Molecular Cloning of a Novel Human Collectin from Liver
(CL-L1)*
Katsuki
Ohtani
§,
Yasuhiko
Suzuki
,
Souji
Eda
,
Takao
Kawai¶,
Tetsuo
Kase
,
Hiroshi
Yamazaki**,
Tsutomu
Shimada**,
Hiroyuki
Keshi§,
Yoshinori
Sakai
,
Atsushi
Fukuoh§,
Takashi
Sakamoto§, and
Nobutaka
Wakamiya§
From the Departments of
Pathology, ¶ Food
Microbiology,
Virology, and ** Pharmaceutical Affairs, Osaka
Prefectural Institute of Public Health, Higashinari, Osaka 537 and the
§ Department of Viral Infections, Research Institute for
Microbial Diseases, Osaka University, Suita, Osaka 565, Japan
 |
ABSTRACT |
Collectins are a C-lectin family with
collagen-like sequences and carbohydrate recognition domains. These
proteins can bind to carbohydrate antigens of microorganisms and
inhibit their infection by direct neutralization and agglutination, the
activation of complement through the lectin pathway, and opsonization
by collectin receptors. Here we report the cloning of a cDNA
encoding human collectin from liver (CL-L1
(collectin liver 1))
that has typical collectin structural characteristics, consisting of an
N-terminal cysteine-rich domain, a collagen-like domain, a neck domain,
and a carbohydrate recognition domain. The cDNA has an insert of
831 base pairs coding for a protein of 277 amino acid residues. The deduced amino acid sequence shows that this collectin has a unique repeat of four lysine residues in its C-terminal area. Northern blot,
Western blot, and reverse transcription-polymerase chain reaction
analyses showed that CL-L1 is present mainly in liver as a cytosolic
protein and at low levels in placenta. More sensitive analyses by
reverse transcription-polymerase chain reactions showed that most
tissues (except skeletal muscle) have CL-L1 mRNA. Zoo-blot analysis
indicated that CL-L1 is limited to mammals and birds. A chromosomal
localization study indicated that the CL-L1 gene localizes
to chromosome 8q23-q24.1, different from chromosome 10 of other human
collectin genes. Expression studies of fusion proteins lacking the
collagen and N-terminal domains produced in Escherichia
coli affirmed that CL-L1 binds mannose weakly. CL-L1 and
recombinant CL-L1 fusion proteins do not bind to mannan columns.
Analysis of the phylogenetic tree of CL-L1 and other collectins
indicated that CL-L1 belongs to a fourth subfamily of collectins
following the mannan-binding protein, surfactant protein A, and
surfactant protein D subfamilies including bovine conglutinin and
collectin-43 (CL-43). These findings indicate that CL-L1 may be
involved in different biological functions.
 |
INTRODUCTION |
Collectins are a family of proteins that have at least two
characteristic structures, a collagen-like domain and a carbohydrate recognition domain (CRD)1
(1). These lectins are found in vertebrates from birds to humans (2).
There are three groups of collectins: the MBP group, which includes
MBP-A and MBP-C (3); the SP-A group (4); and the SP-D group (5), which
includes bovine conglutinin (6) and CL-43 (7). MBP can destroy bacteria
through activation of the complement pathway (8) or by opsonization by
collectin receptors (9). Conglutinin is a
-inhibitor of influenza A viruses that exhibits hemagglutination inhibition and neutralization activities (10, 11). SP-A enhances the phagocytosis of bacteria by
macrophages (12) and opsonizes herpes simplex virus (13). SP-D causes
agglutination of bacteria (14) and exhibits hemagglutination inhibition
against influenza A virus (15). These data indicate that collectins
play an important role in immunoglobulin-independent host defense
(16).
The isolation and functional characterization of novel collectins in
addition to those described above might provide further insights into
their functions. Here we report the cloning and preparation of
recombinant fusion proteins and the characterization of human CL-L1
(collectin liver 1), a
new member of the collectin family. CL-L1 is expressed mainly in liver,
placenta, and adrenal gland and is expressed ubiquitously in most
tissues, except skeletal muscle. Surprisingly, this new collectin is a
cytosolic protein, although other all collectins are secreted proteins.
 |
EXPERIMENTAL PROCEDURES |
Buffers and Media--
Escherichia coli lysis buffer
for the pMAL-c2 system contained 10 mM phosphate, pH 7.2, 30 mM NaCl, 0.25% (w/v) Tween 20, 10 mM
2-mercaptoethanol, 10 mM EDTA, and 10 mM EGTA.
Column buffer contained 10 mM phosphate, pH 7.2, 500 mM NaCl, 1 mM NaN3, 10 mM 2-mercaptoethanol, and 1 mM EGTA, and column
buffer/T contained column buffer and 0.25% (w/v) Tween 20. E. coli lysis buffer A for the His tag system consisted of 6 M guanidine hydrochloride, 0.1 M sodium
phosphate, and 10 mM Tris, pH 8.0. Column buffers B-E
consisted of 8 M urea, 0.1 M sodium phosphate,
and 10 mM Tris, with pH values of 8.0, 6.3, 5.9, and 4.5, respectively. LB medium contained 1% (w/v) Bacto-Tryptone, 0.5% (w/v)
bacto-yeast extract, and 1% (w/v) NaCl. Induction base medium with
glucose contained 0.4% casamino acids, 0.6%
Na2HPO4, 0.3% KH2PO4,
0.05% NaCl, 0.1% NH4Cl, 0.5% glucose, and 1 mM MgCl2. Tris-buffered saline (TBS) consisted
of 20 mM Tris-HCl and 140 mM NaCl, pH 7.4, and
TBS/C was TBS containing 5 mM CaCl2. Coating
buffer contained 15 mM Na2CO3, 35 mM NaHCO3, and 0.05% (w/v) NaN3,
pH 9.6.
