(Received for publication, November 18, 1996, and in revised form, December 27, 1996)
From the Bristol-Myers Squibb, Pharmaceutical Research Institute, Seattle, Washington 98121 and the § Howard Hughes Medical Institute and Department of Genetics, Stanford University Medical School, Stanford, California 94305
CD5 and CD6, two type I cell surface antigens
predominantly expressed by T cells and a subset of B cells, have been
shown to function as accessory molecules capable of modulating T cell activation. Here we report the cloning of a cDNA encoding Sp, a
secreted protein that is highly homologous to CD5 and CD6. Sp
has
the same domain organization as the extracellular region of CD5 and CD6
and is composed of three SRCR (scavenger
receptor cysteine rich) domains.
Chromosomal mapping by fluorescence in situ hybridization
and radiation hybrid panel analysis indicated that the gene encoding
Sp
is located on the long arm of human chromosome 1 at q21-q23
within contig WC1.17. RNA transcripts encoding Sp
were found in
human bone marrow, spleen, lymph node, thymus, and fetal liver but not
in non-lymphoid tissues. Cell binding studies with an Sp
immunoglobulin (Sp
-mIg) fusion protein indicated that Sp
is
capable of binding to peripheral monocytes but not to T or B cells.
Sp
-mIg was also found to bind to the monocyte precursor cell lines
K-562 and weakly to THP-1 but not to U937. Sp
-mIg also bound to the
B cell line Raji and weakly to the T cell line HUT-78. These findings
indicate that Sp
, a novel secreted protein produced in lymphoid
tissues, may regulate monocyte activation, function, and/or
survival.
Leukocyte function is regulated by a discrete number of cell
surface and secreted antigens that govern leukocyte activation, proliferation, survival, cell adhesion and migration, and effector function. Among the proteins that have been shown to regulate leukocyte
function are members of the SRCR1 family.
This family of proteins can be divided into two groups based upon the
number of cysteine residues per SRCR domain, intron-exon organization,
and domain organization (1). Group B includes the cell surface proteins
CD5 (2) and CD6 (3), which are predominantly expressed by thymocytes,
mature T cells, and a subset of B cells, WC1 (4, 5), which is expressed
by T cells in cattle, and M130 (6), which is expressed by
activated monocytes. Of these, only CD5 and CD6 have been studied
extensively. Monoclonal antibody (mAb) cross-linking studies suggest
that both CD5 and CD6 can function as accessory molecules capable of
modulating T cell activation (7, 8). The role of CD5 and CD6 in the regulation of T cell function is further supported by the finding that
following T cell activation, Tyr residues in the cytoplasmic domain of
these two proteins are transiently phosphorylated. This provides a
molecular mechanism whereby the cytoplasmic domains of both CD5 and CD6
can interact with intracellular SH2 containing proteins involved in
signal transduction (9). Furthermore, phenotypic analysis of a
CD5-deficient murine strain showed that its T cells are
hyper-responsive to stimulation (10, 11), suggesting that CD5
expression is required for the normal regulation of T cell receptor
(TCR)-mediated T cell activation.
CD5 and CD6 are structurally the most closely related members of the group B SRCR family of proteins (1). They are both type I membrane proteins whose extracellular region is composed of three SRCR-like domains, each containing eight cysteine residues that are thought to form intrachain disulfide bonds. The extracellular domains of CD5 and CD6 are anchored to the cell membrane via a hydrophobic transmembrane domain and a long cytoplasmic domain. It has been reported that CD5 binds to the B cell antigen CD72 (12) and to CD5L (13), an antigen which is transiently expressed by activated B cells and has yet to be fully characterized. CD6 has been shown to bind to the leukocyte activation antigen ALCAM (activated leukocyte cell adhesion molecule). Unlike CD5 and CD6, which are closely related, CD72 and ALCAM are not homologous. CD72 is a type II membrane protein that is homologous to the C-type lectins; however, a carbohydrate binding activity for CD72 has not been reported. ALCAM is a type I membrane protein whose extracellular region is composed of five Ig-like domains (14). The regions of CD5 and CD72 involved in their interaction have not been identified. Studies with truncated forms of both CD6 and ALCAM have shown that the interaction between these two proteins is primarily mediated by the membrane proximal SRCR domain of CD6 and the amino-terminal Ig-like domain of ALCAM (15, 16).
