Molecular Cloning of a Novel Lipocalin-1 Interacting Human Cell Membrane Receptor Using Phage Display*

Petra Wojnar, Markus Lechner, Petra Merschak, and Bernhard RedlDagger

From the Department of Microbiology (Medical School), University of Innsbruck, Fritz Pregl Strasse 3, A-6020 Innsbruck, Austria

Received for publication, February 26, 2001, and in revised form, April 2, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human lipocalin-1 (Lcn-1, also called tear lipocalin), a member of the lipocalin structural superfamily, is produced by a number of glands and tissues and is known to bind an unusually large array of hydrophobic ligands. Apart from its specific function in stabilizing the lipid film of human tear fluid, it is suggested to act as a physiological scavenger of potentially harmful lipophilic compounds, in general. To characterize proteins involved in the reception, detoxification, or degradation of these ligands, a cDNA phage-display library from human pituitary gland was constructed and screened for proteins interacting with Lcn-1. Using this method an Lcn-1 interacting phage was isolated that expressed a novel human protein. Molecular cloning and analysis of the entire cDNA indicated that it encodes a 55-kDa protein, lipocalin-1 interacting membrane receptor (LIMR), with nine putative transmembrane domains. The cell membrane location of this protein was confirmed by immunocytochemistry and Western blot analysis of membrane fractions of human NT2 cells. Independent biochemical investigations using a recombinant N-terminal fragment of LIMR also demonstrated a specific interaction with Lcn-1 in vitro. Based on these data, we suggest LIMR to be a receptor of Lcn-1 ligands. These findings constitute the first report of cloning of a lipocalin interacting, plasma membrane-located receptor, in general. In addition, a sequence comparison supports the biological relevance of this novel membrane protein, because genes with significant nucleotide sequence similarity are present in Takifugu rubripes, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Bos taurus, and Sus scrofa. According to data derived from the human genome sequencing project, the LIMR-encoding gene has to be mapped on human chromosome 12, and its intron/exon organization could be established. The entire LIMR-encoding gene consists of about 13.7 kilobases in length and contains 16 introns with a length between 91 and 3438 base pairs.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The protein superfamily of lipocalins consists of small, mainly secretory proteins defined on the basis of conserved amino acid sequence motifs and their common structure. Functionally they share several properties including the ability to bind/transport a remarkable array of small hydrophobic molecules, the formation of macromolecular complexes, and the binding to specific cell surface receptors (1-3). Whereas a large number of various lipophilic ligands able to bind to lipocalins are known, only limited data are available concerning the identity of lipocalin receptors. There is clear evidence of a specific receptor for plasma retinol-binding protein (RBP)1 (4-7) and more indirect evidence for receptors for alpha 1-microglobulin (8), major urinary protein (9), beta -lactoglobulin (10), olfactory-binding protein (11), alpha 1-acid glycoprotein (12), and glycodelin (13), but with the exception of megalin, which seems to be an endocytic receptor for a variety of soluble macromolecules including several lipocalins, no specific lipocalin receptor has been fully characterized so far (14). This lack of knowledge is a major disadvantage in understanding the biological function of many lipocalin members.

We have identified Lcn-1 (identical with tear lipocalin or von Ebner's gland protein) as a human member of the lipocalin superfamily (15). It is produced by a number of secretory glands and tissues, including lacrimal and lingual salivary glands, prostate, and mucosal glands of the tracheobronchial tree, nasal mucosa, and sweat glands and by some neuroendocrine tissues (15-21). Lcn-1 is an unusual member of the lipocalin superfamily, because it is able to bind a broad array of various lipophilic ligands in vitro and in vivo (15, 22) and was demonstrated to exhibit cysteine proteinase inhibition and nonspecific endonuclease activity in vitro (23, 24). Although the biological relevance of its multiple activities has still to be established, its main function seems to be scavenging of lipophilic, potentially harmful molecules, thus acting as a protection factor for cells and tissues (25). However, the mechanism of clearance or detoxification of the putative harmful ligands is unknown.

In the present study we have used a phage display-based technique for interaction screening of a complex cDNA expression library with Lcn-1 as bait to isolate proteins that may be involved in the reception or degradation of Lcn-1-specific ligands. Here we describe the identification, molecular cloning, expression, and subcellular localization of a novel Lcn-1 interacting membrane protein. Our findings set the stage for exploring the molecular mechanism of the lipocalin-receptor interaction in more detail. Moreover, this is the first successful attempt of identifying a lipocalin receptor using phage-display techniques.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

General Methods-- Total RNA was extracted by RNazolTMB (Cinna/Biotecx), which is based on the method developed by Chomczynski and Sacchi (26). For Western blot analysis proteins were transferred to nitrocellulose membranes (Sartorius, Göttingen, Federal Republic of Germany), and alkaline phosphatase-conjugated IgG was used as secondary antibody (Sigma). Detection was performed using the ECL system (Amersham Pharmacia Biotech).

