(Received for publication, June 5, 1995)
From the
Binding studies with a CD6 immunoglobulin fusion protein (CD6
Rg) resulted in the identification and cloning of a CD6 ligand. This
ligand was found to be a member of the immunoglobulin supergene family
and was named ALCAM (activated leukocyte cell adhesion molecule). Cell
adhesion assays showed that CD6-ALCAM interactions mediate
thymocyte-thymic epithelium cell binding. ALCAM is also expressed by
activated leukocytes and neurons and may be involved in interactions
between T cells and activated leukocytes and between cells of the
immune and nervous systems, respectively. Herein we describe the
preparation of domain-specific murine CD6 Rg fusion proteins and show
that the membrane-proximal SRCR (scavenger receptor cysteine-rich)
domain of CD6 contains the ALCAM binding site. We also show that mAbs
which bind to this domain preferentially block CD6-ALCAM binding. These
results demonstrate that the membrane-proximal SRCR domain of CD6 is
necessary for CD6 binding to ALCAM and provide the first direct
evidence for the interaction of an SRCR domain with a ligand.
CD6 is a type I cell surface protein and a member of the SRCR ( Antibody
cross-linking studies have shown that CD6 can function as an accessory
molecule in T cell activation. For example, the anti-CD6 mAb 2H1 can
induce T cell activation in conjunction with phorbol 12-myristate
13-acetate or with the anti-CD2 mAb T11 Studies using CD6 Rg, an immunoglobulin
fusion protein consisting of the extracellular domains of CD6 fused to
human IgG Herein we present the results of experiments
designed to determine whether binding to ALCAM can be localized to a
single domain of CD6. Two independent approaches were used: first, a
series of CD6 Rg fusion proteins containing different CD6 extracellular
domains, alone or in combinations, were used in cell and protein
binding assays; second, mAbs directed against different CD6
extracellular domains were used to block CD6 ALCAM binding and provided
independent evidence for which CD6 domain(s) is responsible for ALCAM
binding. The results obtained from both of these experimental
approaches indicated that the membrane-proximal SRCR domain of CD6
contains the ALCAM binding site.
HPB-ALL cells (5
Figure 1:
Murine CD6 Rgs. A, line drawing representations of the chimeric genes encoding
the mCD6 immunoglobulin fusion proteins used in this study. The SRCR
domains in the extracellular domain of mCD6 are represented by boxes which are labeled D1, D2, and D3. The box which represents the membrane-proximal
33-amino-acid stalk domain is labeled S. The human IgG
We had previously shown that hCD6 Rg, an immunoglobulin fusion
protein containing the extracellular domain of human CD6, was capable
of binding to murine cells lines(11) . Here we show that mCD6
Rg is capable of binding ALCAM expressed on the human T cell line
HPB-ALL (Fig. 2A) or to ALCAM Rg, an immunoglobulin
fusion protein which contains the extracellular domain of human ALCAM (Fig. 2B)(13) . The ability of mCD6 to bind to
human ALCAM allowed us to use the mCD6 Rg fusion proteins described
above in two different binding assays to identify the mCD6
extracellular subdomain(s) responsible for ALCAM binding. In the first
assay, the binding of mCD6 Rg to HPB-ALL cells was compared with that
of the different mCD6 Rg domain fusion proteins by flow cytometry. As
shown in Fig. 2A, the binding of mCD6D2-S Rg and
mCD6D3-S Rg to the HPB-ALL cells was comparable to that of mCD6 Rg. On
the other hand, mCD6D1 Rg, mCD6D2 Rg, and mCD6D1-2 Rg as well as
our control fusion protein mCD7 Rg failed to bind to the HBP-ALL cells.
Similar results were obtained when the ability of the mCD6 Rg fusion
proteins to bind to purified ALCAM Rg was examined by ELISA. As shown
in Fig. 2B, fusion proteins containing CD6 SRCR-D3 and
S domains bound ALCAM Rg. In addition, similar results were obtained
when the ability of the mCD6 Rg fusion proteins to bind to COS cells
transfected with a plasmid containing a cDNA encoding ALCAM was
examined (data not shown). These results suggest that the ALCAM binding
site in CD6 is contained in the membrane-proximal region of the protein
which contains the SRCR-D3 and S domains of CD6.
