The Intrinsic Factor-Vitamin B12 Receptor, Cubilin, Is Assembled into Trimers via a Coiled-coil alpha -Helix*

Anders LindblomDagger §, Natascha Quadt§, Tracey Marshparallel , Daniel Aeschlimann**, Matthias MörgelinDagger Dagger , Karlheinz Mann§§, Patrik Maurer, and Mats Paulsson¶¶

From the Dagger  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 parallel  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 Dagger Dagger  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

    ABSTRACT
Top
Abstract
Introduction
References

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 alpha -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 alpha -helix.

    INTRODUCTION
Top
Abstract
Introduction
References

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 alpha -helix.

    MATERIALS AND METHODS

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% beta -mercaptoethanol. Proteins were stained with Coomassie Brilliant Blue R250.

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 -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.

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 beta -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.

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).

    RESULTS

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).


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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. Lalpha and Lbeta gamma designate the migration positions of the alpha 1 (Mr 400,000), beta 1, and gamma 1 (Mr 200,000) chains from Engelbreth-Holm-Swarm tumor laminin.

                              
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Table I
Hydrodynamic parameters of intact cubilin and its subunit obtained after reduction and alkylation

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).


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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).

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.


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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.

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.


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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.

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.

    DISCUSSION

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 beta -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 beta -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.

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 alpha -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 right-arrow i + 3 and i right-arrow 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 alpha -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 alpha -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 right-arrow i + 3 or i right-arrow 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 alpha -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 alpha -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.

    ACKNOWLEDGEMENTS

We are indebted to Dr. Paul Jenö and Ariel Lustig, Biocenter (Basel, Switzerland) for performing initial amino acid sequencing and analytical ultracentrifugation, respectively.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; PBS, phosphate-buffered saline.

    REFERENCES
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Abstract
Introduction
References
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