Construction of the cDNA Phage-display Library-- Poly(A)+ RNA was prepared using Dynabeads oligo(dT)25 (Dynal, Oslo, Norway) exactly as described by the supplier. A portion of pituitary gland mRNA was reverse-transcribed using adapter primer 5'-AATAGGGTACC(T)17-3' and Superscript II reverse transcriptase (Life Technologies, Inc.), according to the manufacturer's specifications. After second strand synthesis using RNaseH and Escherichia coli DNA-polymerase I (Roche Molecular Biochemicals), a portion of the cDNA was digested with KpnI, subjected to agarose gel electrophoresis, and the region between 200 and 3000 base pairs was eluted. Phagemid pJuFo, a generous gift from R. Crameri (27), was cut with XbaI, blunt-ended with Klenow fragment, and successively cut with KpnI. The cDNA was directionally cloned into the blunt end and KpnI site of the vector. Phagemids were then transformed into E. coli strain XL1-Blue, and the cells were grown on Luria-Broth-amp plates.

Biopanning of the Phage-display Library against Lcn-1 as Bait-- E. coli cells were grown to mid-log phase, infected with R408 (Stratagene) helper phage (5 × 1010 plaque-forming units/ml), and grown for an additional 2 h. After centrifugation the supernatant was discarded to remove glucose. The pellet was resuspended in 2× YT supplemented with ampicillin (50 µg/ml), and the culture was grown overnight. Phages were isolated by polyethylene glycol (PEG 8000). Enrichment of phages was performed in MaxiSorb microtiter plates (Nunc, Roskilde, Denmark) under the following conditions: wells were precoated with 2 µg of purified Lcn-1 for 2 h at room temperature, washed, and blocked with 1% BSA. Wells were incubated (1 h, 37 °C) with 50 µl of mixed phages (108 to 109 colony-forming units). Following incubation the wells were washed twice (first round of panning), respectively, five times (second to fifth round of panning) with TBS, 0.05% Tween 20. Trapped phages were rescued by addition of 0.1 ml of log phase XL1-Blue cells and incubation for 30 min at 37 °C. The enriched library was plated on 2× YT/ampicillin/glucose/MgCl2 solid medium for titer determination and estimation of recovery of trapped phages.

Expression of a Recombinant N-terminal Fragment of LIMR in E. coli and Production of an LIMR Antiserum-- For bacterial expression the cDNA insert from phagemid pJuFo.33 was amplified by PCR using primers 5'-TTTATGTCGACATGGA AGCACCTGACTAC-3' and 5'-TTTATAAGCTTGTCACTTGTTGACGGTGGCATC-3', which incorporated a unique SalI and HindIII site, respectively. The PCR product was digested with SalI and HindIII, and after purification by agarose gel electrophoresis and gel extraction it was ligated into the corresponding cloning sites of pQE-9 (Qiagen). E. coli M15 was used as a host. Expression of the recombinant protein was induced by isopropyl-beta -D-thiogalactopyranoside (final concentration 1 mM). For purification of the recombinant protein, E. coli cultures were centrifuged, and the pellet was resuspended in 50 mM Na2HPO4 (pH 8.0)/300 mM NaCl at 2-5 volumes/g wet weight and passed twice through a French pressure cell (at 97 megapascal). The cell extract was centrifuged at 9000 × g for 20 min, and the recombinant protein present in the supernatant was purified by affinity chromatography on Ni-NTA resin (Qiagen) as described by the supplier. The recombinant peptide was further purified by FPLC gel filtration on Superdex-75. The purified peptide was subjected to SDS-polyacrylamide gel electrophoresis, and the first 5 N-terminal amino acids were determined on a gas/liquid phase sequenator. Antibodies against LIMR were raised in rabbit. 0.1 mg of recombinant LIMR peptide in 0.5 ml of buffer was mixed with an equal volume of Freund's complete adjuvant, and subcutaneous injections were performed four times at 3-week intervals.

