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
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ABSTRACT |
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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.
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 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.
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- 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.
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).
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.
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.
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.
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 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.
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
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 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 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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-microglobulin (8),
major urinary protein (9),
-lactoglobulin (10), olfactory-binding
protein (11),
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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-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.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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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.
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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.
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Fig. 3.
Hydrophobicity plot of the deduced amino acid
sequence of LIMR. The analysis was performed using the program
TMpred.
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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 ( ). Controls were precoated
with BSA instead of LIMR (
).
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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.
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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.
-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.
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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
-subunit of the NMDA receptor. Lanes 1 and 2 correspond
to lanes 3 and 4 in A.
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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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
1-acid glycoprotein, a highly
sialylated serum lipocalin. Inhibition of
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.
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).
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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.
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