(Received for publication, August 28, 1996, and in revised form, December 3, 1996)
From the Departments of Partial overlapping cDNA
sequences likely to encode a novel human CC chemokine were identified
from the GenBank Expressed Sequence Tag data base. Using these
sequences, we isolated full-length cDNA encoding a protein of 96 amino acid residues with 20-28% identity to other CC chemokines. By
Northern blot, this chemokine was mainly expressed in liver among
various tissues and strongly induced in several human cell lines by
phorbol myristate acetate. We thus designated this chemokine as LARC
from Chemokines are a large family of small cytokines involved in a
variety of immunoregulatory and proinflammatory responses, primarily by
virtue of their leukocyte chemotaxis activity (1-3). Based on whether
the first two of the four conserved cysteine residues are juxtaposed or
separated by a single amino acid residue, this family is subdivided
into two groups, the CXC and the CC chemokines. Recently, a
new chemokine-like molecule, lymphotactin/SCM-1, which lacks the first
and the third conserved cysteine residues, has been isolated and may
represent a third group C (4-6). In general, CXC chemokines
are potent chemoattractants for neutrophils, whereas CC chemokines are
chemotactic mainly for monocytes and also for basophils, eosinophils,
and lymphocytes with variable selectivity. Lymphotactin/SCM-1 has been
shown to attract lymphocytes. In addition to chemotactic activities,
some chemokines have regulatory roles in hematopoiesis (7, 8).
Recently, three members of the CC chemokines, MIP-1 Until recently, chemokines have been purified according to their
chemotactic activities or have been cloned by subtraction or
differential hybridization. The cell types used in such studies have
been mainly blood lymphocytes or tumor cell lines. However, as shown by
recent Northern hybridization analyses, chemokines are often expressed
constitutively in some normal tissues (6, 13, 15-19). For example, the
mRNA of eotaxin, which is a novel CC chemokine selectively
chemotactic for eosinophils, is expressed at high levels in small
intestine, colon, and heart but at low levels in peripheral blood
leukocytes, spleen, and thymus (15-17). Therefore, there might still
exist uncovered chemokines expressed in other cell types and tissues.
Currently, world-wide efforts to find all human genes, approximately
~100,000, are in progress as part of the human genome project.
Accordingly, cDNAs from libraries of various human tissues are
sequenced from both 5 Here we report the identification of a fifth new CC chemokine, termed
LARC ( The dbEST (21) was searched with
various CC chemokine nucleotide sequences or amino acid sequences as
queries using the data base search and analysis service Search Launcher
(22) available on the World Wide Web. The program used was Basic Local
Alignment Search Tool (23).
Human
histiocytic lymphoma cell line U937 was grown in RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS) and stimulated with
phorbol 12-myristate 13-acetate (PMA) at a concentration of 50 ng/ml
for 6 h. Poly(A)+ RNA was prepared using the QuickPrep
mRNA purification kit (Pharmacia Biotech, Uppsala, Sweden).
cDNA was synthesized by the rapid amplification of cDNA ends
(RACE) method (24) using the Marathon cDNA amplification kit
(Clontech, Palo Alto, CA). Briefly, the double-stranded cDNA was
synthesized from poly(A)+ RNA using a cDNA synthesis
primer supplied in the kit. The cDNA adaptors were then ligated at
both ends of the cDNA. The adaptor-ligated cDNA was then
amplified by polymerase chain reaction (PCR) with one of the
gene-specific primers derived from an EST sequence (GenBank accession
number D31065[GenBank]) (5 A human liver cDNA library, constructed with random hexamers and
cloned in the
Poly(A)+ RNA was
prepared from U937, K562, an erythroleukemia cell line, Jurkat, a T
cell line, and Bowes melanoma cells. Messenger RNAs were extracted from
cells without or with PMA treatment (50 ng/ml, 6 h). After
electrophoresis in a 1% formaldehyde-agarose gel (2 µg per lane),
RNAs were blotted onto a nylon membrane. Northern blot filters
containing human poly(A)+ RNA from various tissues (2 µg
per lane) were purchased from Clontech. Filters were hybridized with
the 32P-labeled LARC cDNA probe in a solution
containing 5 × SSPE, 10 × Denhardt's solution, 100 µg/ml
denatured salmon sperm DNA, 2% sodium dodecyl sulfate, and 50%
formamide at 42 °C overnight and washed at 50 °C for 20 min in
0.1 × SSC, 0.1% SDS. After autoradiography, filters were
reprobed with a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
cDNA probe. The LARC and GAPDH probes were prepared by labeling the
3 Using the 5 According to the radiation hybrid
mapping data, the YAC data base at Whitehead Institute/MIT Center for
Genome Research was searched for CEPH mega-YAC clones containing a
sequence tagged site (STS) D2S159. The YACs containing D2S159 were
grown in AHC medium, and total yeast DNA from individual YAC clones was
prepared as described (28). The presence of a D2S159 STS in these
clones was confirmed by PCR using the primers listed in the Genome Data base (accession ID: 188410). YACs containing the LARC gene
were identified by PCR analysis using 50 ng of these DNAs. The PCR conditions were as described above.
LARC protein was
prepared using a baculovirus expression system. The LARC cDNA
fragment containing the entire coding region was excised with
BglII (5 Spodoptera frugiperda Sf9 cells were cotransfected with
pVL-LARC DNA and the linearized Autographa californica
nuclear polyhedrosis virus DNA (Clontech) using Lipofectin (Life
Technologies, Inc.). In the Sf9 cells, homologous recombination occurs
between the LARC sequence in the transfer vector and the polyhedrin
gene sequence of A. californica nuclear polyhedrosis virus.