Biochemical Assays for Protein-Protein Interaction-- For a solid phase-based assay of LIMR-Lcn-1 interaction wells of a microtiter plate were precoated with purified recombinant LIMR fragment (2 µg per well). The wells were blocked with 2% gelatin in PBS and were then incubated with increasing amounts of Lcn-1. After washing with PBS containing 0.1% Triton X-100 the LIMR-bound Lcn1 was detected by an Lcn-1-specific antiserum and anti-rabbit IgG horseradish peroxidase as secondary antibody. As a control, wells precoated with 2 µg of BSA instead of recombinant LIMR were used. For a binding assay in solution, the 6× His-tagged N-terminal fragment of LIMR (10-20 µg) bound to Ni-NTA resin (50-100 µl) was incubated with 2 µg of purified Lcn-1 in 1 ml of 50 mM NaH2PO4/300 mM NaCl (pH 8.0) at 4 °C for 2 h. The resin was then washed twice with 50 mM NaH2PO4/300 mM NaCl (pH 8.0) buffer containing 1% Triton X-100, and the His-tagged LIMR and any associated proteins were eluted with 250 mM imidazole. 20 µl of each eluate was subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting using an Lcn-1-specific antiserum.

Membrane Preparation-- NT2 cell membranes were prepared after homogenization in 10 mM Tris/HCl (pH 7.0), 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzamidine, and 1 mM iodacetamide essentially as described (28).

Immunocytochemistry-- NT2 cells were grown overnight on coverslips. The cells attached to coverslips were washed with PBS, and the coverslips were immersed immediately in 100% ice-cold acetone for 5 min. The coverslips were then removed from the acetone, rinsed twice in PBS, and blocked for 2 h in PBS/2% BSA. The LIMR-specific primary antibody was added in a dilution of 1:100 for 2 h at room temperature in a humidified chamber. The cells were washed three times with PBS for 15 min, and the fluorescein isothiocyanate-labeled secondary antibody (DAKO, Copenhagen, Denmark) was applied in a 1/35 dilution for 1 h at room temperature. The coverslips were washed in PBS overnight at 4 °C and mounted with a drop of glycerine and sealed with clear nail polish.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of Lcn-1-binding Proteins from the Phage-display Library-- Lcn-1 was found to be expressed in a variety of human glands or tissues. Among them we found pituitary gland to be an excellent source of intact mRNA. Therefore mRNA isolated from this gland was used for constructing a phagemid cDNA library. As cloning vector we used pJuFo (27), a gene pIII fusion-based phagemid system, because it is known that relatively large fusion moieties can be added onto pIII without resulting in steric interference with protein-ligand interaction (29). The recombinant fusion proteins are displayed on the phage surface as a result of the interaction of Fos and Jun. After amplification, the size of the library was about 109 plaque-forming units/ml. Analysis of the insert size of the randomly selected phagemids showed that most inserts had a length between 0.2 and 3 kilobases. Purified Lcn-1 was used to enrich the phage-display library for proteins that interact with Lcn-1. After four rounds of biopanning, 80 individual clones were screened for positive clones by enzyme-linked immunosorbent assay using an antiserum against the phage protein pVIII. 18 of these clones (22%) were positive for apparent binding to Lcn-1 and did not bind to wells that were blocked with BSA in the absence of Lcn-1. DNA sequencing revealed that 11 clones contained the same insert, including a 261-bp open reading frame, which shared identity with a human EST clone (GenBankTM accession numbers AL048902 and DKFZp434J2018) and significant homology with mouse EST clones (GenBankTM accession numbers AI875268, AA611039, and AI594033), encoding a so-far uncharacterized protein. The remaining clones did not share similarity with known sequences and did not contain open reading frames. Therefore we focused our research on one of the eleven identical clones (pJuFo.33), which encode the uncharacterized human protein.

Isolation of the Entire LIMR cDNA by RT-PCR and Determination of the Genomic Structure of the LIMR Gene-- A sequence comparison between clone pJuFo.33 and the human EST clone AL048902 demonstrated that the phage clone isolated contained only a partial fragment encoding the N-terminal part of the putative protein. Because the nucleotide sequence of human EST AL048902 was not complete, we sequenced the entire 2800-bp insert of this clone. However, we could not find an open reading frame. Therefore, in a next step we isolated the LIMR-encoding cDNA by RT-PCR using mRNA isolated from human pituitary gland. As shown in Fig. 1 the 2290-bp cDNA contains one open reading frame encoding a 487-amino acid protein with a predicted molecular mass of 55 kDa. By sequence comparisons using the entire deduced amino acid sequence we found additional putative orthologues in Takifugu rubripes (61% amino acid sequence identity), Drosophila melanogaster (41% identity), and Caenorhabditis elegans (34% identity), indicating that LIMR might be of general biological relevance. Moreover, from recently available sequencing data (GenBankTM accession number AC011603) of a human chromosome 12-specific contig, we were able to deduce the intron/exon structure of the LIMR-encoding gene. From Fig. 2 it is evident that the entire LIMR gene consists of ~13.7 kilobases and is composed of 17 exons ranging in size from 30 bp (exon 3) to 581 bp (exon 17), separated by 16 introns ranging from 91 bp (intron 9) to 3438 bp (intron 1).