Generation of a Probe for Screening by Polymerase Chain
Reaction--
Screening an expressed sequence tag (EST) data base for
potential new collectin genes revealed a novel gene in EST clone
R29493. The partial clone (F1-1006D) from a fetal liver cDNA was
kindly provided by Dr. Hee-Sup Shin (Pohang Institute of Science
and Technology) and used to screen a human liver cDNA library for full-length cDNAs by plaque hybridization. To generate a
digoxigenin-labeled cDNA probe, we used the polymerase chain
reaction (PCR). Primers amplifying the cDNA probe were synthesized
based on the 5'- and 3'-end nucleotide sequences of the insert in clone
F1-1006D. The primers synthesized were 5'-GGCCAACACACTCATCGC-3' for the
reverse primer and 5'-TTACTTTTTTCTTCTTG-3' for the forward primer. PCR was carried out using a PCR DIG labeling kit (Roche Molecular Biochemicals). The reaction mixture (50 µl) consisted of 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 200 mM each dATP, dCTP, and dGTP and 130 mM dTTP (Takara Shuzo Co., Ltd., Tokyo,
Japan); 70 mM digoxigenin-11-dUTP; 1.25 units of
Taq DNA polymerase; a 1 µM concentration of
each primer; and 20 ng of cDNA clone F1-1006D. PCR was performed
for 30 cycles in TaKaRa PCR Model 480 thermal cycler (Takara Shuzo Co.,
Ltd.), with each cycle consisting of denaturation for 45 s at
95 °C, annealing for 1 min at 60 °C, and extension for 2 min at
72 °C. The PCR product was electrophoresed on a 1% (w/v) agarose
gel (Wako Pure Chemical Industries) and then extracted from the gel
using a Sephaglas BandPrep kit (Amersham Pharmacia Biotech).
Determination of a cDNA Encoding CL-L1 by Screening a Human
Liver cDNA Library and "Cap Site Hunting"--
A phage library
was screened essentially as described previously (17). In brief,
~1 × 106 plaque units of a human liver
gt11
cDNA library (CLONTECH) were plated with
E. coli Y1090r
and incubated at 42 °C for
4 h. Nylon filters (Nytran 13N, Schleicher & Schüll) were
prehybridized in hybridization buffer (5× SSC, 1% blocking reagent
(Roche Molecular Biochemicals), 0.1% N-lauroylsarcosine, and 0.02% SDS) for 1 h at 68 °C and then hybridized for
16 h at 55 °C with a digoxigenin-labeled probe in hybridization
buffer. The filters were washed twice for 5 min in 2× SSC and 0.1%
SDS at room temperature and then twice for 15 min in 0.5× SSC and 0.1% SDS at 55 °C. The hybridized probe was detected by 30 min of
incubation at room temperature with alkaline phosphatase-conjugated anti-digoxigenin antibody (Fab; Roche Molecular Biochemicals) diluted
1:5000. The enzyme-catalyzed color reaction was carried out using a
nitro blue tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate system
(Wako Pure Chemical Industries) in buffer consisting of 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2. The cDNA inserts in the positive
clones were amplified using the primers described above and then
directly subcloned in the pCR2.1 vector of a TA cloning kit
(Invitrogen). The subclones were sequenced using an Autoread DNA
sequencing kit and an A.L.F. Autosequencer (Amersham Pharmacia Biotech).
To identify the sequence including the transcriptional start site, we
took the cDNA including the transcriptional start site from the Cap
Site cDNATM (Nippon Gene Co., Ltd., Tokyo) of human
liver by nested PCR (18). This procedure is named cap site hunting
(18). The primer set for the first PCR was 5'-CAAGGTACGCCACAGCGTATG-3'
(primer 1RC2; Nippon Gene Inc., Ltd.) and 5'-TCTTCAGTTTCCCTAATCCC-3'
(primer PR1), synthesized by the manufacturer indicated. The primer set for the second PCR was 5'-GTACGCCACAGCGTATGATGC-3' (primer 2RC2; Nippon
Gene Co., Ltd.) and 5'-CATTCTTGACAAACTTCATAG-3' (primer PR2),
synthesized by the manufacturer indicated. The reaction mixture (50 µl) consisted of long and accurate (LA) PCR Buffer II
(Mg2+-free); 2.5 mM MgCl2; 200 µM each dATP, dCTP, dGTP, and dTTP; 1 µl of Cap Site
cDNATM from human liver; 1.25 units of TaKaRa LA
Taq DNA polymerase (Takara Shuzo Co., Ltd.); and 0.5 µM each primer 1RC2 and PR1 for the first PCR and primer
2RC2 and PR2 for the second PCR. The first PCR was performed for 35 cycles in the TaKaRa PCR Model 480 thermal cycler, with each cycle
consisting of denaturation for 20 s at 95 °C, annealing for
20 s at 60 °C, and extension for 20 s at 72 °C. The
second PCR was performed for 25 cycles in same buffer using 1 µl of
the first PCR products as a probe. The final PCR products were
extracted from the agarose gel after gel electrophoresis and directly
subcloned into the pT7Blue vector (Novagen). The subclones were
sequenced using the Autoread DNA sequencing kit and the A.L.F. Autosequencer.
Southern Blot Analysis--
Genomic DNA was purified from human
placenta by a standard method. Routinely, 4 µg of genomic DNA was
completely digested with restriction enzymes, separated on 0.7%
agarose gel, and vacuum-transferred to the Nytran 13N nylon
filters. Blots were prehybridized in ExpressHyb hybridization solution
(CLONTECH) at 68 °C for 30 min and then hybridized for 1 h at 68 °C with a 10 ng/ml concentration of
the digoxigenin-labeled probe corresponding to a fragment of the CRD in
the same buffer as used for prehybridization. The filters were washed
for 5 min in 2× SSC and 0.1% SDS at room temperature and then for 15 min in 0.2 × SSC and 0.1% SDS at 68 °C. The hybridized probe
was detected by a chemiluminescent technique (Roche Molecular Biochemicals) as described by the manufacturer.