Here we report the cloning, chromosomal mapping, and cell binding
properties of Sp, a novel member of the SRCR family of proteins.
Sp
is expressed in lymphoid tissues and has the same domain
organization as the extracellular regions of both CD5 and CD6.
Binding studies with an Sp
Ig fusion protein were carried out to
identify cells expressing a putative receptor for Sp
.
Cloning of Sp
An expressed sequence tag (EST) data base screen for potential
new SRCR domain-containing genes revealed a novel gene in EST clone
number 201340. The partial (Sp) clone from a fetal liver-spleen was
purchased from Research Genetics and used to screen a human spleen
library (Clontech HL5011a) by plaque hybridization for full-length
cDNAs. Approximately 1 × 106 clones were plated
onto 20 150-mm plates and transferred to Hybond N+ nylon membranes
(Amersham Life Science, Inc., rpn132b) as per manufacturer
instructions. Membranes were UV cross-linked and hybridized by the
method of Church (17). The hybridization probe was a radiolabeled
EcoRI fragment digested from the EST clone 201340. The
EcoRI fragment contained base pairs 1-1594 and was radiolabeled with [32P]dCTP (Amersham Life Science, Inc.)
using a random labeling kit (Boehringer Mannheim). Membranes were
washed at 60 °C using high stringency wash buffer and exposed to
Kodak x-ray film (X-Omat AR). A subset of positive plaques were then
replated and rescreened. After three rounds of screening, ten
individual clones were obtained, of which two were full-length. Both of
these clones were sequenced in both directions using the dideoxy
method.
Northern Blot
One cell line and two tissue Northern blots were purchased from
Clontech (Nos. 7757-1, 7766-1, and 7754-1, respectively) and hybridized in 50% formamide at 42 °C according to manufacturer instructions. Radiolabeled Northern blot probes were prepared as
outlined above. mRNA normalization probes were either GAPDH or
-actin. Positive blots were washed under high stringency conditions. Blots were exposed to Kodak x-ray film (X-Omat AR).
Chromosomal Mapping
Somatic Cell Hybrids and PCR Amplifications of SpHuman
Sp was localized to a human chromosome using a panel of 17 human-Chinese hamster hybrid cell lines derived from several independent fusion experiments (18). PCR primers used to amplify the
human Sp
gene sequence were derived from the 3
untranslated region,
and they are 5
-GAGTCTGAACACTGGGCTTATG (forward at nucleotide 1231-1252) and 5
-GTAATGGTCTGCACATCTGACC (reverse primer at nucleotide 1431-1452). The PCR conditions were 94 °C for 3 min, 35 cycles of
94 °C for 30 s, 55 °C for 40 s, and 72 °C for 1 min
followed by 72 °C for 7 min.
Two human radiation hybrid (RH) mapping panels, GeneBridge 4 (Whitehead/MIT Genome Center) and Stanford G3 (Stanford Human Genome
Center), were used to confirm and further define the localization of
the Sp gene. Typing was carried out using the primers and PCR
conditions described above.2
The chromosomal location
of the human Sp gene was independently determined by fluorescence
chromosomal in situ hybridization (FISH) (22). Briefly, a
genomic DNA clone containing a 2.4-kbp insert of the human Sp
genomic sequence in a TA cloning vector was labeled with biotin-16-dUTP
by nick-translation using commercial reagents (Boehringer Mannheim).
Labeled probe was hybridized at a concentration of 300 ng/µl/slide to
pretreated and denatured human lymphocyte metaphase chromosomes.
Hybridizations were performed in the presence of salmon sperm DNA and
human genomic DNA.