View larger version (62K):
[in this window]
[in a new window]
 
Fig. 1.   Nucleotide and deduced amino acid sequence of LIMR. The start codon and the putative polyadenylation signal are in boldface. Underlined amino acids indicate the predicted central transmembrane helix segments. The two single underlined letters in boldface mark the insert of phage pJuFo.33.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Structural organization of LIMR on human chromosome 12. A, intron/exon structure deduced from the LIMR cDNA sequence found in this paper and the genomic sequence obtained from Homo sapiens chromosome 12 clone RP11-386G11 (GenBankTM accession number AC011603). Exons are presented by black boxes. kb, kilobase. B, length of exons and amino acids (aa) encoded by each exon. C, length of introns.

Hydropathy analysis of the deduced amino acid sequence using TMbase (30) revealed nine putative transmembrane helices (Fig. 3), indicating that LIMR is a putative membrane receptor. Therefore, this novel protein was called LIMR. A model of LIMR, obtained by several computer programs (31, 32), strongly suggests a transmembrane topology with the N terminus outside. This model is in good agreement with the fact that the Lcn-1 interacting phage clone contained only the cDNA encoding the N-terminal part and the first transmembrane region of LIMR, indicating that the N terminus is the region interacting with the extracellular Lcn-1.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3.   Hydrophobicity plot of the deduced amino acid sequence of LIMR. The analysis was performed using the program TMpred.

Production of a 6.2-kDa Recombinant LIMR Peptide and Confirmation of the LIMR-Lcn-1 Interaction-- The LIMR-encoding DNA fragment of phage clone pJuFo.33 was expressed as a His-tagged peptide in E. coli and was purified on an Ni-NTA column. This N-terminal peptide of LIMR was used for further confirmation of the LIMR-Lcn-1 interaction. In a first approach, the recombinant LIMR fragment was attached to the solid phase of a microtiter plate and incubated with various amounts of purified Lcn-1. The LIMR-bound Lcn-1 was detected by an Lcn-1-specific antiserum and horseradish peroxidase-conjugated secondary antibody. From Fig. 4 it is evident that the recombinant LIMR fragment binds Lcn-1 in a specific and saturable fashion. Wells precoated with BSA instead of LIMR did not show specific, saturable binding of Lcn-1. The LIMR-Lcn-1 interaction was also investigated by binding in solution. For this purpose the His-tagged recombinant LIMR fragment was incubated with purified Lcn-1, and the complex was precipitated using Ni-NTA-agarose beads. The positive results depicted in Fig. 5 again demonstrate a LIMR-Lcn-1 interaction. Thus, these biochemical studies clearly confirm the results from phage display.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Analysis of the interaction between Lcn-1 and LIMR investigated by enzyme-linked immunosorbent assay. Microwells precoated with recombinant LIMR peptide and blocked with gelatin were incubated with increasing amounts of Lcn-1, and the bound protein was analyzed by an Lcn-1-specific antiserum (black-diamond ). Controls were precoated with BSA instead of LIMR (black-square).


View larger version (78K):
[in this window]
[in a new window]
 
Fig. 5.   LIMR-Lcn-1 interaction analyzed in solution. A 6× His-tagged LIMR peptide was incubated with Lcn-1 in solution, and the protein complex bound to Ni-NTA resin was analyzed. M, marker. A, SDS-polyacrylamide gel electrophoresis on 18% polyacrylamide gels. Lane 1, flow-through of the Ni-NTA column; lanes 2 and 3, first and second wash with 1% Triton X-100/phospate buffer (pH 8.0); lane 4, last wash with 1% Triton X-100/phosphate buffer; lanes 5 and 6, elution of bound proteins by 250 mM imidazole; lane 7, purified Lcn-1; lane 8, recombinant 6× His-tagged LIMR peptide. B, Western blot analysis using an anti-Lcn-1 antiserum. Lane numbers correspond to those in A.

Expression Analysis of LIMR-- The expression of LIMR in various tissues was investigated by Northern dot blot analysis using a human RNA master blot (CLONTECH), which contained dots of poly(A)+ RNA from 50 human tissues. As seen in Fig. 6A a significant expression of LIMR is detected in testis, pituitary gland, adrenal gland, trachea, placenta, thymus, cerebellum, stomach, mammary gland, spinal cord, fetal kidney, and fetal lung. A weaker expression is also seen in colon, pancreas, and prostate. It should be mentioned that many of these tissues, including trachea, testis, prostate, pituitary gland, thymus, adrenal gland, trachea, placenta, spinal cord, fetal lung, and fetal kidney are known to express Lcn-1, as well.