Zoo-blot analysis was performed with the modified method described
above using completely EcoRI-digested genomic DNAs (5 µg) from human, rhesus monkey, cow, dog, rabbit, rat, mouse, chicken, and
yeast (Saccharomyces cerevisiae). Prehybridization,
hybridization, and washing were performed at 58 °C for the same time
periods. The probe (which corresponded to the CRD) for the zoo-blot
analysis was made with the PCR DIG labeling kit.
Northern Blot and RT-PCR Analyses--
A human multiple-tissue
Northern blot membrane was purchased from CLONTECH.
It contained 2 µg of poly(A)+ RNAs from human heart,
brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas.
The membranes were prehybridized at 65 °C for 3 h in a solution
containing 5× SSC, 10× Denhardt's solution, 10 mM sodium
phosphate, pH 6.5, 0.5% SDS, 50% formamide, and 0.1 mg/ml denatured
salmon sperm DNA. Hybridization was then performed for 18 h at
65 °C with RNA synthesized in vitro and labeled with
digoxigenin using a DIG RNA labeling kit (Roche Molecular Biochemicals). The template for the RNA probe was a whole cDNA insert subcloned into pSPT18. The filters were washed twice for 5 min
in 2× SSC and 0.1% SDS at room temperature and then for 15 min in
0.1× SSC and 0.1% SDS at 68 °C. The hybridized probe was detected
as described above.
Reverse transcription was carried out using total RNAs (1 µg) from
brain, heart, kidney, liver, lung, trachea, bone marrow, colon, small
intestine, spleen, stomach, thymus, mammary gland, prostate, skeletal
muscle, testis, uterus, placenta, adrenal gland, pancreas, salivary
gland, and thyroid. Oligo(dT)-adaptor primers (RNA LA PCR kit (avian
myeloblastosis virus), Version 1.1, Takara Shuzo Co., Ltd.) were used
for the reverse transcription reaction. The reverse transcription
products were amplified by 28 or 35 cycles of PCR using degenerate
first primers (0.2 µg), TaKaRa LA Taq polymerase (1.25 units), and reverse transcription reaction products in a TaKaRa PCR
Model MP thermal cycler. Nested PCRs were performed using the PCR
products as a probe after 35 cycles of RT-PCR. The primer set for the
first PCR was 5'-TAGCAAATACGTAGGATGAG-3' for the reverse primer and
5'-CCACAGCAATGAATGGCTTT-3' for the forward primer. The inner primer set
for nested PCR was 5'-TTACTTTTTCTTCTTGATGA-3' for the reverse primer
and 5'-ATGAATGGCTTTGCATCCTT-3' for the forward primer. These primers
were located in the neck and CRD regions, which spanned an intron in
genomic DNA. Amplicons were separated on 1.0% agarose gels.
Chromosomal Localization of the CL-L1 Gene--
The
~10-kilobase pair human CL-L1 genomic DNA fragment from the neck
domain to the CRD, biotin-labeled with a nick translation kit (Roche
Molecular Biochemicals), was used as a probe. The gene was localized by
fluorescence in situ hybridization and PCR analysis using
DNAs from human monochromosomal fusion cells kindly provided by Dr.
Hashimoto (National Institute of Infectious Diseases). Map position was
determined by inspection of fluorescent signals on
4,6-diamidino-2-phenylindole-stained chromosomes. 25 metaphase preparations were analyzed.
Expression of Neck and CRD Fragments of Cloned cDNA in E. coli--
The recombinant fusion protein with the maltose-binding
protein of E. coli was expressed using the expression vector
pMAL-c2 system (New England Biolabs Inc.). E. coli XL1-Blue,
which carries the plasmid containing the proper insert of neck and CRD
fragments as described previously (19), was grown to
A600 nm ~ 0.5 in 200 ml of LB medium
supplemented with 0.5% glucose. After addition of
isopropyl-
-D-thiogalactopyranoside to a final
concentration of 1 mM, the culture was incubated for an
additional 3 h, and the cells were harvested. The cell pellets
were suspended in 10 ml of lysis buffer and lysed by sonication (15 s,
70% output, 10 times). After centrifugation at 9000 × g for 30 min, the supernatant was diluted with 5 volumes of
column buffer/T and applied to an amylose resin column (New England
Biolabs Inc.). The column was washed with column buffer/T and column
buffer, and the recombinant fusion protein (CL-L1-CRDmal) was eluted
with column buffer containing 10 mM maltose. The eluate was
dialyzed against three changes of 1000 volumes of TBS/C and used for
further characterization. To obtain another truncated CL-L1 protein
(CL-L1-CRDhis), a new expression vector (pPLH3) was constructed and
used. This vector consisted of a
PL-Trp fusion promoter and
histidine hexamer coding sequence just downstream from the ATG
initiation codon. Recombinant proteins expressed by this vector system
will have N-terminal MHHHHHH sequences. E. coli GI724,
containing the plasmid with the neck and CRD fragments, was grown at
30 °C to A600 nm ~ 0.5 in 200 ml of
induction base medium with glucose. After addition of tryptophan to a
final concentration of 0.1 mg/ml, the culture was incubated for 3 h at 37 °C, and the cells were harvested. The cells were suspended
in 20 ml of lysis buffer A and lysed by sonication (15 s, 70% output,
10 times). After centrifugation at 9000 × g for 30 min, the supernatant was incubated with nickel-nitrilotriacetic acid-agarose (QIAGEN Inc.) for 15 min, and the gel was loaded onto a
column. The column was washed with column buffers B and C. The
histidine-tagged recombinant protein was eluted with column buffers D
and E. The eluate was dialyzed against 1000 volume of TBS, followed by
two changes of 1000 volumes of TBS/C. This CL-L1 fusion protein
(CL-L1-CRDhis) was used to produce antisera in New Zealand White
rabbits. Purification and identification of the recombinant CL-L1 CRDs
were confirmed by SDS-PAGE and Western blotting using the rabbit
anti-CL-L1 CRD serum.