After hybridization at 37 °C overnight, the slides were washed in 50% formamide in 2 × SSC at 42 °C. To detect and amplify specific hybridization signals, slides were reacted with avidin-FITC (Vector Laboratories), washed, and treated with biotinylated goat anti-avidin D antibody (Vector Laboratories) followed by another round of incubation with avidin-FITC. Metaphase chromosomes were analyzed under an Axiophot (Carl Zeiss, Inc.) epifluorescence microscope. Specific hybridization signals were counted only when the fluorescence staining was observed on both chromatids of a chromosome. Digital images were generated using a cooled charge-coupled device camera (Photometrics PM512)/Macintosh computer system, with software supplied by Tim Rand (Yale University). Photographs were produced from PICT files.
Fusion Protein Constructs
DNA corresponding to the translated region of Sp was obtained
by PCR using full-length Sp
cDNA as template. Primers were designed with restriction sites enabling Sp
C-terminal ligation to
the hinge, CH2, and CH3 domains of murine IgG2a (see Fig.
4). All constructs were sequenced to verify correct sequence and
correct reading frames. Sp
-mIg (in the CDM8 expression vector) was
transiently expressed in COS cells (23). The soluble Sp
-mIg was
purified from the COS cell supernatant by protein A column
chromatography. Following protein A binding, the column was washed
extensively with PBS (pH 7.0) and eluted with 4.0 M
imidazole (pH 8.0) containing 1 mM each MgCl2
and CaCl2. Proteins were dialyzed extensively with PBS.
Cell Culture
Human cell lines were grown to 0.5-0.9 × 106 cells/ml in Iscove's modified Dulbecco's medium (Life Technologies, Inc.) containing 10% fetal bovine serum. Human peripheral blood T, B, and monocytes cells were separated by counterflow centrifugal elutriation.
Flow Cytometry
Approximately 5 × 105 cells were incubated on
ice for 1 h in 100 µl of stain buffer (PBS containing 2% bovine
serum albumin fraction V, 0.05% sodium azide, 1 mM each
MgCl2 and CaCl2) containing 20 µg/ml
Sp-mIg fusion protein and 200 µg/ml human IgG
(Sigma I-8640). Cells were then washed with stain
buffer, centrifuged, and aspirated. Following a second wash, cells were
then incubated on ice for 1 h in 100 µl of stain buffer
containing 1:100 diluted FITC-labeled rabbit anti-mouse
IgG2a antibody (Zymed Laboratories, Inc. 61-0212). Cells
were then washed twice and resuspended in 0.5 ml of stain buffer.
Samples were run on a Becton Dickinson Facscan. Prior to running
samples, propidium iodide was added to a final concentration of 1 µg/ml. Dead cells were identified as propidium iodide-positive and
were gated out and not used in the analysis. Mouse antibodies specific
for CD3 (64.1 generously donated by Jeff Ledbetter, Ph.D., T cell,
Bristol-Myers Squibb), CD19 (B cell, IOB4a Amak 1313), and CD14
(monocytes, MY4 Coulter 6602622) were used to verify elutriated cells.
Second step staining for these antibodies was an FITC-labeled goat
anti-mouse IgG (Biosource 4408).
We have taken two approaches to isolating novel members
of the SRCR family of proteins. The first involves a low stringency DNA
hybridization technique, and the other involves a screening of the DNA
data bases. This latter approach resulted in the identification of a
cDNA fragment from the human EST data base that showed extensive sequence homology with members of the SRCR group B proteins, including CD5, CD6, M130, and WC1. The EST sequence (from fetal liver-spleen) was
used as a probe to screen a cDNA library prepared from mRNA isolated from a human spleen. This resulted in the isolation of ten
cDNA clones. The two longest clones, 1804 and 2152 bp,
respectively, were sequenced in both orientations and found to contain
a long open reading frame that encoded a 347-amino acid polypeptide, which had the features of a secreted protein (Fig.