The recombinant viruses were subsequently isolated by limiting
dilution. For expression of the recombinant LARC, Trichoplusia
ni BTI-TN-5B1-4 cells were infected with the recombinant viruses
at m.o.i. of 10-20. The Sf9 and BTI-TN-5B1-4 cells were maintained at
27 °C in EX-CELL 400 medium (JRH Biosciences, Lenexa, KS).
The culture supernatants collected 2 days after infection were mixed
with 1/10 volume of 500 mM MES (pH 6.5) and applied
to a 1-ml cation-exchange HiTrap-S column (Pharmacia Biotech Inc.) equilibrated with a buffer containing 50 mM MES (pH 6.5)
and 100 mM NaCl. The column on a fast protein liquid
chromatographic system (Pharmacia Biotech) was eluted at a rate of 1 ml/min with a 45-ml linear gradient of 0.1-1.0 M NaCl in
50 mM MES. The fractions containing recombinant LARC were
pooled and injected into a reverse-phase high performance liquid
chromatography column (4.6 × 250 mm Cosmocil 5C4-AR-300) (Cosmo
Bio, Tokyo, Japan) equilibrated with 0.05% trifluoroacetic acid. The
column was eluted with a 100-ml linear gradient of 20-40%
acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 1 ml/min.
Fractions containing recombinant LARC were pooled and lyophilized.
Protein concentrations were determined by the BCA kit (Pierce).
Endotoxin levels were determined by the Limulus amoebocyte
lysate assay (QCL-1000) (Bio Whitaker, Walkersville, MD) and were <4
pg/µg recombinant LARC. NH2-terminal sequence analysis
was performed on a protein sequencer (Shimazu, Tokyo, Japan).
Polymorphonuclear neutrophils and
mononuclear cells in heparinized human peripheral blood from single
donors were separated by gradient centrifugation (30 min, 400 × g) on Ficoll/sodium metrizoate (Lymphoprep, Nyegaard, Oslo,
Norway). The cell pellets, containing granulocytes and erythrocytes,
were suspended in hydroxyethyl starch (Plasmasteril, Fresenius AG, Bad
Homburg, Germany) for 30 min to remove erythrocytes by sedimentation.
Residual erythrocytes were lysed by hypotonic shock in double distilled
water (30 s). The total mononuclear cell fraction was used as a source
for monocytes and lymphocytes. Blood lymphocytes were further purified
by incubating mononuclear cells for 30 min at 4 °C with paramagnetic
microbeads conjugated with monoclonal antibody against CD14 expressed
on monocytes. The cell suspension was passed over a column placed in a
magnetic field (VarioMACS, Miltenyl Biotec, Bergisch, Germany). Alternatively, monocytes and lymphocytes were isolated by magnetic cell
sorting (VarioMACS) using positive selection with anti-CD14 or
anti-CD3, respectively. After positive or negative magnetic cell
sorting, a cell purity (analyzed by fluorescence-activated cell sorter)
of more than 80% was reached for monocytes (CD14+) and
lymphocytes (CD3+). Purified granulocytes, mononuclear
cells, lymphocytes, and THP-1 cells were washed, counted, and
resuspended at 1 × 106, 2 × 106,
1 × 107, and 0.5 × 106 cells/ml,
respectively, in HBSS (Life Technologies, Inc.) supplemented with
pyrogen-free human plasma protein (1 mg/ml albumin, Cohn fraction V).
The monocytic THP-1 cells, grown in Dulbecco's modified Eagle's
medium with 10% FCS (Life Technologies, Inc.), were used in the
chemotaxis assay as an alternative for fresh monocytes.
Migration of cells was assessed in a microchamber (NeuroProbe, Cabin
John, MD) as described previously (29). Briefly, the lower compartments
of the microchamber were filled with dilutions of chemokine (27 µl)
or with control buffer and the upper compartments with 50 µl of cell
suspension. The two compartments were separated by a 5-µm pore size
polycarbonate filter (Nuclepore, Pleasanton, CA).
Polyvinylpyrrolidone-free filters were used for neutrophils and
lymphocytes and polyvinylpyrrolidone-treated filters for monocytes and
THP-1 cells. For lymphocyte chemotaxis, the membranes were coated with
20 µg/ml fibronectin (Life Technologies, Inc.) for 24 h at
4 °C. After incubation at 37 °C for 45 min (neutrophils), 2 h (monocytes, THP-1 cells), or 4 h (lymphocytes), the filters were
removed from the chambers, fixed, and stained with Diff-Quick (Harleco,
Gibbstown, NJ). Finally, the cells of 10 oil immersion fields were
counted. The chemotactic index was calculated from the number of cells
migrated to the test sample divided by the number of cells migrated to
the control. Synthetic human MCP-3 and natural IL-8, purified to
homogeneity (29), were used as positive controls.
LARC was expressed
as a soluble fusion protein with secreted alkaline phosphatase (SEAP)
containing a (His)6 COOH-terminal tag. For this purpose, a
SEAP(His)6 tag vector, termed
pDREF-SEAP(His)6-Hyg, was constructed as follows. Using the
pSEAP-Enhancer (Clontech) as a template, the (His)6 coding
sequence was introduced at the COOH-terminal region of SEAP by PCR
using the 5 To produce the LARC-SEAP fusion protein, 293/EBNA-1 cells (Invitrogen)
were transfected with the expression vector using lipofectamin (Life
Technologies, Inc.). The 293/EBNA-1 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% FCS. The
transfected cells were incubated for 3-4 days in Dulbecco's modified
Eagle's medium containing 10% FCS. The culture supernatant was
centrifuged, filtered (0.45 µm), and stored at 4 °C with 20 mM HEPES (pH 7.4) and 0.02% sodium azide. For
NH2-terminal sequence analysis, the fusion protein was
purified by affinity chromatography on nickel agarose (QIAGEN, Hilden,
Germany).