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 6.   Human tissue expression of LIMR. A, dot blot hybridization. Human poly(A)+ mRNA on the blot was hybridized with a 32P-labeled cDNA probe. B, RT-PCR of total RNA isolated from NT2 cells amplified using LIMR-specific primers. Lane 1, PCR product using LIMR-specific primers; lane 2, control without template; lane 3, PCR using Lcn-1-specific primers (Lcn-1/1, 5'-TTGGAGGACTTTGAGAAAGC-3'; Lcn-1/2, 5'-GCTGGATGGTGCCGTCC-3'); lane 4, control without template; lane 5, PCR using glyceraldehyde-3-phosphate dehydrogenase-specific primers (5'-ACGTCGTGGAGTCCACTG-3' and 5'-GGGCCATCCACAGTCTTC-3'); lane 6, control without template. M, marker.

So far, Lcn-1 was found to be produced by the serous glands of several tissues exclusively. However, in search for a cell line that produces Lcn-1 we have recently detected production of this protein by human teratocarcinoma NT2 cells. Therefore, we have investigated the expression of LIMR by these cells using RT-PCR analysis. From Fig. 6B it is evident that NT2 cells express both Lcn-1 and LIMR. Thus, the NT2 cells were chosen as a reliable object for further characterization of the cellular distribution of LIMR.

Western Blot Analysis of NT2 Cell Membrane Fractions-- Membranes of NT2 cells were prepared by subcellular fractionation, and the proteins contained within the different fractions were analyzed by Western blotting using the LIMR-specific antiserum. As a marker for the plasma membrane fraction an antiserum against the typical membrane-located zeta -submit of the NMDA receptor was used. As seen in Fig. 7 the LIMR antiserum stained a protein band with an approximate molecular mass of 60 kDa, which was present in the same fractions as the NMDA receptor, thus supporting a plasma membrane localization of LIMR. The molecular mass of the immunoreactive band is in good agreement with that calculated from the deduced amino acid sequence.


View larger version (70K):
[in this window]
[in a new window]
 
Fig. 7.   Western blot analysis of NT2 cell membrane fractions. A, Western blot of subcellular fractions incubated with an LIMR-specific antiserum. Lane 1 and 2, 25 µl of cytoplasmatic fractions; lanes 3 and 4, 25 µl of membrane fractions. B, Western blot of NT2 cell membrane fractions incubated with an antibody against the zeta -subunit of the NMDA receptor. Lanes 1 and 2 correspond to lanes 3 and 4 in A.

Immunofluorescence Localization of LIMR in NT2 Cells-- The subcellular distribution of LIMR was also probed by indirect immunofluorescence using an antiserum raised against an N-terminal peptide of LIMR (amino acids 1-52) consisting of the outside N terminus, the first membrane domain, and part of the first intracellular loop. These analysis showed that the immunoreactivity was associated with the plasma membrane of NT2 cells, with no staining in other compartments of the cells (Fig. 8A). To demonstrate the specificity of staining, the LIMR antibodies in the antiserum were blocked/saturated with 20 µg/ml recombinant LIMR peptide at 37 °C for 1 h. In this case the cell staining was almost completely abolished (Fig. 8B). These experiments clearly indicate that LIMR is a cell plasma membrane-located protein. In addition, they confirmed the proposed model of LIMR with the N terminus orientated outside the cell membrane.


View larger version (54K):
[in this window]
[in a new window]
 
Fig. 8.   Immunocytochemical localization of LIMR on NT2 cells. A, NT2 cells were stained with an anti-LIMR antibody followed by analysis using fluorescence microscopy (magnification × 400; exposure time 45 s). B, preincubation of the anti-LIMR antibody with the recombinant peptide used as antigen (magnification × 400, exposure time 90 s). Note the presence of immunoreactivity exclusively in cell plasma membrane and plasma membrane protrusions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lcn-1 is of considerable interest, because it was supposed to be a member of the immunocalin subfamily of lipocalins, which are involved in modulation of immune and inflammatory responses, and is one of the lipocalins used as a biochemical marker of disease in man (33, 34). Functionally, it seems to act as a physiological scavenger of lipophilic, potentially harmful compounds. In search for identification of Lcn-1 interacting proteins involved in the reception or detoxification of Lcn-1 ligands we have applied a phage-display technology using a complex cDNA library and expression screening against Lcn-1 as a bait. Using this method we have identified and characterized a novel human cell membrane protein, LIMR, which is likely to be a receptor for lipophilic ligands physiologically bound to Lcn-1. The predicted topology of LIMR, consisting of 487 amino acids, is complex. It contains nine putative transmembrane domains, with the N terminus orientated outside of the cell. The outside orientation of the N terminus is supported by the result from phage display and the fact that an antiserum raised against this part of the protein reacted with NT2 cells. From phage display it is also evident that the N terminus of LIMR represents the Lcn-1 interacting domain. A notable feature of LIMR is a large central cytoplasmatic loop consisting of 82 amino acids, whereas all of the other loops are rather small (between 18 and 27 amino acids). Both immunocytochemical analysis and cell fractionation experiments gave clear evidence of LIMR being located within the cell plasma membrane. Therefore, it has to be characterized as the first human cell surface membrane-located lipocalin receptor.

