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INTRODUCTION |
gC1qR is a biologically important, widely distributed,
multiligand-binding and multifunctional protein (1). Numerous reports have claimed that gC1qR and its homologue could be isolated or identified in various cellular compartments, including plasma membrane,
cytoplasm, mitochondria and nucleus. gC1qR isolated from the plasma
membrane was originally characterized as a high affinity C1q-binding
protein (2), and later many reports showed that gC1qR could interact
with several proteins of the intrinsic coagulation/bradykinin-forming
cascade, including high molecular weight kininogen (3), Factor XII (4),
fibrinogen (5), and multimeric vitronectin (6). Conversely,
intracellular gC1qR was shown to interact and subsequently
down-regulate the surface expression of the
1b-adrenergic receptor (7) as well as bind to the kinase
domain of protein kinase Cµ and thus prevent its substrate
phosphorylation activity (8). In addition, gC1qR was also reported to
bind to a nuclear splicing factor, SF2, and to many viral proteins,
including HIV-1 Tat (9) and Rev (10), core protein V of adenovirus
(11), EBNA-1 of the Epstein-Barr virus (12), and open reading frame P
of the herpes simplex virus (13), implying that gC1qR may play a role
in virus-host interaction.
The full-length cDNA of gC1qR encodes a pre-pro-protein of 282 residues from which a 73-residue-long N-terminal segment is removed by
site-specific cleavage to generate the mature gC1qR (2). It was shown
that the fusion of the residues 1-81 or 1-33 of the pre-pro-protein
to the N terminus of the green fluorescent protein directed the fusion
protein to mitochondria (14). However, the findings of Dedio
et al. (14) do not exclude the possibility that gC1qR, like
many other proteins, could be exported from the mitochrondria by an
unknown mechanism (15). This possibility is supported by a
recent report showing that anti-gC1qR monoclonal antibody can reverse
the anti-proliferation effects of hepatitis virus C core antigen on
activated T cells (16).
The human C1qBP gene was assigned to human chromosome
17q13.3 (17). A high degree of amino acid identity exists between the
human, rat, and mouse gC1qR cDNA sequences (18). In this study, the
full-length gene of human gC1qR was cloned, and its exon-intron
boundaries were revealed. Furthermore, the transcription start site and
its promoter elements were also mapped and characterized.
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MATERIALS AND METHODS |
Screening of the Human Genomic Library--
A human genomic
library in bacteriophage EMBL3 was purchased from
CLONTECH (cat. no. HL1067J). The human cDNA of
the C1qBP gene was used as the probe for library screening
(2). The cDNA insert was released from its vector and gel-purified
before radiolabeling with [32P]dATP by the random priming
method. Positive plaques were picked, replated, and rescreened until
single positive plaques were picked. The insert in a positive plaque
was digested with various restriction enzymes, and the positive
fragments were mapped by Southern blot analysis. Six overlapping
subclones (Fig. 1) were obtained by inserting the positive fragments
into the plasmid pBluescript. Each subclone was sequenced by the primer
walking method using the ABI Prism 310 Genetic Analyzer (Applied
Biosystems, Hong Kong Limited, Hong Kong).
Sequence Assembly and Analysis--
Sequence data were assembled
and analyzed by DNA processing software including MAC DNASIS (Hitachi,
Japan) and DNA Strider (Christian Marck, Service de Biochimie,
Department de Biologie, Institut de Recherche Fondamentale, CEA,
Paris, France). Promoter analysis was performed using
MatInspector software (version 2.2) (19) obtained from the
German Research Center for Biotechnology, Braunschweig, D-38124,
Germany. A transcription factor data base (TRANSFAC, version 4.0) was
employed for the search of promoter elements (20).
Construction of Expression Plasmids for Promoter
Assay--
Various DNA fragments of the 5'-upstream region of the
human gC1qR gene (C1qBP) were obtained by polymerase chain
reaction using subclone gI as the template. Restriction sites
BglII or SacI were added to the 5'-end of the
primers (Table I) so that the
polymerase chain reaction products could easily be subcloned into the
corresponding restriction sites in the dual-luciferase reporter vector,
pGL3-Basic (Promega, Hong Kong). A nested family of 5' and 3' deletion
clones (Table II) was generated in
this manner (Fig. 4).