The concentration of LARC-SEAP was estimated by sandwich enzyme-linked
immunosorbent assay. Briefly, 96-well microtiter plates (Maxsorb, Nunc,
Roskilde, Denmark) were coated with anti-placental alkaline phosphatase
(anti-PLAP) monoclonal antibody (Medix Biotech, Foster City, CA) (2 µg/ml in 50 mM Tris-HCl (pH 9.5)). After blocking nonspecific binding sites with 1 mg/ml bovine serum albumin in phosphate-buffered saline (PBS), the samples were titrated in T-PBS
(0.02% Tween 20 in PBS). After incubation for 1 h at room temperature, the plates were washed with T-PBS, incubated with biotinylated rabbit anti-PLAP antibody diluted 1:500 for 1 h at room temperature, washed again, and incubated with
peroxidase-conjugated streptavidin (Vector Laboratories, Burlingame,
CA) for 30 min. After washing, bound peroxidase was detected by
3,3 Alkaline phosphatase (AP) activities were determined by a
chemiluminescent assay using the Great EscApe Detection Kit (Clontech). Purified PLAP (Cosmo Bio) was used to generate standard curves. AP
activities are expressed here as relative light unit/s; 1 pmol of
SEAP(His)6 and LARC-SEAP(His)6 corresponds to
approximately 8.7 × 107 and 1.7 × 108 relative light unit/s, respectively.
Peripheral blood mononuclear cells were
isolated from venous blood obtained from healthy adult donors using
Ficoll-Paque (Pharmacia Biotech). Monocytes and lymphocytes were
incubated with fluorescein isothiocyanate-conjugated anti-CD14 antibody
and separated by MACS (Miltenyi Biotec, Bergisch, Germany). The purity
of the cells was >98% as determined by flow cytometry on a FACStar
Plus (Beckton Dickinson, Mountain View, CA).
For displacement experiments, 2 × 105 cells were
incubated for 1 h at room temperature with diluted supernatant
containing 1 nM of SEAP(His)6 or
LARC-SEAP(His)6 in the presence of increasing concentrations of unlabeled chemokines in 200 µl of RPMI 1640 containing 20 mM HEPES (pH 7.4), 1% bovine serum albumin,
and 0.02% sodium azide. MCP-1 and TARC were prepared as described (18). MIP-1 To find new members of the human CC
chemokine family, we have searched the dbEST with various CC chemokine
amino acid sequences and nucleotide sequences. This search identified
five novel CC chemokine-like molecules encoded by single-pass cDNA
sequences. Four of these were mapped within a YAC contig containing the
human CC chemokine gene cluster on chromosome 17q11.2, providing
additional evidence that they are indeed CC chemokine members (20).
Another molecule, later termed LARC ( To clone the full-length cDNA of LARC, we first utilized the RACE
method. Since a preliminary reverse transcriptase-PCR analysis showed
that the LARC mRNA is expressed in PMA-stimulated macrophage-like U937 cells, we used the cDNA prepared from the PMA-stimulated U937
mRNA for the RACE reaction. Two LARC gene-specific
primers, one for 5 Nucleotide sequence of human LARC cDNA.
A, cDNA and predicted amino acid sequence of human LARC.
The arrow indicates the cleavage site of the signal
sequence. Filled circles above the nucleotides indicate
those that are missing in some EST records (D82589[GenBank], T27336[GenBank], and T27433[GenBank];
Fig. 1). The ATTTA sequences that have been shown to decrease the
mRNA stability (33) are overlined. The putative
polyadenylation signals are underlined. The predicated
open reading frame is indicated below the nucleotide
sequence. *, termination codon. B, alignment of the LARC
amino acid sequence with other human CC chemokines. The amino acid
sequence of LARC was compared with the amino acid sequences of 12 other
human CC chemokines MIP-1
The LARC cDNA is approximately 0.8 kb long and contains the longest
open reading frame of a 96-amino acid protein that starts at the first
methionine codon (Fig. 2A). The nucleotide sequence around
this methionine codon conforms to the consensus sequence shared by many
mRNAs of higher eukaryotes (30). The NH2-terminal end
of the deduced amino acid sequence is highly hydrophobic and is
consistent with a typical signal peptide sequence (31). The cleavage
site is predicted to be between Ala-26 and Ala-27. The primary sequence
of LARC contains no putative N-glycosylation site. The
3 LARC
shows relatively low sequence similarity with other human CC
chemokines, having the highest similarity to MIP-1 We
examined the expression of LARC mRNA in various human cell lines
and tissues. Poly(A)+ RNAs were extracted from Jurkat,
K562, U937, and Bowes melanoma without and with PMA stimulation for
6 h and analyzed by Northern hybridization. While there was no
detectable LARC mRNA in unstimulated cells, LARC mRNA was
strongly induced in U937 and Bowes melanoma after stimulation with PMA
(Fig. 3A). No such induction was, however, observed in Jurkat and K562. The detected LARC mRNA was
approximately 0.9 kb long, which corresponded to the size of the LARC
cDNA plus a poly(A) tail.
We then examined expression of LARC mRNA in various human tissues.