Although there is clear experimental evidence that many lipocalins bind to specific cell surface receptors, isolation of these proteins or cloning of the encoding genes has failed so far. Because of its important role in retinol transport and retinol supply of cells, a large number of investigations have dealt with the receptor for the classical lipocalin RBP. Thereby, the gene encoding a 63-kDa protein, suspected to be a receptor of bovine plasma retinol-binding protein, was cloned (35). However, several consecutive reports demonstrated that it is not an integral membrane receptor but rather a membrane-associated protein involved in enzymatic processing of retinol (36, 37). Because no other specific RBP receptor has been identified so far, there is still no consensus whether retinol transfer into the cell is receptor-driven or proceeds via passive diffusion (7, 38). There is also convincing evidence that in some cells RBP undergoes internalization by a receptor-mediated endocytotic process (39).

Another well characterized protein that is supposed to interact with lipocalins is megalin. This protein, also called gp330, is an epithelial endocytic receptor with a single transmembrane region and shares some characteristic features with the low density lipoprotein receptor family (40, 41). From studies on a megalin knockout mouse it was concluded to interact with different lipocalins, including RBP, major urinary protein, olfactory-binding protein, and alpha 1-microglobulin (42). However, because it also binds a variety of other macromolecular ligands, such as thyroglobulin, apolipoprotein A, clusterin, albumin, and insulin, its specific role in lipocalin binding remains unclear.

With exception of these two defined molecules the interaction of other lipocalins with putative receptors is only marginally characterized, and a number of interactions described are likely to be of no physiological relevance. However, those receptors that have been characterized in some detail show no consensus in biochemical or biophysical properties, suggesting a great diversity of receptor structure (14).

There is also no consensus in the mechanism of interaction between lipocalins and their receptors. In principle, lipocalin receptors have been found in both groups, carbohydrate-binding receptors and receptors binding via protein-protein interaction. An example for the first group seems to be the receptor for alpha 1-acid glycoprotein, a highly sialylated serum lipocalin. Inhibition of alpha 1-acid glycoprotein binding to several cells could be inhibited by simple sugars, like mannose and GlcNAc, suggesting that this interaction is mediated by carbohydrates and that the receptor is of a lectin-type (43). Our investigations clearly show that LIMR is an example for a receptor-type binding via direct protein interaction, because it was isolated by means of prokaryotic expression, and a recombinant LIMR produced in E. coli interacts in vitro with Lcn-1.

Apart from its interaction with Lcn-1 the precise physiological function of LIMR is unknown to date. Supposing that it is involved in detoxification of ligands bound to Lcn-1, LIMR could be directly involved in the transfer of ligands to the cell, where they undergo detoxification. Alternatively, it could act as a detoxification protein per se, probably by displaying an enzymatic activity. Indeed, membrane-associated enzymes involved in modification of lipid molecules have been described, e.g. human enzymes involved in sterol or cholesterol modification (44, 45). Interestingly, a recently identified human Delta 7-sterol reductase, involved in reduction of 7-dehydrocholesterol to cholesterol, shows a similar overall topography to LIMR, including nine transmembrane regions and a molecular mass of 55 kDa (46).

Although many aspects concerning the precise function of LIMR are speculative at the moment, our investigations might have important consequences for the isolation and characterization of lipocalin receptors, in general. First, we could demonstrate that the phage-display strategy applied is a reliable method for isolating such receptors. Second, our findings constitute the first report of identifying a lipocalin receptor that is, in contrast to the RBP-associated p63, clearly a plasma membrane-located protein. Furthermore, the availability of a cloned gene is a vital first step for elucidating the structural requirements necessary for lipocalin-receptor interaction. Finally, the identification of an Lcn-1-specific plasma membrane receptor will add new hints to the precise physiological function of Lcn-1 and may help to dissect putative detoxification pathways for the ligands bound.

    ACKNOWLEDGEMENTS

We are grateful to R. Crameri for providing the vector pJuFo and to C. Enzinger for the antiserum against NMDA. We thank M. Schöser, F. Marx, and B. Hörtnagl for helpful comments in preparing the manuscript.

    FOOTNOTES

* This work was supported by Austrian Science Foundation (FWF) Grant P14850.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF260728 and AF351620.