Cell Culture, Transient Transfection, and Promoter
Assays--
The human cell lines PANC-1 (ATCC no. CRL-1469),
MDA-MB-231 (ATCC no. HTB-26), SVG P12 (ATCC no. CRL-8621), HuTu80 (ATCC
no. HTB-40), and 293-EBNA (Invitrogen, no. R620-07) were selected for
transient transfection studies. SVG P12 and HuTu80 cells were cultured
in minimum essential medium (Life Technologies, Inc.); PANC-1,
MDA-MB-231, and 293-EBNA cells were cultured in high glucose Dulbecco's modified Eagle's medium (Life Technologies, Inc.). All
culture media were supplemented with 10%(v/v) fetal bovine serum (50 ml in 500 ml of medium) and 1× antibiotic-antimycotic mixture (Life
Technologies, Inc.) at 37 °C with 5% CO2. Plasmid DNA
used for transfection was prepared using the Quantum prep Kit (Biorad,
Hong Kong), and treated with phenol-chloroform before transfection. For
transient transfection, cells were seeded onto 12-well plates (Costar,
Corning Glass Inc.) at a density of 1.0 × 105
cells/well. Co-transfection was performed 19 h later using the LipofectAMINE Plus® Reagent (Life Technologies, Inc.) with
procedures performed according to the manufacturer's protocol. The
molar ratio of the recombinant plasmid to be assayed and the internal
control pRL-SV40 plasmid (Promega, Hong Kong) was kept at 1:1 in all
transfection. Immediately after transfection, the cells were incubated
in the medium without serum and antibiotics at 37 °C with 5%
CO2 for 3.5 h before changing to complete medium. The
cells were lysed at 39 h post-transfection by washing the cells
twice with 1× phosphate-buffered saline followed by the addition of
reporter passive lysis buffer (Promega, Hong Kong). The dual-luciferase
reporter assay was performed according to the manufacturer's protocol
(Dual-Luciferase reporter assay kit, Promega, Hong Kong) using a
luminometer (Lumat LB-9507, EG&G) as the measuring apparatus.
RNA Isolation and Primer Extension Analysis--
Total cellular
RNA was isolated from human cell line MDA-MB-231 by TRIzol reagent
(Life Technologies, Inc.). Poly(A)+ RNA was extracted from
2 mg of total RNA using the Poly(A)Tract mRNA isolation kit
(Promega). Labeling of the primer and primer extension reaction was
performed using 1 µg of poly(A)+ RNA with 10 pmol of the
antisense primer G1A2Bgl following the instructions for the
Primer Extension System (Promega). 4 µl of the primer extension
reaction product was analyzed on a 6% (w/v) acrylamide, 7 M urea sequencing gel. The size of the primer extension product was determined by comparison with a DNA sequence ladder generated with the same oligonucleotide primer using ERES3 plasmid as a template.
Gel Mobility Shift Assays--
The human cell line PANC-1 was
selected for gel shift assays. Cells were cultured to exponential phase
as mentioned above and harvested. End-labeling of double-stranded
oligos (Table I) was done using the Ready-To-Go T4
polynucleotide kinase labeling kit and
[
-32P]ATP (5000 Ci/nmol) (Amersham Pharmacia Biotech).
Nuclear extract preparation and gel shift assays were performed as
described previously (21, 22).
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RESULTS |
Genomic Organization of the Human C1qBP Gene--
Fig.
1 is a schematic representation of the
genomic organization of the gene, which, including its 5'- and
3'-flanking regions, spans about 7.8 kb.1 From the first codon of
the initiation methionine to the stop codon of the gene, the gene spans
6055 bp (Fig. 2). By alignment of the
cDNA and the genomic sequences, intron-exon boundaries were
defined. There are 6 exons and 5 introns in the C1qBP gene. The size of the exons range from 94 (exon 3) to 232 bp (exon 1), and
that of the introns range from 128 (intron 5) to 3156 bp (intron 2).
Amino acid codons are split by introns 1 and 2 at the junctions of
their adjacent exons. (Table III) A
poly(A) signal is located 369 bp from the stop codon. The entire
genomic sequence has been deposited in the GenBankTM under
accession no. AF338439.

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Fig. 1.
The genomic organization of the human
C1qBP gene. Exons are highlighted as
dark boxes. Dark, bold lines below the gene indicate the
overlapping subclones. The arrows indicate the positions of
sequencing primers and the approximate length sequenced by each primer.
Restriction enzyme sites are shown at locations above the
gene.
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Fig. 2.