LARC mRNA was found to be constitutively expressed at high levels
in liver and, to a lesser extent, in lung (Fig. 3B). When another batch of the commercial Northern filters was examined, expression of LARC mRNA in lung was quite low compared with that in
liver (data not shown). Very low levels of expression were also seen in
thymus, prostate, testis, small intestine, and colon. In testis, two
transcripts, approximately 1.0 and 1.3 kb long, were observed instead
of the 0.9-kb one. Transcripts were not detected in spleen and
peripheral blood leukocytes. Although three LARC ESTs (D82589[GenBank], T27336[GenBank],
and T27433[GenBank]) were derived from the pancreatic islet cDNA library, no
constitutive expression was detected in pancreas even using different
batches of filters.
We tested whether
the LARC gene was present in the YAC contig of several mega
bases corresponding to human chromosome 17q11.2. All CC chemokines
reported so far and four newly identified CC chemokine genes
(NCC-1~-4) have been mapped to this
YAC contig (20). Surprisingly, however, the result was negative (data
not shown). We therefore examined the chromosomal location of the LARC gene by using the DNA panel of the somatic cell hybrids
containing human monochromosomes. The LARC gene was
localized to chromosome 2 (Fig. 4A). To map
the location of the LARC gene more precisely, radiation
hybrid mapping was performed using the GeneBridge 4 panel (27) (Fig.
4B). The resulting PCR data were analyzed at the Radiation
Hybrid mapper server at the Whitehead Institute/MIT Center for Genome
Research. The results showed that the LARC gene is located
0.0 centi-Ray (3.7 centi-Ray is approximately 1 mega base pairs) apart
from a STS marker D2S159 that is mapped between the bands q33 and q37
of chromosome 2 (47). We therefore analyzed 20 YAC clones containing
D2S159 for the LARC gene by PCR. Two clones, 770_f_5 (1540 kb) and 933_c_7 (1690 kb), were positive for LARC (Fig. 4C).
One of the clones, 933_c_7, has already been used for fluorescence
in situ hybridization analysis and has been mapped to a
locus 93-95% from the top of chromosome 2 (48). This fluorescence
in situ hybridization result is consistent with our
radiation hybrid mapping data.
To obtain LARC protein,
recombinant baculovirus was prepared. Insect cells BTI-TN-5B1-4 were
infected with the virus, and recombinant LARC was purified from the
culture supernatants by cation-exchange chromatography and
reverse-phase high performance liquid chromatography. LARC was eluted
from the reverse-phase column as a single major peak (Fig.
5A). The yield of purified protein was ~0.4
µg per ml of starting culture supernatant. When analyzed by
SDS-polyacrylamide gel electrophoresis and silver staining, the
purified protein migrated as a single band of 8 kDa (Fig.
5B). Amino acid sequence analysis demonstrated that the
NH2 terminus of recombinant LARC started at Ala-27 of the predicted sequence. These results agreed with the predicted signal cleavage site and molecular weight of the mature protein.
LARC was not significantly chemotactic for THP-1
cells (Fig. 6A) (n = 4, maximal stimulation index of 2.2 ± 0.7 at 1 µg/ml) or monocytes
(Fig. 6B) (n = 4, maximal index of 1.0 ± 0.4 at 1 µg/ml), whereas MCP-3 induced strong chemotaxis in THP-1
and monocytes at 1 and 10 ng/ml, respectively. However, LARC was found
to be chemotactic for lymphocytes (Fig. 6C) from 100 ng/ml
(n = 8, maximal index of 3.3 ± 0.5) onwards and
at 1 µg/ml the chemotactic response (n = 8, maximal
index of 8.2 ± 2.1) was almost as strong as with MCP-3 at 100 ng/ml. LARC was also slightly chemotactic for neutrophils (Fig.
6D) from 100 ng/ml (n = 6, maximal index of
4.0 ± 0.7) onward (at 1 µg/ml: n = 6, maximal
index of 5.7 ± 0.9). The chemotactic index for neutrophils,
however, remained much below that observed with IL-8.
We next
investigated the specific binding of LARC to human blood leukocytes. An
expression vector was constructed to produce LARC fused with a
SEAP-(His)6 tag. The LARC-SEAP(His)6 protein was secreted as a single major protein with an expected apparent molecular mass of 74 kDa (data not shown). This fusion protein has an
AP activity for quantitative tracing and a (His)6 tag in its COOH terminus for easy purification. Amino acid sequence analysis of the purified fusion protein demonstrated that the NH2
terminus of LARC-SEAP(His)6 started at Ala-27. As LARC has
chemotactic activity for lymphocytes, we characterized the receptor for
LARC on these cells. When binding was performed with increasing
concentrations of LARC-SEAP(His)6 (Fig.
7A), a single class of receptor with a
Kd of 0.4 nM and 2100 sites/cell was
observed (Fig. 7B). Competition experiments showed that LARC
fully inhibited binding of LARC-SEAP(His)6 to lymphocytes
with an IC50 of 3.2 nM (Fig. 7C). On
the other hand, similar competition experiments showed monocytes to
have only low affinity binding sites for LARC with an IC50
of about 200 nM (data not shown). Other CC and
CXC chemokines, TARC, MCP-1, RANTES, MIP-1
In the present report we have described a novel human CC
chemokine, designated LARC, which was first identified from the EST data base. LARC is only distantly related to other CC chemokine members
so far identified, although it has certain characteristics of the CC
chemokines (Fig. 2). For example, LARC retains three out of five amino
acid residues found in most CC chemokines in addition to the conserved
four cysteine residues. However, the well conserved tyrosine and
threonine residues present between the second and the third cysteine
residues in other CC chemokines are replaced in LARC with phenylalanine
and alanine, respectively. The well conserved tyrosine residue has been
shown to be critical for monocyte chemotaxis (45, 46). The recently
cloned CC chemokine TARC (18), which is chemotactic for T cells but not
for monocytes, does not contain the tyrosine residue either. The
presence of the tyrosine residue, however, may not be sufficient for
monocyte chemotaxis since eotaxin contains the tyrosine residue but
does not attract monocytes (15-17).