1A). This protein was named Sp. Sp
contains 19 hydrophobic amino acids at its amino-terminal that act as a
secretory signal sequence and are removed from the mature protein as
determined by N-terminal sequence of the Sp
immunoglobulin fusion
protein produced by COS cells. This secretory signal sequence is
followed by three cysteine-rich domains, each approximately 100 amino
acids in length. These domains are significantly homologous to the
cysteine-rich domains found in the SRCR group B family of proteins
(Fig. 1B) (1). The third SRCR domain of Sp
is followed by
an in-frame stop codon. The predicted amino acid sequence of Sp
contained no putative N-linked glycosylation sites. The two
clones differ in the length of their 3
-untranslated regions, where one
clone is 348 bp longer. The shorter clone has a poly(A) sequence
starting 18 bases downstream from a consensus polyadenylation sequence.
The longer clone has two polyadenylation consensus sequences; the first
one is identical to the one found in the shorter clone, and the second
is located 351 bp downstream from the first site. The longer clone also
contains three adenylate/uridylate-rich elements (AREs) in the
3
-untranslated sequence located between the two polyadenylation
sites.
Amoung the SRCR group B members, the SRCR domain organization of
Sp most closely resembles CD5 and CD6 (Fig. 1C).
Genomic
DNAs from a panel of 17 human-Chinese hamster hybrid cell lines were
analyzed by PCR using primers that specifically amplified human Sp
sequence. The expected 222-bp PCR products containing the
3
-untranslated region sequence were obtained from human control DNA
and from hybrid cell lines that had retained human chromosome 1. As
shown in Table I, except for chromosome 1, all other
human chromosomes were excluded by this panel. These results indicated
that the human Sp
gene is located on chromosome 1. Fluorescence
in situ hybridization confirmed the Sp
assignment to
chromosome 1 and refined the physical map position. Based on the
localization of the signal on R-banded chromosomes in 22 metaphase spreads, the human Sp
gene was assigned to human chromosome bands q21-q23 (Fig. 2).
|
To confirm this assignment and to map the Sp locus more precisely,
two human RH mapping panels were typed by PCR amplification with the
Sp
specific primers. In the Stanford G3 mapping panel, 9 of 83 RH
cell lines were positive for the human-specific Sp
gene signal. By
maximum likelihood analysis, Sp
was placed 45.8 centiRays10000 (cR) from the STS marker D1S3249. In the
GeneBridge 4 mapping panel, 30 of 93 RH cell lines were positive, and
Sp
was placed 3.0 cR3000 and 3.1 cR3000 from
the chromosome 1 markers WI-8330 and CHLC.GATA43A04,
respectively. The order of markers in this region from centromere to
telomere is D1S305-WI-8330-Sp
-CHLC.GATA43A04-D1S2635. D1S305,
WI-8330, CHLC. GATA43A04, and D1S2635 are known markers in the WC1.17
contig (Whitehead Institute/MIT Center for Genome Research), while
D1S3249 and D1S2635 are clustered as chromosome 1 bin 69 in the
Stanford Human Genome Center RH map. A more distal marker D1S196, which
is in Stanford Human Genome Center chromosome 1 bin 75 and WC1.17
contig, was previously mapped to the q22-q23 region (24). These results
are consistent with our FISH mapping data that placed Sp
at q21-q23.
The insertion of Sp
into the linkage map will enable the evaluation
of this locus as a candidate for genetic disorders.
RNA blot analysis using
a Sp cDNA fragment as a probe indicated that mRNA encoding
Sp
is expressed in the spleen, lymph nodes, thymus, bone marrow, and
fetal liver but not in peripheral blood leukocytes (PBL) nor
appendix (Fig. 3). Hybridizing bands to Sp
were also
not detected in prostate, testis, uterus, small intestine, and colon
(separate blot, data not shown). In all cases, tissues expressing
mRNA transcripts encoding Sp
expressed three hybridizing
transcripts. Three bands in the spleen (Fig. 3) are seen with shorter
film exposure. These transcripts are ~2.4, 2.1, and 1.8 kbp in
length. The 1.8- and 2.1-kbp transcripts correspond in length to the
two longest cDNAs isolated from the spleen cDNA library.
