more than one binding site for cubilin at some
distance from each other may interact at the same time with two or more
arms of the molecule. A trimeric structure similar to that of cubilin
has been found for the macrophage scavenger receptor, which is,
however, anchored in the plasma membrane by a transmembrane domain
(33). In this receptor, a membrane-spanning domain is followed by a
coiled coil -helix consisting of 16 heptads and, further toward the
C terminus, by 23-24 Gly-X-Y triplets forming a
collagenous triple helix. At the C terminus, each chain contains a
scavenger receptor cysteine-rich domain. Even though the binding
partners for these domains in the scavenger receptor are not known,
homologous domains in other proteins are involved in extracellular
ligand interactions (33). Thus, both cubilin and the macrophage
scavenger receptor are examples of trimeric proteins facilitating
endocytosis of ligands, and for some other such receptors, the
oligomeric state is not yet known. Possibly both a multivalency of
binding within each subunit and an oligomeric arrangement of subunits
are mechanisms to facilitate endocytosis by concentrating ligands to a
small area of the membrane.
Birn et al. (2) have demonstrated that cubilin binds to the
intrinsic factor-vitamin B12 complex with high affinity and that labeled vitamin B12 is in vivo endocytosed
by a cubilin-mediated process into the epithelial cells of proximal
tubules. Although intrinsic factor is mainly acting in the
gastrointestinal uptake of vitamin B12 via cubilin, the
renal uptake of filtered intrinsic factor-vitamin B12
complex might prevent losses of filtered complexes to the urine. The
human cubilin gene has been mapped to the gene locus for a rare form of
congenital vitamin B12 deficiency,
Imerslund-Gräsbeck disease (5). Patients with this condition
suffer not only from megaloblastic anemia due to vitamin
B12 deficiency but also from proteinuria. This hints
at a further role of cubilin in renal protein reabsorption.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. Paul Jenö and Ariel Lustig, Biocenter (Basel, Switzerland) for performing initial amino acid sequencing and analytical ultracentrifugation, respectively.
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FOOTNOTES |
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* This work was supported by Grant Kr 558/10-1 from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work and share the first authorship.
¶¶ To whom correspondence should be addressed. Tel.: 49-221-478-6997; Fax: 49-221-478-6977; E-mail: mats.paulsson{at}uni-koeln.de.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; PBS, phosphate-buffered saline.
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REFERENCES |
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