Among the five ESTs containing the coding region of LARC, three encode
proteins lacking the NH2-terminal alanine residue that is
present in the other two ESTs and the isolated cDNAs. If the signal
sequence cleavage site of this LARC variant (LARCvar) is the same as
that of LARC, the NH2-terminal residue of its mature protein is serine (Fig. 2). The NH2-terminal sequences of
chemokines have been shown to markedly affect their activities and
binding to receptors (49-52). In the case of MCP-1, deletion of one
NH2-terminal residue has been shown to change the target
cell specificity (53). Therefore, it may be interesting to see whether
LARC and LARCvar have the same activities and cell specificities.
The LARC gene has been localized close to a marker D2S159
that has been mapped to the bands between q33 and q37 on chromosome 2 (Fig. 4). This was unexpected since all the other CC chemokine genes
and four newly identified putative CC chemokine genes
(NCC-1~-4) have been mapped to
chromosome 17q11.2 (20). Another such example is the CXC
chemokine SDF-1/PBSF gene that has
been localized on human chromosome 10 instead of chromosome 4 (13). The
CC and CXC chemokines on human chromosomes 17 and 4, respectively, have been presumably generated from successive gene
duplication events. Since LARC is only distantly related to other CC
chemokines, LARC may have been generated before amplification of the CC
chemokines on chromosome 17. This also suggests that there might be
LARC-related chemokines on chromosome 2.
We have identified two YAC clones from chromosome 2 containing the
LARC gene. These two YAC clones and the clones from the CC
chemokine gene cluster on chromosome 17 (20) may be useful tools for
localizing new CC chemokine genes. Since ESTs are only partial
sequences, it may be sometimes helpful to know whether or not a
particular EST is mapped to one of these YAC clones by PCR. An EST
mapped to one of these YAC clones is more likely to be a CC chemokine
cDNA. Furthermore, such YAC clones may be useful for cloning new CC
chemokine genes by, for example, exon trapping.
Because of its unorthodox chromosomal location, the question remained
whether LARC was a true chemokine. Recombinant LARC was prepared and
tested on various cell types. Whereas monocytes and THP-1 cells did not
respond to LARC, lymphocytes reacted positively to LARC with a typical
bell-shaped dose-response curve (Fig. 6). Granulocytes were also weakly
attracted at a concentration of 1 µg/ml. These chemotaxis results
were confirmed by binding experiments using a LARC-alkaline phosphatase
fusion protein. This method was previously employed for identification
of a specific receptor for a CXC chemokine IP-10 (54).
Lymphocytes showed a single class of high affinity receptor for LARC
with a Kd of 0.4 nM that was not shared
by other CC and CXC chemokines (Fig. 7). On the other hand,
monocytes possessed only a substantial level of low affinity binding
sites for LARC (data not shown). We extended the above analysis and
found that LARC did not bind to the CC chemokine receptors CCR1, 2B, 3, 4, and 5 and also to the chemokine receptor-like orphan receptors EBI1
and BLR1 (data not shown).
Compared with the concentration required for LARC binding to the
receptor (Kd = 0.4 nM, Fig.
7B), considerably higher concentrations (~100 ng/ml that
corresponds to approximately 12 nM, Fig. 6) were required
for the significant chemotactic responses of lymphocytes. Since
chemokines are now known as pleiotropic cytokines, this discrepancy may
indicate that LARC has physiological functions other than chemotactic
activity. For example, CC chemokine I-309 has been shown to be
chemotactic for monocytic cell line THP-1 at ~50 ng/ml but has an
anti-apoptotic activity at 0.1-2 ng/ml (55). Cloning of the LARC
receptor will demonstrate its distribution among various types of cells
and will help to elucidate the biological functions of LARC.
In conclusion, LARC is a novel inducible CC chemokine that is mainly
expressed in liver and mainly attracts lymphocytes through a single
class of high affinity receptors unique to LARC. The LARC
gene is on human chromosome 2. The biological roles of LARC in
inflammatory and immunological responses as well as its physiological functions in the liver remain to be established.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D86955[GenBank]. We thank M. Takiguchi for the human
liver cDNA library, M. Nishimura and M. Kakizaki for excellent
technical assistance, and J. A. Egan for helpful comments on the
manuscript.
Biochemistry and
§ Internal Medicine,
Laboratory of Molecular Immunology, Rega Institute for Medical
Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium, the
Laboratory of
Genetic Resources,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
iver and
ctivation-
egulated
hemokine. We mapped the LARC gene close to the
chromosomal marker D2S159 at chromosome 2q33-q37 by somatic cell and
radiation hybrid mappings and isolated two yeast artificial chromosome
clones containing the LARC gene from this region. To
prepare LARC, we subcloned the cDNA into a baculovirus vector and
expressed it in insect cells. The secreted protein started at Ala-27
and was significantly chemotactic for lymphocytes. At a concentration
of 1 µg/ml, it also showed a weak chemotactic activity for
granulocytes. Unlike other CC chemokines, however, LARC was not
chemotactic for monocytic THP-1 cells or blood monocytes. LARC tagged
with secreted alkaline phosphatase-(His)6 bound
specifically to lymphocytes, the binding being competed only by LARC
and not by other CC or CXC chemokines. Scatchard analysis
revealed a single class of receptors for LARC on lymphocytes with a
Kd of 0.4 nM and 2100 sites/cell. Collectively, LARC is a novel CC chemokine, which may represent a new
group of CC chemokines localized on chromosome 2.