Presently there is no information as to the nature of these
transcripts; however, the finding that two of the cDNAs which we
isolated have sizes that are consistent with those seen on the RNA blot
suggest that they may all encode Sp
but differ from one another in
the length of their untranslated regions. It should be noted that we
cannot exclude the possibility that one or more of these transcripts
may encode closely related proteins.
In an effort to determine which cells might produce Sp, we have
analyzed several cell lines by Northern blot. The RNA message for Sp
was not detected in the following cell lines: HL60, K562, Raji, Molt4,
A549, SW480, GA361, HeLa S3, and peripheral blood leukocytes (data not
shown).
Previously, we had successfully used an Ig fusion
approach to identify cells expressing a CD6 ligand (25). These studies eventually led to the isolation of a cDNA encoding a CD6 ligand named ALCAM (14). The successful application of this technique in the
isolation of a CD6 ligand and the characterization of the CD6-ALCAM
interaction led us to use the same approach to identify cells that
express Sp receptors. We prepared a full-length Sp
murine
IgG2a (Sp
-mIg) fusion protein by transient expression in
COS cells (Fig. 4).
We began a systematic examination of the ability of Sp-mIg to
bind to human cell lines using flow cytometry. We observed that the
myeloid cell line K-562 bound to Sp
-mIg but not to a control protein
(WC1-mIg) containing the amino-terminal three SRCR domains of bovine
WC1 fused to the same constant domain of murine IgG2a
(Fig. 5A, panel A). Binding of
Sp
to the K-562 cells was concentration-dependent and
saturable (Fig. 5B). Sp
-mIg also displayed weaker binding
to the myeloid cell line THP1 (Fig. 5A, panel B)
but not to U-937 cells (Fig. 5A, panel C).
Binding of Sp
-mIg was also observed on the lymphoma B cell line Raji
(Fig. 6, panel A) and also the T cell line
Hut78 (Fig. 6, panel C). Binding was not seen with the
control protein on these two cell lines (Fig. 6, panel B and
D).
These observations led us to examine if the Sp-mIg fusion protein
could bind peripheral blood mononuclear cells. As shown in
Fig. 7, Sp
-mIg (panels A and
D), but not WC1-mIg (panels B and E),
bound to peripheral blood monocytes. Binding of Sp
-mIg was not seen
on elutriated peripheral blood T cells (Fig. 8,
panels A and D) nor on elutriated B cells (data
not shown). The binding of Sp
-mIg to elutriated monocytes from
different donors could always be detected but showed some degree of
variability (Fig. 7, panels A and D).
We have been interested in studying the structure and
function of CD5 and CD6 and their regulatory role in the immune system. A large body of in vitro data suggests that these proteins
play an important role in regulating T cell activation and, in the case
of CD6, T cell development. The isolation and functional characterization of novel proteins that are closely related to CD5 and
CD6 might provide further insights on the function and structure of
this class of proteins. We screened the human EST data base for
cDNA fragments that encoded polypeptides, which were homologous to
CD5 and CD6, and identified a cDNA fragment encoding Sp.
Analysis of full-length cDNA clones encoding Sp
suggests that
Sp
is a secreted protein that has the same domain organization as
the extracellular region of CD5 and CD6. However, a detailed comparison
of the amino acid sequence of SRCR domains of Sp
with all members of
the SRCR protein family revealed a closer homology to WC1 and M130.
This suggests that Sp
may be more closely related to WC1 than CD5 or
CD6. Further evidence that points to a more distant evolutionary
relationship between Sp
and CD5 or CD6 than that between CD5 and CD6
comes from the finding that the genes encoding CD5 and CD6 are found in
close proximity on chromosome 11 (26-29), whereas the gene encoding
Sp
is located on chromosome 1. Presently there is no information of
the genomic localization of the human equivalent of WC1 or M130.