DNA sequences of the human C1qBP
gene. The first nucleotide of the translation start codon
ATG is designated as 1. Exon-intron boundaries are shown. All
intronic sequences, the 5'-flanking region of the gene, and the
3'-flanking region as defined by the cDNA amino acid coding
sequences are shown as lowercase letters. A poly(A) signal
located 369 bp from the stop codon is underlined.
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Table III
Exon-intron boundaries of the human C1qBP gene
The exon-intron boundaries follow the GT/AG rule (as indicated by the
bold letters).
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Characterization of the 5'-Flanking Region of the Human C1qBP
Gene--
A 1.3-kb nucleotide sequence located upstream of the ATG
initiation codon of the gene was analyzed (Fig.
3) using the TRANSFAC transcription
factor data base. Putative promoter and enhancer elements including
TATA boxes, CCAAT boxes, octamers, Sp1 binding sites, GATA sequences,
E-boxes, and AP elements were identified by the software MatInspector.
There are four putative TATA boxes (
806 to
811,
614 to
617,
446 to
449, and
399 to
406) and three putative CCAAT boxes
(
1033 to
1037,
460 to
463, and
410 to
414). The 8-bp
consensus TATA box with the sequence TATATATA located at
399 to
406
is the longest TATA element found in the region, and it is in close
proximity to a CCAAT box located at
410 to
414. Sp1 binding GC-rich
motifs are also found throughout the 5'-flanking region of the
gene at positions
1309 to
1314,
959 to
965,
535 to
540,
519 to
524,
337 to
342,
177 to
182,
148 to
154,
139 to
144,
111 to
118,
83 to
90, and
51 to
61.

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Fig. 3.
Analysis of the 5'-flanking region of the
human C1qBp gene. The first nucleotide of the
translation start codon ATG (bolded) is designated
as +1 and the preceding nucleotide as 1. All potential transcription
factor binding sites, promoters, and elements that may be responsible
for transcription control are underlined and labeled
below the line.
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A variety of consensus elements for transcription factors are also
present, including an octamer site for the homeobox domain factor Oct-1
(
28 to
34) and several recognition sites for the putative zinc
transcription factor GATA, which is expressed in high levels in the
pancreas and gut-derived cells (23). Other putative sites for enhancer
elements AP1, AP2, AP3, and AP4, HSF (heat-shock factor) (24), NFAT
(nuclear factor of activated T cells) (25), AML transcription
factor (26), and c-Ets-1 transcription factor (27) are also identified
in the 5'-flanking region.
Determination of the Transcription Start Sites--
A primer
extension analysis was performed to determine the transcription start
site of the gene. As shown in Fig. 4,
there are three regions detected with stronger intensities, and each region was found with a number of individual start sites. These regions
are the major transcription start sites. The farthest extension band
was observed at 49 bp upstream of the ATG translation initiation codon
and is an adenine residue. This nucleotide is positioned just
downstream to an SP1 binding site found in the promoter region. All of
these regions lie within 50 bp upstream to the translation initiation
codon.

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Fig. 4.
Primer extension analysis of the human
C1qBP gene promoter. Lanes 1-4 are
sequencing ladders generated by the same primer (G1A2). Lane
5 is the primer extension reaction product. The position of the
nucleotides marked on the left side of the gel was
designated according to the translation initiation codon ATG, which was
designated as +1. Three regions of strong signals were identified. The
farthest extension product was mapped at 49 bp upstream of the ATG
codon.
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Mapping of the Human gC1qR Gene (C1qBP) Promoter Element in PANC-1
cells--
To localize the essential promoter region for human gC1qR
gene expression, several 5' and 3' deletion clones were constructed by
cloning various restriction fragments produced by polymerase chain
reaction into a reporter vector, pGL3-Basic. Because these fragments
were cloned upstream to a luciferase reporter gene, transcriptional
activities of the promoter-luciferase cartridge could be studied by
transfecting various clones to the human cell line PANC-1 (Fig.
5). It was found that the essential
region for promoting transcription is very close to the start site of
translation. By comparing luciferase activities between constructs
ERES2 to ERES7 and that of constructs ERES7 with ERES-2H and -2P, the
essential promoter region could be mapped to a region spanning the
translation start site to
364 bp. This observation is supported by
the fact that no significant difference in the luciferase activities
was observed upon deletion of the 5' sequence from
364 to
1319
(ERES7). However, in the two constructs in which the 3' region was
deleted, the luciferase activities were completely abolished. Because
no 5' deletion studies were carried out beyond
364, we cannot
conclude and finely map the essential promoter elements in the
1 to
365 region upstream of the translation start site. Experimental data also showed that inverting the 1.3-kb 5'flanking region (ERES1) greatly
decreased the transcription activities.