Characterization of Recombinant CL-L1 CRDs Produced in E. coli--
The binding of recombinant CL-L1-CRDmal to mannose
(mannose-biotin probe) was measured by ELISA with avidin-biotinylated
peroxidase (Vector Labs, Inc.). Microtiter plates were coated at
4 °C overnight with CL-L1-CRDmal, maltose-binding protein as a
negative control, and recombinant human MBP from Chinese hamster ovary
cells as a positive control (10 µg of each in 100 µl of coating
buffer (15 mM Na2CO3, 35 mM NaHCO3, and 0.05% (w/v) NaN3,
pH 9.6)). The plates were washed three times with 20 mM
Tris-HCl, pH 7.4, 140 mM NaCl, 0.05% NaN3, 5 mM CaCl2, and 0.05% (v/v) Tween 20 after each
step. After coating, the plates were blocked with Block Ace (Dainippon Seiyaku, Tokyo) for 1 h at room temperature. After blocking, the samples were supplemented with an
-D-mannose BP-probe (Seikagaku Kogyo, Tokyo) at various
concentrations (0.01, 0.1, 1, and 10 µg/ml) with or without mannan
(10 mg/ml) or EDTA (10 mM). The lectins,
-D-mannose BP-probe, and VECTASTAIN Elite ABC reagents (Vector Labs, Inc.) were added sequentially and incubated for
1 h at 4 °C at each step. Finally, 100 µl of
3,3',5,5'-tetramethylbenzidine substrate solution
(3,3',5,5'-tetramethylbenzidine microwell peroxidase substrate,
Kirkegaard and Perry Laboratories) was applied to each well. Before and
after addition of 100 µl of 1 M phosphoric acid, the
A450 nm was read with a Model 450 microplate reader (Bio-Rad). The monosaccharide specificities of CL-L1 were analyzed using the same ELISA system. The microplates were coated with CL-L1-CRDmal and maltose-binding protein (10 µg of each).
Saccharide-biotin probes were used with the
-D-mannose,
-D-fucose,
-D-galactose,
-N-acetylglucosamine, and
-N-acetylgalactosamine BP-probes (Seikagaku Kogyo). The
inhibition studies were done with EDTA (10 mM) and mannan
(10 mg/ml). The staining procedure was described above.
Another characterization was performed using a sugar-blot method (20).
Recombinant CL-L1 CRDs, maltose-binding protein, and recombinant human
MBP (1 µg of each) were dissolved in SDS sample buffer, separated by
SDS-PAGE (10-20% gradient polyacrylamide gel), and transferred to
BioBlot-NC membranes (Coster Co.) by standard procedures. After
treating the membranes with TBS/TC (20 mM Tris-HCl, 140 mM NaCl, 0.1% Triton X-100, and 5 mM
CaCl2) with or without EDTA (10 mM), they were
incubated with the
-D-mannose BP-probe alone or together
with EDTA (10 mM) or mannan (100 µg/ml) at 4 °C for 60 min, washed with TBS/TC, and then incubated with the VECTASTAIN
Elite ABC kit in TBS/TC for 60 min. After washing in TBS/TC,
the membranes were stained with 3,3'5,5'-tetramethylbenzidine substrate solution.
Western Blotting of Liver Tissue Extracts and Immunofluorescence
Analyses in Primary Hepatocyte Cells--
Cytosolic and microsomal
fractions from human liver were prepared as described previously (21).
Solutions of recombinant CL-L1-CRDhis, CL-L1-CRDmal, and
maltose-binding proteins were dissolved with SDS sample buffer in the
presence of 2-mercaptoethanol and applied to a 10-20% gradient
polyacrylamide gel. After electrophoresis, gels were transferred to
BioBlot-NC membranes. The blots were blocked with Block Ace in TBS
containing 0.1% Triton X-100 and then treated with a 1:1000 dilution
of rabbit anti-CL-L1-CRDhis serum or rabbit anti-human MBP serum. The
bound antibodies were visualized using alkaline phosphatase-conjugated
goat anti-rabbit IgG (Chemicon International, Inc.) diluted 1:5000
and 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium
(Kirkegaard and Perry Laboratories).
The primary hepatocyte cells (Cell Systems Corp.) were plated and
cultured at a density of 3 × 104 cells/0.2 ml in a
14-mm hole of 35-mm plastic culture dishes (Matsunami Glass Industries,
Ltd., Kishiwada, Japan) in CS-C complete medium (Cell Systems Corp.).
They were fixed in PBS containing 4% paraformaldehyde, pH 7.4, and
treated with PBS containing 0.1% Triton X-100, followed by incubation
with rabbit anti-MBP serum or rabbit anti-CL-L1-CRDhis serum with or
without PBS containing 400 µg/ml recombinant CL-L1-CRDmal and 25%
Block Ace and fluorescein-conjugated anti-rabbit IgG (Chemicon
International, Inc.) diluted 1:200. The cells were then treated with 1 drop of SlowFade antifade reagent (Molecular Probes, Inc.), mounted,
and sealed. The fluorescence images were observed with a Nikon
Optiphot-2 microscope, and photographs were prepared with Filing
Imaging Software U6341 (Hamamatsu Photonics K.K.).
Sequence Analysis and Construction of a Phylogenetic
Tree--
The comparison of the amino acid sequence of the CRD of
CL-L1 with the GenBankTM sequences below was done using the
DNASIS Sequence Analysis Program (Hitachi). A phylogenetic tree was
constructed by the neighbor-joining method (22) using the amino acid
sequences of the CRD of CL-L1, human MBP (23), rhesus MBP-A (24),
rhesus MBP-C (24), bovine MBP (17), rabbit MBP (25), rat MBP-C (3), rat
MBP-A (3), mouse MBP-A (26), mouse MBP-C (26), bovine conglutinin (27), bovine SP-D (28), bovine CL-43 (29), rat SP-D (30), and human SP-A (4).