/LD78
,
MIP-1
, and RANTES, have been shown to block entry of the human
immunodeficiency virus type 1 into macrophages (9-12). The genes of
the CXC and CC chemokines are clustered on human chromosomes
4 and 17, respectively (1), except for the CXC chemokine
SDF-1/PBSF whose gene has been mapped to human chromosome 10 (13). The
lymphotactin/SCM-1 gene has been
localized to human chromosome 1 (5, 14).
- and 3
-ends, and their partial, "single
pass" cDNA sequences are deposited as expressed sequence tags
(ESTs)1 in the public data bases. Recently
we found four ESTs encoding CC chemokine-like proteins in the EST data
base (dbEST), a division of GenBank (20). The genes of these four ESTs
have been mapped in a yeast artificial chromosome (YAC) contig covering
the CC chemokine gene cluster on human chromosome 17q11.2 (20). This demonstrates that the dbEST is a good source for new chemokines. Furthermore, the YAC contig may be useful to quickly assign new chemokine genes within the cluster.
iver and
ctivation-
egulated
hemokine)
from the dbEST. This new CC chemokine is different from other CC
chemokine members in several respects. 1) LARC is only distantly
related to other CC chemokines, and its gene is located on a different chromosome than other CC chemokines. 2) Unlike other CC chemokines, LARC is not chemotactic for monocytes but is primarily a lymphocyte chemoattractant. 3) Lymphocytes display a single class of receptors for
LARC that is not shared by other CC or CXC chemokines.
EST Data Base Search
oligomer: GTACTCAACACTGAGCAGATCT and 3
oligomer:
AGGTGGAGTAGCAGCACT) and an AP1 primer which is complementary to part of
the cDNA adaptor and supplied in the kit. PCR was performed in a
50-µl reaction mixture containing 0.25 mM each of the
dNTPs, 50 pmol of each of the primers, 2.5 units of TAKARA LA
Taq (Takara, Kyoto, Japan), 1 × buffer supplied with
the polymerase, and 0.55 µg of TaqStart antibody (Clontech). The PCR
conditions were as follows: 94 °C, 30 s; 60 °C, 30 s; 68 °C, 3 min, for 30 cycles. The products were inserted into a pGEM-T vector (Promega, Madison, WI) and sequenced on both strands using gene-specific and commercial primers.
gt11 vector, was screened using the LARC 3
-RACE fragment (Fig. 1) as a probe according to standard methods (25). The
library was kindly provided by Dr. M. Takiguchi of Kumamoto University
Medical School. The cDNA fragments of the positive clones were
excised with EcoRI and inserted into pBluescript II KS+
(Stratagene, La Jolla, CA) and sequenced.
Fig. 1.
Alignment of the cloned LARC cDNAs and
ESTs. Below the LARC mRNA are the 5- and 3
-RACE cDNA
fragments with the gene-specific primers used at one of their ends and
the LARC cDNA cloned from a human liver
gt11 library. The
arrows indicate the positions, lengths, and orientations of
the LARC ESTs. GenBank accession numbers and the cDNA libraries
from which each EST was derived are shown. HepG2, hepatocyte
cell line.
[View Larger Version of this Image (14K GIF file)]
-RACE cDNA fragment (Fig. 1) and the HindIII-XbaI fragment (0.5 kb) of the human GAPDH
cDNA clone pKS321 (26), respectively.
and
3
oligomers described as above as a primer pair, PCR was performed on
the National Institute of General Medical Sciences (NIGMS) human
monochromosomal somatic cell hybrid mapping panel No. 2 version 2 (Coriell Cell Repositories, Camden, NJ) and on the GeneBridge 4 Radiation Hybrid panel (27) consisting of 91 hybrid DNAs (Research
Genetics, Huntsville, AL), for chromosomal mapping and radiation hybrid
mapping, respectively. The PCR conditions for amplification of the
LARC-specific sequence were 35 cycles of 94 °C, 30 s; 60 °C,
30 s; 72 °C, 1 min, in a 5-µl reaction buffer containing 0.25 mM of each of the dNTPs, 50 pmol of each of the primers,
and 0.125 units of TAKARA Taq (Takara). PCR products were
electrophoresed on 2% agarose gels. Using these primers, PCR is
expected to generate a 100-base pair fragment. The radiation hybrid
mapping data were sent to the Radiation Hybrid server at the Whitehead
Institute/MIT Center for Genome Research for analysis.
-noncoding region) and NotI (cDNA
synthesis primer sequence) from the 3
-RACE cDNA and subcloned into
the BamHI-NotI sites of the pVL1393 baculovirus
transfer vector (Invitrogen, San Diego, CA), downstream of the
polyhedrin gene promoter. The resulting recombinant plasmid, termed
pVL-LARC, was identified by restriction mapping.