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Fig. 5.
Promoter deletion assay in PANC-1 cell.
Schematic diagrams of various promoter-luciferase constructs were shown
on the left. The arrows indicate the orientation
of the sequences. Values indicated above the
arrows show the respective positions of the
constructs at the 5'-flanking region of the gene. The relative promoter
activities of various constructs, after normalization by the activity
of the pGL3-Basic vector (Basic), are shown on the
right. This figure represents the mean of three individual
transfection experiments.
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Deletion Analysis on the Other Human Cell Lines--
Three
constructs, ERES2, ERES7, and ERES-2P were transfected to the other
human cell lines to test the cell specificity of the promoter element
as well as to map the element. All of the four cell lines, MDA-MB-231,
SVG P12, 293-EBNA, and HuTu80, showed enhanced levels of luciferase
activities for constructs ERES2 and ERES7, ranging from 10- to 100-fold
depending on the cell lines (Fig. 6).
However, similar to the results obtained in PANC-1, deletion of the 3'
region of the promoter (ERES-2P) completely abolished the
promoter-reporter luciferase activities in all of these four cell
lines. It was surprising that the removal of the TATA box at
399 to
406 and the CCAAT box at
410 to
414 in ERES7 did not
significantly affect the transcription efficiency of the promoter. This
finding implied that there are strong promoter elements in the
1 to
364 region of the 5'-flanking region.

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Fig. 6.
Promoter assays in different human cell
lines. The relative promoter activities of all cell lines were
normalized by the pGL3-Basic activity. The figure represents the mean
of two individual transfection experiments.
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Interaction between DNA-specific Nuclear Protein Factors and
Promoter Elements--
In the
1 to
364 regions, seven
GC-rich sequences with high homology to the consensus SP1 binding site
were identified (Fig. 3). To test whether there is any nuclear factor
interacting with these GC-rich sequences, gel shift assays (Fig.
7A) were performed with
nuclear extracts of PANC-1 cells. Seven pairs of synthetic oligonucleotides in both sense and antisense orientations (Table I) were designed based on these GC-rich motifs. Of the eight oligo pairs (including a negative control that is not GC-rich), only
the L6 probe formed DNA-protein complexes with the nuclear extract. In
the competitive assay, a decreased intensity of the hybridization bands
was observed (Fig. 7B) when the concentrations of the
unlabeled L6 probe were 10-100-fold that of the labeled L6 probe. When
an unrelated DNA probe was used for the competition assay (Fig.
7B), one (complex 1) of the four bands also showed a reduced
signal, indicating that complex 1 is a nonspecific signal. To reveal
the identity of the complexes, monoclonal anti-Sp1 antibodies (Research
Diagnostics Inc.) and bovine serum albumin were mixed with the
complexes in a supershift assay. It was observed that the DNA-protein
complex 4 was shifted only by anti-Sp1 antibodies but not by bovine
serum albumin, indicating that complex 4 is a complex between
oligonucleotide L6 and Sp1 (Fig. 8).

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Fig. 7.
Gel-shift assays. 8 µg of PANC-1
nuclear extracts were used for each individual reaction. A,
gel shift was observed only in the L6 DNA probe, corresponding to the
sequence 96 to 76 of the human C1qBP gene. B,
a competition assay was performed using 8 µg of the PANC-1 nuclear
extracts in the presence of an increasing concentration of unlabeled
DNA probe (0-, 10-, and 100-fold). The formation of DNA-protein
complexes was gradually inhibited only by the specific oligo but not by
an unrelated oligo. In the negative control experiments
( ve) using an unrelated oligo, only the formation of
DNA-protein complex 1 was inhibited, but the formation of the other
three complexes was not disturbed; this showed that DNA-protein complex
1 was an artifact.
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Fig. 8.
Supershift assay was performed using the L6
oligo as a probe. When 4 µg of anti-Sp1 antibodies were added to
the mixture, the band of DNA-protein complex 4 shifted upward from its
original position (as shown when no antibody was added), whereas the
other three bands were unaffected. DNA-protein complex 4 should be a
complex of oligo L6 and the transcription factor Sp1. Bovine serum
albumin was used as the negative control in this assay, which was
unable to display DNA-protein complex 4.