The phylogenetic relationships were analyzed using the computer program
PHYLIP Version 3.57c package (25).
 |
RESULTS |
Identification of a New Human Liver Collectin (CL-L1)--
We
screened DNA data bases to identify novel members of the collectin
family. This resulted in the identification of a cDNA fragment from
human EST data bases that showed carboxyl-terminal sequence homology to
the collectins. The EST clone F1-1006D from a fetal liver cDNA
library was used to screen a human liver cDNA library, and two
positive clones (HL11-3M and HL11-9) were isolated. Furthermore, cap
site hunting was performed to determine the complete 5'-terminal
sequence including the transcriptional start site of CL-L1 mRNA
using Cap Site cDNATM (18). Restriction map analysis
and sequencing of the clones revealed that they contained an open
reading frame encoding a sequence of 277 amino acid residues. The
cDNA contained a 75-nucleotide 5'-nontranslated sequence, followed
by 831 nucleotides corresponding to a whole protein and a
759-nucleotide 3'-nontranslated sequence with an AATAA incomplete
polyadenylation signal (Fig. 1). The deduced amino acid sequence from the cDNA revealed a collectin structure consisting of an N-terminal region with cysteine residues, a
collagen-like region, a neck region, and a CRD. A hydrophobic signal
peptide sequence was not evident at the N-terminal region, and the
amino-terminal residue of the mature protein was unknown.

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Fig. 1.
Nucleotide and deduced amino acid sequences
of human CL-L1 cDNA. Nucleotide residues are numbered in the
5' to 3' direction. Amino acid residues are numbered in the N- to
C-terminal direction, beginning with the first Met residue and ending
with Lys. The underlined portions were identified by cap
site sequencing. PR1 is the reverse primer for the first PCR, and PR2
was used for the second PCR for cap site sequencing.
|
|
Northern Blot and RT-PCR Analyses--
To examine the distribution
of CL-L1 mRNA, Northern blot analyses were performed with mRNAs
from various human tissues. The Northern blot was hybridized at high
stringency with a probe derived from a CL-L1 cDNA clone. Two bands
of ~1.2 and 3.8 kilobases were detected in liver and also at low
levels in placenta (Fig. 2A). The 1.2-kilobase mRNA was considerably more abundant than the 3.8-kilobase mRNA. RT-PCR analyses showed that most tissues, except skeletal muscle, expressed CL-L1 mRNAs. Slightly high expression levels of CL-L1 mRNAs were found in liver, placenta, adrenal gland, lung, small intestine, and prostate (Fig. 2B).

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Fig. 2.
Detection of CL-L1 mRNA by Northern blot
and RT-PCR analyses of poly(A)+ and total RNAs from various
human tissues. A, for Northern blot analysis,
poly(A)+ RNAs (2 µg) from heart, brain, placenta, lung,
liver, skeletal muscle, kidney, and pancreas were subjected to 1%
agarose gel electrophoresis. Calculated sizes of detected RNAs are
indicated by arrowheads. B, RT-PCR analyses using
total RNAs (1 µg) from brain (lane 1), heart (lane
2), kidney (lane 3), liver (lane 4), lung
(lane 5), trachea (lane 6), bone marrow
(lane 7), colon (lane 8), small intestine
(lane 9), spleen (lane 10), stomach (lane
11), thymus (lane 12), mammary gland (lane
13), prostate (lane 14), skeletal muscle (lane
15), testis (lane 16), uterus (lane 17),
placenta (lane 18), adrenal gland (lane 19),
pancreas (lane 20), salivary gland (lane 21), and
thyroid (lane 22) were carried out (28 or 35 cycles of
RT-PCRs and nested PCR after 35 cycles of RT-PCR). kb,
kilobases.
|
|
Southern Blot Analysis--
Genomic DNA analyses were performed
with several animal DNAs. Zoo blotting showed that all mammalian and
avian species have the CL-L1 gene, but it is absent in yeast
(Fig. 3). Genomic Southern blotting with
several restriction enzyme fragments did not show whether or not
CL-L1 is a single copy gene (data not shown).

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Fig. 3.
Zoo blotting of genomic DNAs from animals and
yeast probed with CL-L1 cDNA. The blot contains
EcoRI-digested genomic DNAs from human, rhesus monkey, cow,
dog, rabbit, rat, mouse, chicken, and yeast (S. cerevisiae).
kbp, kilobase pairs.
|
|
Localization of the CL-L1 Gene--
The CL-L1 gene was
localized to chromosome 8q23-q24.1 by fluorescence in situ
hybridization (Fig. 4). In 25 of 25 metaphase preparations, hybridization signals were observed to the long arm of chromosome 8 in band q23-q24.1. In 22 samples, both copies of
chromosome 8 were labeled, and in three samples, a signal was detected
on one copy of chromosome 8. Its position was confirmed by PCR analysis
using DNA from human monochromosomal hybrid cells (data not shown).

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Fig. 4.
Chromosomal localization of the human
CL-L1 gene by fluorescence in situ
hybridization. Shown is a photograph of human metaphase
chromosomes counterstained with 4,6-diamidino-2-phenylindole.
Arrows point to the site of hybridization of the
biotin-labeled human CL-L1 genomic probe on both copies of chromosome 8 in band q23-q24.1; both sister chromatids show hybridization at that
site. Hybridization was observed with a Nikon Optiphot-2 microscope,
and photographs were prepared with the fluorescence in situ
hybridization Imaging Software U6744 (Hamamatsu Photonics K.K.).
|
|
Characterization of Recombinant CL-L1 Sequences of the Neck and CRD
Domains in E. coli--
Although CL-L1 has collectin organizations
that predict mannose or N-acetylglucosamine binding, we
sought to verify this lectin activity using recombinant CL-L1 CRDs.