XbaI-AP primer (5
-CGC
AGCTCCGGAATCATCCCAGTTGAGGAGGAGAAC) and the 3
AP(His)6-NotI primer
(5
-CGC
TCAGTGATGGTGATGGTGATGACCCGGGTGCGCGGCGTCGGT). The PCR product containing the
XbaI-SEAP-(His)6-NotI sequence was
digested with XbaI and NotI and inserted into the
XbaI-NotI sites of pDREF-Hyg (18). Prior to
subcloning of the LARC cDNA into this vector, the 5
-RACE LARC
cDNA was amplified by PCR using the 5
SalI-LARC primer
(5
-CGC
AAAACCATGTGCTGTACCAAG) and the 3
LARC-XbaI primer
(5
-CGC
CATGTTCTTGACTTTTTTACT). After digestion with
SalI and XbaI, the product was ligated into the SalI-XbaI sites of the
pDREF-SEAP(His)6 vector. The vector is a derivative of the
signal sequence trap vector pDREF-CD4ST (18), and the signal-deleted
CD4 coding sequence in the pDREF-CD4ST was replaced with
SEAP(His)6. This subcloning engineered a five-amino acid
linker (Ser-Arg-Ser-Ser-Gly) between the NCC-7 and the
SEAP(His)6.
-5,5
-tetramethylbenzidine. The reaction was stopped by adding 1 N H2SO4, and the absorbance was
read at 450 nm.
/LD78
, RANTES, and interleukin-8 (IL-8) were purchased from PeproTech (Rocky Hill, NJ). For saturation experiments, cells were
incubated for 1 h at 15 °C with increasing concentrations of
LARC-SEAP(His)6 in the presence or absence of 1 µM unlabeled LARC. After incubation, cells were washed
five times and lysed in 50 µl of 10 mM Tris-HCl (pH 8.0),
1% Triton X-100. Samples were heated at 65 °C for 10 min to
inactivate cellular phosphatase and then centrifuged. Bound AP activity
in 25 µl of the lysate was determined by the chemiluminescence assay
as described above. All samples were assayed in duplicate. The binding
data were analyzed by the LIGAND program.
Cloning of LARC cDNAs
iver and
ctivation-
egulated
hemokine (see
below)), was encoded by EST sequences, D17181, D31065[GenBank], D82589[GenBank], T27336[GenBank],
and T27433[GenBank] (GenBank accession numbers), but its gene was not present in
the YAC contig (data not shown). Sequence length, orientation, and the
cDNA library of each EST clone are shown in Fig.
1.
-RACE and one for 3
-RACE, were designed from one of the EST sequences, D31065[GenBank]. The RACE products were cloned and sequenced
(Fig. 2A). To exclude the base substitutions
that might be incorporated during PCR, three cDNA clones were
further isolated from a human liver cDNA library with the 5
-RACE
cDNA fragment as a probe. The three cDNA clones were identical.
The cDNA sequence was also identical to those of the RACE products in overlapping regions and in agreement with the ESTs. Fig. 1 schematically shows the alignment of the RACE products, the phage three
identical cDNA clones, and the five ESTs. The 5
-RACE product extended 7 nucleotides beyond the 5
-end of D31065[GenBank] that was the longest
of the five ESTs. The 5
-ends of the three phage cDNA clones were
identical with the 5
-RACE product. Interestingly, three ESTs, D82589[GenBank],
T27336[GenBank], and T27433[GenBank], had a 3-base pair deletion that causes deletion of
the NH2-terminal residue of the mature protein (see
"Discussion"). By searching the dbEST with the full-length LARC
sequence as a query, we identified one more EST, D17012[GenBank], which contains
the sequence of the 3
-noncoding region (Fig. 1).
Fig. 2.
/LD78
(34), LD78
(34), MIP-1
(25),
HCC-1/NCC-2 (36), RANTES (37), MCP-1 (38, 39), MCP-3 (40, 41), eotaxin
(15-17), MCP-4/NCC-1 (42), MCP-2 (43), I-309 (44), and TARC (18) using
the Clustal W program. Cysteines and the other conserved residues among
LARC and the other family members are indicated in boxed form. Filled circles indicate the residues conserved in
the other family members but not in LARC. An arrow shows the
cleavage site of the signal sequence of LARC. Hyphens are
inserted to maximize the homology. Percent homology compared with LARC
is indicated on the right. C, phylogenetic tree.
According to the alignment data in B, evolutionary distances
between the human CC chemokines were estimated using the GeneWorks
(IntelliGenetics, Mountain View, CA). The location of the branch points
is not to scale.
[View Larger Version of this Image (33K GIF file)]
-noncoding region does not contain the typical AATAAA polyadenylation
signal but contains two copies of the AATAAG sequence that is also used
as a polyadenylation signal in the human
-globin gene (32). The
3
-noncoding region also contains three copies of the mRNA
destabilization signal (ATTTA) that is often present in the
3
-noncoding regions of many cytokine mRNAs (33).
(28% identity)
(Fig. 2B). LARC contains the four cysteine residues characteristic of CC chemokines, the relative distances between these
cysteine residues being well conserved. In addition, three other amino
acid residues (Phe-65, Trp-81, and Val-82) that are highly conserved in
CC chemokines are conserved in LARC. However, two additional residues
that are conserved in other CC chemokines are replaced in LARC (Tyr
Phe-49, Thr
Ala-54; indicated by filled circles in Fig.
2B). One of these residues, Tyr, has been shown to be
important for monocyte chemotaxis (45, 46). A phylogenetic tree based
on the similarity scores highlights the low relatedness of LARC to
other CC chemokine family members (Fig. 2C).
Fig. 3.
Northern blot analysis of
poly(A)+ RNAs from human cell lines and tissues.