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DISCUSSION |
Like the mouse gene, the human gC1qBP gene
contains six exons and five introns, and the sizes of the exons do not
differ much except for exon 6 (28). Exon 6 in the human
C1qBP gene is about 100 bp larger in than the mouse gene
because of a longer 3'-untranslated region defined by the
polyadenylation sites. Even though the sizes of the exons
between the human and mouse genes are almost the same, three of the
introns of the human C1qBP gene have expanded sizes in
comparison with their mouse counterparts. The size of intron 2 in the
human and mouse genes is 3156 and 2071 bp, respectively, which makes
the human gene approximately 1 kb larger than the mouse gene.
The promoter elements of the human gene were identified by computer
analysis and transfection studies. Several TATA boxes and CCAAT boxes
were found on the 1.3-kb 5'-flanking region. The TATA box is a crucial
positioning component of the core promoter and is usually located about
25 bp upstream of the transcription start site; it constitutes the only
upstream promoter element that has a relatively fixed location with
respect to the transcription start site. The CCAAT boxes are often
located close to the TATA boxes, but they can function at
distances that vary considerably from the transcription start
site in either orientation. Mutation studies have suggested that the
CCAAT box plays a strong role in determining the efficiency of the
promoter, and its inclusion increases promoter strength.
The transcription start sites are usually located downstream in close
proximity to the TATA elements in the majority of promoters. However,
the transcription start sites (
49 to
29) of the human C1qBP gene are around 350 bp from the closest TATA element
(
399) found in the 5'-flanking region of the gene. The 5'-flanking
region of the human gene contains an 8-bp consensus TATA box with the sequence TATATATA at
399 to
406 and a CCAAT box at
410 to
414, whereas in the mouse gene, the TATA box closest to the ATG start codon
is located approximately at
306 to
309 (29). However, in the
promoter studies, the construct ERES7, which did not carry any TATA or
CCAAT boxes, still gave more than 90% transcription efficiency,
indicating that none of the TATA boxes or CAAT boxes was essential for
the transcription of luciferase mRNA in the cell lines under test.
Instead, deletion studies carried out in all cell lines showed complete
abolishment of the promoter-reporter activity when the 364 bp
adjacent to the initiation methionine was deleted (ERES-2P). This
364-bp promoter sequence, similar to the large stretch of GC-rich
sequence in the mouse gene, especially from nucleotides
1 to
200
(75% G + C content) (28), contains seven GC-rich Sp1 sites, which are
frequently linked to the transcriptional control of genes lacking a
functional TATA box. As no TATA element was found to be essential for
the activity of the gC1qBP promoter, Sp1 sites in the
upstream region close to the transcription start site are thought to be
very important for the promoter activity of the gene. In fact, one of
these seven GC-rich Sp1 sites (
96 to
76) was found to bind
specifically to PANC-1 nuclear proteins in gel mobility shift assays,
and one of these nuclear factors was further proved to be Sp1 binding
factor in supershift assays employing anti-Sp1 antibodies. These
results show that binding of Sp1 to the SP1 binding site located at
around 80 bp upstream to the translation initiation codon may play an
important role in transcription control in human gC1qBP
gene. With regard to the number of various transcription control
elements in the 5'-flanking region, it may be possible that different
transcription control elements are essential for the gene expression in
different human cells.
The three-dimensional structure of gC1qR was revealed by x-ray
crystallography. The mature protein molecule has one N-terminal
-helix followed by seven consecutive antiparallel
-strands and two C-terminal
-helices (29). Three molecules form a doughnut-shaped quaternary structure with an internal channel 10 Å in diameter. By aligning the exon boundaries and the three-dimensional structure, it
is observed that: exon 1 encodes the first 77 amino acid residues containing the mitochondria-targeting sequence (14); exon 2 encodes the
N-terminal
-helix and the first two
-strands; exon 3 encodes
-strand 3; exon 4 encodes
-strands 4 and 5; exon 5 encodes
-strands 6 and 7; and the last exon encodes for the C-terminal
-helices. As gC1qR was shown to be a multiligand-binding protein, it
would be interesting to map the binding sites on gC1qR to various ligands.
More recently, gC1qR was found to function as a receptor for the
internalin B (InlB) invasion protein of Listeria
monocytogenes (30) and to bind to protein A of
Staphylococcus aureus (31). Another report showed that the
hepatitis virus C core antigen can interact with gC1qR, and the
anti-proliferation effects of hepatitis virus C core antigen on
activated T cells can be reversed by anti-gC1qR monoclonal antibodies
(16). All of these reports further suggest that gC1qR is a biologically
important, widely distributed, multiligand-binding and multifunctional
protein (1).