Previously, we made collectins lacking the collagen domain in E. coli and characterized their biological activities (19, 31). In
this experiment, we made two recombinant CL-L1 CRD fusion proteins:
CL-L1 CRD-histidine tag fusion protein (CL-L1-CRDhis) and CL-L1
CRD-maltose binding fusion protein (CL-L1-CRDmal). After
solubilization, the proteins were applied to mannan or maltose columns,
but they did not bind (data not shown). Both recombinant CL-L1 fusion
proteins were purified by tag ligands to be used for sugar-blot assays;
only CL-L1-CRDmal was used for ELISA because CL-L1-CRDhis is insoluble in TBS. SDS-PAGE showed that the fusion proteins have different molecular sizes of 22 kDa (CL-L1-CRDhis) and 60 kDa (CL-L1-CRDmal) (Fig. 5). Both proteins were
immunostained by rabbit polyclonal anti-CL-L1-CRDhis serum (see Fig.
8A). Sugar-blot analyses showed that the two recombinant
CL-L1 CRDs from E. coli and recombinant human MBP from
Chinese hamster ovary cells were stained; only maltose-binding protein
used as a negative control was not stained (Fig. 5). CL-L1-CRDhis was
stained more strongly than CL-L1-CRDmal and human MBP. Mannan (100 µg/ml) and 10 mM EDTA inhibited the collectins from
binding to the
-D-mannose BP-probe.

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Fig. 5.
Sugar-blot analysis of recombinant CL-L1 CRDs
and human MBP with the mannose-biotin probe. CL-L1-CRDhis
(lanes 1), CL-L1-CRDmal (lanes 2),
maltose-binding protein as a negative control (lanes 3), and
recombinant human MBP produced in Chinese hamster ovary cells as a
positive control (lanes 4) were separated by SDS-PAGE and
transferred to the membrane. The membrane was treated with the
-D-mannose BP-probe with or without mannan (100 µg/ml)
or EDTA (10 mM). The binding of the proteins to sugar was
determined as described under "Experimental Procedures." Lane
M, molecular mass markers; CBB, Coomassie Brilliant
Blue.
|
|
On the other hand, ELISA analyses showed that the
-D-mannose BP-probe bound to CL-L1-CRDmal coated on
96-well microwells at its high concentration (Fig.
6). EDTA (10 mM) and mannan
(10 mg/ml) inhibited MBP from binding to the
-D-mannose
BP-probe completely, but inhibited CL-L1-CRDmal binding only slightly. EDTA inhibited more strongly than mannan. A comparison of saccharide specificities by ELISA showed that CL-L1 has affinity for mannose, fucose, and galactose; a lower affinity for
N-acetylglucosamine; and the lowest affinity for
N-acetylgalactosamine (Fig.
7). The maltose fusion core protein
itself at a high concentration exhibited weak affinity for sugar
BP-probes (Fig. 7). Therefore, we understand that the difference
between the binding activity of recombinant CL-L1-CRDmal and that of
maltose fusion core protein would reveal the real lectin activity of
CL-L1.

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Fig. 6.
Binding of recombinant CL-L1-CRDmal and human
MBP to the mannose-biotin probe. The plates were coated with 10 µg each of CL-L1-CRDmal ( ), maltose-binding protein ( ), and
human MBP ( ). After blocking, the -D-mannose BP-probe
was added at various concentrations (0.01, 0.1, 1, and 10 µg/ml) with
or without mannan (10 mg/ml) or EDTA (10 mM). The binding
of the proteins to sugar was determined as described under
"Experimental Procedures."
|
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Fig. 7.
Comparison of the binding of CL-L1-CRDmal to
several sugar-biotin probes. The plates were coated with 10 µg
each of CL-L1-CRDmal (black and white bars) and
maltose-binding protein (shaded and hatched
bars). EDTA (10 mM) was used as an inhibitor
(white and hatched bars). The saccharide-biotin
probes used were -D-mannose BP, -D-fucose
BP, -D-galactose BP, -N-acetylglucosamine
BP, and -N-acetylgalactosamine BP. The binding of the
proteins to sugars was determined as described under "Experimental
Procedures."
|
|
Expression of CL-L1 in Human Tissues
--
To examine the
expression of CL-L1 at the protein level, we performed immunoblot
analyses of cytosolic and microsomal fractions from liver using rabbit
anti-CL-L1-CRDhis serum. CL-L1 was detected only in the cytosol, but
not in microsomes (Fig. 8A) or
mitochondria or extracts of nuclei (data not shown). On the other hand,
MBP existed in the cytosol and microsomes. This antiserum reacted with
CL-L1-CRDhis and CL-L1-CRDmal, but not with MBP. The antiserum detected
a band corresponding to ~40 kDa in liver. The immunofluorescence analyses in human primary hepatocyte cells showed that CL-L1 was expressed in the cytoplasm, as was MBP (Fig. 8B). Its
staining was inhibited by addition of another recombinant CL-L1-CRDmal fusion protein. Immunoblotting of cell culture medium from hepatocyte cells showed only the band of MBP, but no specific bands of CL-L1.

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Fig. 8.
Detection of CL-L1 in liver tissue extracts
by immunoblotting and in hepatocyte cells by microscopic study.
A, the recombinant fusion proteins CL-L1-CRDhis (lanes
1) and CL-L1-CRDmal (lanes 2), native human MBP
(lanes 3), and microsomal (lanes 4) and cytosolic
(lanes 5) fractions from human liver tissue were subjected
to SDS-PAGE, Western blotting, and probing with rabbit anti-human MBP
serum (panel a) and rabbit anti-CL-L1-CRDhis serum
(panel b). The bound antibody was visualized with alkaline
phosphatase-conjugated secondary antibody and the nitro blue
tetrazolium salt/5-bromo-4-chloro-3-indolyl phosphate substrate system.