Dagger To whom correspondence should be addressed. Tel.: 43-512-507-3603; Fax: 43-512-507-2866; E-mail: bernhard.redl@uibk.ac.at.

Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101762200

    ABBREVIATIONS

The abbreviations used are: RBP, retinol-binding protein; LIMR, lipocalin-1 interacting membrane protein; Lcn-1, lipocalin-1 (also called tear lipocalin or von Ebner's gland protein); Ni-NTA, nickel-nitrilotriacetic acid; NMDA, N-methyl-D-asparate; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; bp, base pair(s); TBS, Tris-buffered saline; BSA, bovine serum albumin; EST, expressed sequence tag; RT, reverse transcription; contig, group of overlapping clones.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Flower, D. R. (1996) Biochem. J. 318, 1-14[Medline] [Order article via Infotrieve]
2. Akerstrom, B., Flower, D. R., and Salier, J-P. (2000) Biochim. Biophys. Acta 1482, 1-8[Medline] [Order article via Infotrieve]
3. Flower, D. R., North, A. C. T., and Sansom, C. E. (2000) Biochim. Biophys. Acta 1482, 9-24[Medline] [Order article via Infotrieve]
4. Bavik, C. O., Busch, C., and Eriksson, U. (1992) J. Biol. Chem. 267, 23035-23042[Abstract/Free Full Text]
5. Smeland, S., Bjerknes, T., Malaba, L., Eskild, W., Norum, K. R., and Blomhoff, R. (1995) Biochem. J. 305, 419-424[Medline] [Order article via Infotrieve]
6. Sivaprasadarao, A., Boudjelal, M., and Findlay, J. B. C. (1994) Biochem. J. 302, 245-251[Medline] [Order article via Infotrieve]
7. Sundaram, M., Sivaprasadarao, A., DeSousa, M. M., and Findlay, J. B. C. (1998) J. Biol. Chem. 273, 3336-3342[Abstract/Free Full Text]
8. Wester, L., Michaelsson, E., Holmdahl, R., Olofsson, T., and Akerstrom, B. (1998) Scand. J. Immunol. 48, 1-7[CrossRef][Medline] [Order article via Infotrieve]
9. Böcskei, Z., Groom, C. R., Flower, D. R., Wright, C. R., Phillips, S. E., Cavaggioni, A., Findlay, J. B., and North, A. C. (1992) Nature 360, 186-188[CrossRef][Medline] [Order article via Infotrieve]
10. Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C., Findlay, J. B., Sivaprasadarao, R., Jones, T. A., Newcomer, M. E., and Kraulis, P. J. (1986) Nature 324, 383-385[Medline] [Order article via Infotrieve]
11. Boudjelal, M., Sivaprasadarao, A., and Findlay, B. B. (1996) Biochem. J. 317, 23-27[Medline] [Order article via Infotrieve]
12. Andersen, U. O., Bog-Hansen, S., and Kirkeby, S. (1999) Acta Histochem. 101, 113-119[Medline] [Order article via Infotrieve]
13. Miller, R. E., Fayen, J. D., Chakraborty, S., Weber, M. C., and Tykocinski, M. L. (1998) FEBS Lett. 436, 455-460[CrossRef][Medline] [Order article via Infotrieve]
14. Flower, D. R. (2000) Biochim. Biophys. Acta 1482, 327-336[Medline] [Order article via Infotrieve]
15. Redl, B., Holzfeind, P., and Lottspeich, F. (1992) J. Biol. Chem. 267, 20282-20287[Abstract/Free Full Text]
16. Lassagne, H., and Gachon, A. M. (1993) Exp. Eye Res. 55, 605-609[CrossRef]
17. Bläker, M., Kock, K., Ahlers, C., Buck, F., and Schmale, H. (1993) Biochim. Biophys. Acta 1172, 131-137[Medline] [Order article via Infotrieve]
18. Holzfeind, P., Merschak, P., Rogatsch, H., Culig, Z., Feichtinger, H., Klocker, H., and Redl, B. (1996) FEBS Lett. 395, 95-98[CrossRef][Medline] [Order article via Infotrieve]
19. Redl, B., Wojnar, P., Ellemunter, H., and Feichtinger, H. (1998) Lab. Invest. 78, 1121-1129[Medline] [Order article via Infotrieve]
20. Scalfari, F., Castagna, M., Fattori, B., Andreini, I., Maremman, C., and Pelosi, P. (1997) Comp. Biochem. Physiol. B Biochem. Mol. Biol. 118, 819-824[CrossRef]
21. Lacazette, E., Gachon, A. M., and Pitiot, G. (2000) Hum. Mol. Genet. 9, 289-301[Abstract/Free Full Text]