The subgroup of SRCR family members, which contains CD5, CD6, WC1 and
M130 (Group B), can be distinguished from other members of the family
based on the number of cysteine residues contained within the SRCR
domains and the observation that the extracellular domains of each of
these proteins are composed exclusively of SRCR domains. More recently,
analysis of the genomic organization of the genes encoding some of the
members of this subfamily has indicated that a third distinguishing
feature of this group of proteins is that each of its SRCR domains is
encoded by a separate exon (27, 30, 31). This is in contrast to the
type I macrophage scavenger receptor and related proteins (Group A).
The SRCR domains of group A proteins have fewer Cys residues (six
instead of eight), and each SRCR domain is encoded by two exons.
Preliminary data on the genomic organization of Sp indicates that
the second SRCR domain is encoded by a single
exon.3 Based on these criteria, we propose
that Sp
be considered a member of the SRCR Group B family of
proteins.
RNA blot analysis indicates that transcripts encoding Sp are
exclusively expressed in lymphoid tissues. However, it appears that
leukocytes do not express this protein. This finding indicates that
Sp
may be produced by specialized epithelial and or endothelial cells in lymphoid tissues. The observation that Sp
is expressed in
bone marrow, thymus, and fetal liver, as well as in the spleen and
lymph nodes, implicates this protein in processes responsible for both
the development and maintenance of the lymphoid compartment. Studies
are currently underway to identify the cells that make this protein and
factors that are involved in regulating its expression. The Northern
blot probed with Sp
showed three bands. Based on our analysis of two
different cDNAs encoding Sp
, it appears that at least two of
these transcripts correspond to mRNAs encoding Sp
and differ in
the length of their 3
-untranslated regions. We also observed a
significant difference in the 3
-untranslated region of these Sp
mRNAs. We found that the longer clone contained three consensus ARE
elements (AUUUA). ARE elements are located within the 3
-untranslated
region of mRNAs and have been found to be the most common
determinant of RNA stability (32, 33). Messenger RNAs encoding
cytokines and transcription factors, among others, contain these
elements, which provide an additional mechanism for the regulation of
protein expression by directing the stability and, therefore, half-life
of the mRNA encoding the protein. The finding that at least one of
the mRNAs encoding Sp
contains ARE motifs suggests that the
expression of this protein might be tightly regulated.
Preliminary studies designed to identify cells that bind Sp and are
the target of its activity revealed that some resting myeloid cell
lines, as well as peripheral blood monocytes, are capable of binding
Sp
. Sp
-mIg was also found to bind to the B cell line Raji and
also the T cell line Hut78. These studies were carried out using an
Sp
immunoglobulin fusion protein, and thus, the possibility existed
that the interaction between this protein and the myeloid cell lines
and monocytes, which are known to express high levels of Fc receptors,
was mediated via the Ig portion of the molecule rather than the Sp
moiety. This is unlikely for the following reasons. 1) Two Ig fusion
control proteins, WC1-mIg (SRCR Group B member) and human ALCAM-mIg,
showed no binding; and 2) the interaction between the Sp
-mIg and
myeloid cell lines and peripheral blood monocytes was detected in the
presence of a vast excess of human IgG (up to 2 mg/ml) present in the
binding studies.
The isolation of cDNAs encoding Sp, the preparation of Sp
immunoglobulin fusion proteins, and the identification of cells that
express putative receptors will provide the basis for future studies on
the structure and function of this new member of the SRCR family of
proteins. The finding that this protein is expressed in lymphoid organs
involved in the development of the lymphoid compartment as well as in
immune surveillance, in conjunction with the observation that
peripheral blood monocytes are capable of binding Sp
, suggests that
this protein may play an important role in regulating the immune
system.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U82812[GenBank].
We thank Dr. H. Clevers for providing us with
cDNAs encoding bovine WC1 and Debby Baxter for help in the
preparation of this manuscript. We also thank Patty Davis
(Bristol-Myers Squibb PRI) for providing us with some of the elutriated
peripheral blood lymphocytes, Alison Wallace (Bristol-Myers Squibb PRI)
for N-terminal sequencing of the Sp fusion protein, and the DNA
sequencing department (Bristol-Myers Squibb Seattle) for help in
sequencing the Sp
clones.