B, immunofluorescence analysis showed that CL-L1
(panel c) as well as MBP (panel b) are localized
in the cytoplasm. The control was reacted with nonimmunized rabbit
serum (panel a). The inhibition study was carried out by
addition of recombinant CL-L1-CRDmal to anti-CL-L1 rabbit serum
(panel d).
|
|
Sequence Alignment with Collectins from Other Animal
Species--
To compare the amino acid sequences of CL-L1, MBPs, SP-D,
conglutinin, and SP-A, the sequences were aligned with that of CL-L1 (Fig. 9A). This new collectin
has the four major domains: an N-terminal cysteine-rich domain, a
collagen-like domain, a neck domain, and a carbohydrate recognition
domain (Fig. 1). It is composed of 277 amino acids, whereas human,
rabbit, and bovine MBPs are composed of 248, 247, and 249 amino acids,
respectively. Collectins usually have two or more cysteines in their
N-terminal domains that are conserved in all species and are involved
in oligomerization. However, this new collectin has only a single
cysteine in its N-terminal domain. A collagen domain of 24 Gly-X-Y amino acid repeats is found in CL-L1,
without interruption at the eighth repeat, whereas this interruption is
conserved in most MBPs. The collagen domain also has many prolines
(five residues) and lysines (12 residues) for hydroxylation, like other
collectins. The neck region is a variable domain that has hydrophobic
amino acids, causing the triple helical structure (32, 33). The four
cysteine residues and 14 amino acid residues that form the CRD frame in CL-L1 are conserved and found in all collectins (Fig. 9A).
The four repeated lysine residues constitute the most characteristic motif that is not found in any other protein examined to date. Analyses
using the DNASIS Sequence Analysis Program show that CL-L1 has 29%
homology to human MBP (23) when 10 gaps are allowed in the
alignment.

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Fig. 9.
Alignments and phylogenetic tree of CL-L1 and
several collectins. A, the amino acid sequences of
CL-L1, human MBP, rabbit MBP, bovine MBP, rhesus MBP-A and MBP-C, mouse
MBP-A and MBP-C, rat MBP-A and MBP-C, human SP-A, rat SP-D, bovine
SP-D, bovine CL-43, and bovine conglutinin were aligned to look for
comparisons. Shaded residues are identical.
Dashes indicate gaps included for better alignment of the
sequences to obtain maximal matching. B, the phylogenetic
relationships of collectins were determined by the neighbor-joining
method using amino acid sequences of CRD fragments of CL-L1, human MBP
(hMBP), rhesus MBP-C (rheMBP-C), rabbit MBP
(rabMBP), bovine MBP (bMBP), mouse MBP-A
(mMBP-A), rat MBP-A, rhesus MBP-A (rheMBP-A),
mouse MBP-C (mMBP-C), rat MBP-C, bovine conglutinin
(bKg), bovine CL-43, bovine SP-D (bSP-D), rat
SP-D, and human SP-A (hSP-A).
|
|
Phylogenetic Tree of CL-L1 and Other Collectins--
The
phylogenetic relationships between the amino acid sequence of the CRD
of CL-L1 and those of other collectins were analyzed (Fig.
9B). The tree shows that the collectin family is made up of
four classes: the MBP class, consisting of MBP, MBP-A, and MBP-C; the
SP-D class, including SP-D, conglutinin, and CL-43; the SP-A class; and
finally, CL-L1, which may be the first member of a new group. These
data suggest that CL-L1 is a unique group in the collectin family.
 |
DISCUSSION |
We have been interested in studying the function and structure of
collectins and their role in the immune system. Much recent data
suggest that collectins play an important role in innate immunity (16).
The isolation and functional characterization of novel collectins in
addition to MBP, SP-D, and SP-A might provide further insights on the
functions of these collectins. We screened the human EST data base for
cDNA fragments that showed sequence homology to most of the
collectins in their carboxyl-terminal amino acid residues and
identified a cDNA fragment encoding CL-L1. Analyses of the cDNA
encoding CL-L1 suggest that CL-L1 has the same domain organization as
collectins, namely an N-terminal cysteine region, a collagen-like
domain, a neck domain, and a CRD (34). Furthermore, a comparison of the
amino acid sequence of the CL-L1 CRD with those of collectins suggests
that CL-L1 has a basic frame of CRD (four cysteines and 14 amino acid
residues). The four C-terminal repeated lysine residues constitute the
most characteristic motif in this collectin and are not found in other
collectins or any other proteins examined to date. The phylogenetic
relationship between CL-L1 and other collectins suggests that CL-L1 may
belong to a novel group in the collectin family. The CL-L1
gene was located on chromosome 8q23-q24.1. Other collectins are located
on chromosome 10q (35), and we are very interested in the genomic
localization of CL-L1.
The genomic organization of the collectin gene family, which includes
the MBP, SP-A, and SP-D groups, shows differences. The CRD and neck
domains are encoded by a single exon in all collectins (34), whereas
the collagen domain is encoded by two (MBP and SP-A) or five (SP-D)
exons. Preliminary data on the genomic organization of CL-L1 indicate
that the CRD and neck domains are also encoded by a single exon, but
the collagen-like domain is encoded by five exons (data not shown), and
the number of amino acids in each exon is different from that in other
collectins. These data and chromosome mapping results indicate that
this new collectin developed differently than other collectins. More
detailed genomic studies of its organization are needed.
Northern and Western blot analyses indicated that CL-L1 is expressed in
ubiquitous organs (mainly expressed in liver, placenta, and adrenal
gland), whereas SP-D and SP-A are expressed in lung and
gastrointestinal tracts, and RT-PCR studies show that MBP is expressed
in murine kidney and liver. RNA blotting showed that CL-L1 is expressed
only faintly in placenta, and Western blotting of placenta showed bands
similar to those in liver, but in trace amounts (data not shown).
RT-PCRs and nested PCR after RT-PCR showed more sensitive results.
CL-L1 gene expression is found in most tissues, except
skeletal muscle. The expression level is varied in individual tissues.
The high expression organs are liver, placenta, and adrenal gland. This
ubiquitous expression pattern is different from that of other
collectins and galectins.
Western blot and immunofluorescence analyses indicated that CL-L1
protein is localized in cytosolic fractions from liver. Usually,
collectins are secreted into the extracellular space through the
endoplasmic reticulum pathway. Using anti-MBP serum with the same
sample blot shows that MBP is found mainly in microsomal fractions and
less in cytosolic fractions. The secreted collectins are considered to
play an important role in innate immunity against pathogens invading
from outside the organism (16). CL-L1 may react with internal ligands
in contrast to other collectins.