22. Glasgow, B. J., Abduragimov, A. R., Farahakhsh, Z. T., Faull, K. F., and Hubbell, W. L. (1995) Curr. Eye Res. 14, 363-372[Medline] [Order article via Infotrieve]
23. van't Hof, W., Blankenvoorde, M. F. J., Veerman, E. C. I., and Nieuw Amerongen, A. V. (1997) J. Biol. Chem. 272, 1837-1841[Abstract/Free Full Text]
24. Yusifov, T. N., Abduragimov, A. R., Gasymov, O. K., and Glasgow, B. J. (2000) Biochem. J. 347, 815-819[CrossRef][Medline] [Order article via Infotrieve]
25. Redl, B. (2000) Biochim. Biophys. Acta 1482, 241-248[Medline] [Order article via Infotrieve]
26. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
27. Crameri, R., and Suter, M. (1993) Gene 137, 69-75[CrossRef][Medline] [Order article via Infotrieve]
28. Mitterdorfer, J., Froschmayr, M., Grabner, M., Striessnig, J., and Glossmann, H. (1994) FEBS Lett. 352, 141-145[CrossRef][Medline] [Order article via Infotrieve]
29. Kang, A. S., Barbas, C. F., Janda, K. D., Benkovic, S. J., and Lerner, R. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4363-4366[Abstract]
30. Hofmann, K., and Stoffel, W. (1993) Biol. Chem. Hoppe-Seyler 347, 166
31. von Heijne, G. (1992) J. Mol. Biol. 225, 487-494[Medline] [Order article via Infotrieve]
32. Sonnhammer, E. L. L., von Heijne, G., and Krogh, A. (1998) in Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (Glasgow, J. , Littlejohn, T. , Major, F. , Lathrop, R. , Sankoff, D. , and Sensen, C., eds) , pp. 175-182, American Association for Artificial Intelligence Press, Menlo Park, CA
33. Lögdberg, L., and Wester, L. (2000) Biochim. Biophys. Acta 1482, 284-297[Medline] [Order article via Infotrieve]
34. Xu, S., and Venge, P. (2000) Biochim. Biophys. Acta 1482, 289-307
35. Bavik, C. O., Levy, F., Hellmann, U., Wernstedt, C., and Eriksson, U. (1993) J. Biol. Chem. 268, 20540-20546[Abstract/Free Full Text]
36. Nicoletti, A., Wong, D. J., Kawase, K., Gibson, L. H., Yang-feng, T. L., Richards, J. E., and Thompson, D. A. (1995) Hum. Mol. Genet. 4, 641-649[Abstract]
37. Redmond, T. M., Yu, S., Lee, E., Bok, D., Hamasaki, D., Chen, N., Goletz, P., Ma, J. X., Crouch, R. K., and Pfeifer, K. (1998) Nat. Genet. 20, 344-351[CrossRef][Medline] [Order article via Infotrieve]
38. Noy, N., and Xu, Z. J. (1990) Biochemistry 29, 3613-3619
39. Senoo, H., Smeland, S., Malaba, L., Bjerknes, T., Stang, E., Roos, N., Berg, T., Norum, K. R., and Blomhoff, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3616-3620[Abstract]
40. Saito, A., Pietromonaco, S., Loo, A. K., and Farquhar, M. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9725-9729[Abstract/Free Full Text]
41. Korenberg, J. R., Argraves, K. M., Chen, X. N., Tran, H., Strickland, D. K., and Argraves, W. S. (1994) Genomics 22, 88-93[CrossRef][Medline] [Order article via Infotrieve]
42. Leheste, J. R., Rolinski, B., Vorum, H., Hilpert, J., Nykjaer, A., Jacobsen, C., Aucouturier, P., Moskaug, J. O., Otto, A., Christensen, E. I., and Willnow, T. E. (1999) Am. J. Pathol. 155, 1361-1370[Abstract/Free Full Text]
43. Andersen, U. O., Kirkeby, S., and Bog-Hansen, T. C. (1996) J. Mol. Recognit. 9, 364-367[CrossRef][Medline] [Order article via Infotrieve]
44. Robinson, G. W., Tsay, Y. H., Kienzle, B. K., Smith-Monroy, C. A., and Bishop, R. W. (1993) Mol. Cell. Biol. 13, 2706-2717[Abstract]
45. Silve, S., Dupuy, P. H., Labit-Lebouteiller, C., Kaghad, M., Chalon, P., Rahier, A., Taton, M., Lupker, J., Shire, D., and Loison, G. (1996) J. Biol. Chem. 271, 22434-22440[Abstract/Free Full Text]
46. Moebius, F. F., Fitzky, B. U., Lee, J. N., Paik, Y. K., and Glossmann, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1899-1902[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.