A, leukemic cell lines, Jurkat, K562, and U937, and Bowes
melanoma cell lines. Poly(A)+ RNAs were prepared from the
cells that had been stimulated with PMA for 6 h (+) or left
unstimulated () and transferred onto a nylon membrane. B,
normal tissues. The filters were obtained from Clontech. These filters
were hybridized with 32P-labeled LARC 3
-RACE fragment
(Fig. 1). After autoradiography, the filters were rehybridized with the
GAPDH cDNA probe. PBL, peripheral blood leukocyte.
[View Larger Version of this Image (43K GIF file)]
Fig. 4.
Chromosomal localization of the
LARC gene and identification of YAC clones containing the
LARC gene. A, PCR analysis of somatic cell
hybrid DNAs containing a single human chromosome. Hybrid DNAs from the
NIGMS human × rodent somatic cell mapping panel No. 2 version 2 were analyzed by PCR with LARC primers. Lanes are labeled
1-22, X, and Y to indicate the human chromosome retained in each hybrid. Human (H), Chinese hamster
(C), and mouse (M) genomic DNA controls were used
to identify bands generated by the parental cells. N,
negative control (PCR reaction without DNA). S, size marker.
B, localization of the LARC gene by radiation hybrid mapping. The GeneBridge 4 Radiation Hybrid panel consisting of
91 hybrid DNAs (Research Genetics, Whitehead Institute Center for
Genomic Research order) was used. The result of radiation hybrid
screening was 1000000000 0000000011 1110100100 0100000010 0010011001 1000000000 0111000001 0010000010 0000000000 0. 0 and 1 represent
negative and positive PCR assays, respectively. C, YAC
identification. Twenty YACs containing a STS D2S159 were analyzed by
PCR with LARC primers.
[View Larger Version of this Image (49K GIF file)]
Fig. 5.
Purification of recombinant LARC expressed in
the baculovirus system. A, reverse-phase high performance
liquid chromatogram of the pooled HiTrap-S fractions. The culture
supernatant of BTI-TN-5B1-4 cells infected with the LARC recombinant
virus was loaded on a HiTrap-S column and eluted with a salt gradient.
The pooled fractions were then loaded on a Cosmocil 5C4-300 column and
eluted with a gradient of acetonitrile. B, silver stain of
purified recombinant LARC. Proteins were separated on a 15%
SDS-polyacrylamide gel electrophoresis gel and detected by silver
staining. Positions of size markers (kDa) are shown on the
right.
[View Larger Version of this Image (14K GIF file)]
Fig. 6.
Chemotaxis assay. Homogeneous
recombinant LARC was tested for a dose-dependent
chemotactic activity (microchamber assay) on THP-1 cells
(A), freshly isolated peripheral blood monocytes (B), lymphocytes (C), and granulocytes
(D). Chemotactic responses are expressed as stimulation
indexes (significant chemotaxis corresponds to an index >2). For each
cell type, a positive control chemokine was included for comparison
(MCP-3 and IL-8).
[View Larger Version of this Image (22K GIF file)]
/LD78
, and
IL-8, had virtually no inhibitory effect on LARC-SEAP(His)6
binding to lymphocytes (Fig. 7D), indicating that the LARC
receptor is highly specific for LARC.
Fig. 7.
Binding characteristics of
LARC-SEAP(His)6 to lymphocytes. A, binding of
LARC-SEAP(His)6 to lymphocytes (5 × 105
cells) with increasing concentrations of LARC-SEAP(His)6.
B, Scatchard analysis of the binding data in A.
The calculated Kd is 0.4 nM.
C, displacement of LARC-SEAP(His)6 with
unlabeled LARC. Lymphocytes (2 × 105 cells) were
incubated with 1 nM LARC-SEAP(His)6 in the
presence of increasing concentrations of unlabeled LARC. The
IC50 of LARC-SEAP binding is 3.2 nM of LARC.
D, displacement of LARC-SEAP(His)6 with other
chemokines. Lymphocytes (2 × 105 cells) were
incubated with 1 nM LARC-SEAP(His)6 in the
absence or presence of 200 nM unlabeled LARC, TARC, MCP-1,
RANTES, MIP-1/LD78
, or IL-8. The results represent three
independent experiments and are shown as mean ± standard
error.
[View Larger Version of this Image (19K GIF file)]
*
This work was supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Inamori 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.
**
Supported by the General Savings and Retirement Fund (ASLK), the
Belgian Charcot Foundation, the Belgian Cancer Association, and the
National Fund for Scientific Research (NFWO), Belgium.
¶¶
Present address: Kitano Hospital, Tazuke Kofukai
Medical Research Institute, 13-3 Kamiyama, Kita-ku, Osaka 530, Japan.
||
To whom correspondence should be addressed: Dept. of
Biochemistry, Kumamoto University Medical School, Honjo 2-2-1, Kumamoto 860, Japan. Tel.: 81-96-373-5063; Fax:
81-96-372-6140; E-mail: nomiyama{at}gpo.kumamoto-u.ac.jp.
1
The abbreviations used are: ESTs, expressed
sequence tags; YAC, yeast artificial chromosome; dbEST, expressed
sequence tag data base; FCS, fetal calf serum; PMA, phorbol
12-myristate 13-acetate; RACE, rapid amplification of cDNA ends;
PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; STS, sequence tagged site; MES,
2-morpholinoethanesulfonic acid; kb. kilobase pair(s); SEAP, secreted
alkaline phosphatase; PLAP, placental alkaline phosphatase; ; PBS,
phosphate-buffered saline; AP, alkaline phosphatase; IL-8, interleukin
8.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.