Western blotting showed that MBP has molecular mass of 32 kDa, whereas
its calculated molecular mass is 24.5 kDa. Other collectins showed
slightly larger molecular masses than those estimated. These results
indicate that CL-L1 of ~40 kDa on SDS-PAGE is not inconsistent. The
amino acid residues in the neck domain of CL-L1 can form
-helices
like other collectins (34). The
-helical bundle is very stable
against denaturation by heat (Tm > 55 °C) or pH
(pH 3.0-8.5), indicating that CL-L1 might maintain dimer and trimer
structures under the reducing conditions of the cytoplasm, like other collectins.
The expression studies using CL-L1-CRDhis and CL-L1-CRDmal indicated
that lectin activity is preserved in CL-L1, but it is very weak. Two
analyses (ELISA and sugar-blot) of weak lectin activity suggested that
CL-L1 can bind to mannose at high concentrations and that this can be
inhibited by mannan and EDTA. The carbohydrate-binding specificities of
most collectins are for mannose-type saccharides, and saccharide
specificities of the CL-L1 CRD include galactose as well as mannose,
fucose, and N-acetylglucosamine. Previously, the recombinant
collectin fusion proteins produced in E. coli were used in
the analysis of carbohydrate-binding specificities in recombinant
collectins. All of these lectin fusion proteins attached to saccharide
columns due to their high affinity. However, CL-L1-CRDmal cannot bind
to saccharide columns under any buffer conditions. CL-L1-CRDmal at a
high concentration (10 µg/ml) has binding activity. At such a high
concentration, the maltose fusion core protein itself exhibits weak
affinity for sugar BP-probes. Therefore, we understand that the
difference between the binding activity of recombinant CL-L1-CRDmal and
that of the maltose fusion core protein would reveal the real lectin
activity of CL-L1.
The two amino acid residues of the five in collectins responsible for
complexing the calcium ion involved in carbohydrate binding, namely
Glu-185 and Asn-187 (36), when changed to Gln-185 and Asp-187, resulted
in an increased affinity for galactose. The two amino acids in CL-L1
are Glu-238 and Ser-240, indicating that CL-L1 is a hybrid type, like
SP-A, between collectins and other lectins specific for galactose (34).
These sugar specificities and weak lectin activities are involved in
various functions of the collectins. Furthermore, the polycharge
islands of repeated lysine residues in the carboxyl-terminal area might
indicate an alternative role for this new collectin. A crystallographic
study and computer graphic model suggest that conserved
KGQKGEKGS sequences in the collagen
domain of the macrophage scavenger receptor make a "charged
collagen" molecule with a coiled groove surrounded by lysine residues
(37). These lysine clusters are considered to be the receptor site of
the macrophage scavenger receptor. Scavenger receptors have
characteristic broad ligand specificities and are able to bind various
substances including degenerated lipoproteins, lipopolysaccharide, and
microorganisms (38). These findings suggest that CL-L1 may play a role
as a scavenger or chaperonin in the cytoplasm.
The searches of the EST data base with the CL-L1 sequence
can identify related molecules already described in the Unigene data
base. From the first hit to the third hit are fragments of the
CL-L1 gene. The related collectin genes are hit 18 times. The SP-A gene is hit 12 times, the SP-D gene is hit five times, and MBP
is hit once. Other hit sequences are collagen genes. The searches
showed that the related genes with high homology are hit from the
first. These data support the relationship between the CL-L1
gene and other groups (SP-A, SP-D, and MBP genes) in the phylogenetic
tree (Fig. 9B). The EST data base is made from mRNA. The
covering how much mRNAs is different in individual tissues. The
expression of mRNA varies in different tissues. The high expression gene is easily picked up by the EST database. The highly developed organ's EST data base might be able to pickup the CL-L1 gene.
The isolation of cDNA encoding CL-L1, the preparation of CL-L1 CRD
fusion proteins, and the cytoplasmic localization of CL-L1 will provide
the basis for future studies on the function and structure of this new
collectin. The findings that CL-L1 has a novel carboxyl-terminal lysine
cluster and weak lectin activities for galactose as well as mannose,
fucose, and N-acetylglucosamine and the ubiquitous
expression in RT-PCR analysis suggest that this protein might play some
role in regulating cell functions.
 |
ACKNOWLEDGEMENTS |
We thank Dr. H.-S. Shin for providing the
cDNA clone F1-1006D. We also thank the late Veterinary Dr. M. Naiki
and Professors T. Kinoshita, T. Azuma, and H. Nakada for useful
discussions and encouragement; Drs. K. Inoue and K. Hashimoto for
technical help with the chromosomal localization of the
CL-L1 gene; Dr. T. Yasunaga for preparing the phylogenetic
tree; and Dr. H. Schulman for critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in aid 09672356 and 10178210 for Scientific Research (to N. W. and Y. S.) from the Ministry of Education, Science, Sports, and Culture of Japan; the Fuso
Pharmaceutical Industry; the Sankyo Foundation of Life Science; and the
Japan Health Sciences Foundation.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 reported in this paper has been submitted
to the DDBJ/GenBankTM/EMI Data Bank with accession
number AB002631.

To whom correspondence should be addressed. Tel.:
81-6-879-8288; Fax: 81-6-875-3894; E-mail:
wakamiya{at}biken.osaka-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
CRD, carbohydrate
recognition domain;
MBP, mannan-binding protein;
SP-A, surfactant
protein A;
SP-D, surfactant protein D;
TBS, Tris-buffered saline;
EST, estimated sequence tag;
RT-PCR, reverse transcription-polymerase chain
reaction;
PAGE, polyacrylamide gel electrophoresis;
ELISA, enzyme-linked immunosorbent assay;
BP-probe, biotinylated polymeric
probe.
 |
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