Figure 2:
Binding of the mCD6 Rgs to HPB-ALL cells
or ALCAM Rg. A, flow cytometry profiles of the binding of the
mCD6 Rgs (mCD6 Rg, mCD6D1-2 Rg, mCD6D2-S Rg, mCD6D3-S Rg, mCD6D1 Rg,
mCD6D2 Rg, mCD6D1-3 Rg, mCD6D2-3 Rg, and mCD6D3 Rg (dark
profiles) and the control fusion protein, CD7 Rg (empty
profile), to HPB-ALL cells. Mean channel fluorescence values for
each mCD6 Rg are noted on each histogram. The mean channel fluorescence
value for CD7 Rg was 2.4. These binding profiles are a representative
set selected from a group of three independent binding assays. B, binding of increasing concentrations of the mCD6 Rg fusion
proteins listed in A of this figure legend and the control CD7
Rg fusion protein to ALCAM Rg immobilized in the wells of a 96-well
plastic dish. Standard deviations were calculated on the basis of three
binding measurements.
To further examine
the contribution of the CD6 S domain to CD6-ALCAM binding, we prepared
three additional mCD6 Rg fusion proteins: mCD6D1-3 Rg, mCD6D2-3 Rg, and
mCD6D3 Rg (Fig. 1). HPB-ALL (FACS) and ALCAM Rg (ELISA) binding
studies with this set of fusion proteins showed that the
membrane-proximal SRCR domain of CD6 alone can bind ALCAM (Fig. 2). Taken together these data suggest that the CD6 SRCR-D3
domain is necessary for CD6-ALCAM binding.
Figure 3:
Blocking of the CD6-ALCAM interaction by
domain-specific mAbs. Flow cytometry profiles showing the binding of
ALCAM Rg to murine thymocytes which had been pretreated with hybridoma
supernatants containing the rat anti-mCD6 mAbs M6-1A.1, M6-3A.1, and
M6-3B.1 or an isotype-matched rat anti-human gp39 mAb (dark
profile) compared to binding at the negative control, CD7Rg (empty profiles). These profiles are a representative set
derived from a group of two independent blocking
assays.
Using immunoglobulin fusion proteins containing single or
multiple extracellular domains of mCD6, we have mapped the ALCAM
binding site to the membrane-proximal SRCR domain of CD6. Fusion
proteins containing this domain are capable of binding ALCAM, and mAbs
directed against this CD6 domain were found to block CD6-ALCAM binding.
Taken together these results demonstrate that mCD6 SRCR-D3 is necessary
for CD6 binding to ALCAM. However, these results do not rule out the
possible contribution of other CD6 domains in CD6-ALCAM binding. Indeed
we observed that some of the truncated fusion proteins containing
SRCR-D3, such as mCD6D3 Rg and mCD6D1-3 Rg, bound ALCAM Rg less
efficiently than the others (Fig. 2B. This might be the
result of a loss of residues in other CD6 domains which directly
contribute to ALCAM binding and/or due to structural perturbations of
SRCR-D3 caused by the truncation of adjacent domains. In this regard,
we also observed that mCD63-S Rg bound ALCAM as well as mCD6 Rg. This
suggests that the S domain may play a more important role in ALCAM
binding than SRCR-D1 or -D2 by either contributing directly to ALCAM
binding, playing a structural role in facilitating CD6-ALCAM
interactions, and/or by providing an appropriate spacer between the
SRCR domain and the Ig portion of the fusion protein which allows
efficient binding. These findings leave open the possibility that the
other extracellular domains of mCD6, mCD6 SRCR-D1, and -D2, have other
ligands. Indirect evidence for additional CD6 ligands is provided by
immunoprecipitation studies of CD6 binding proteins from HBL-100 cells (11) . These studies showed that CD6 Rg bound to three
proteins, only one of which had a molecular weight consistent with the
predicted molecular weight of ALCAM(11, 13) . Also,
anti-CD6 mAbs which bind to different CD6 epitopes have different
functional characteristics(4, 6, 20) . This
has led to the suggestion that CD6 has multiple ligands which might
differentially regulate CD6 function(20) . There are a number
of examples of leukocyte receptors which have multiple ligands. Perhaps
one of the best studied is CD2 which has been found to interact with
CD48, CD58, and CD59(21, 22) . The CD6 fusion proteins
described here may be useful for the identification of the biological
function of the other CD6 subdomains. Binding studies with mCD6D1-2 Rg
on different cell types are ongoing to explore the possibility that the
SRCR-D1 and -D2 extracellular subdomains of CD6 interact with other
ligands. Ligands for other members of the SRCR family of proteins
have been reported. In particular, CD5 has been reported to bind
CD72(23, 24) , the type I macrophage scavenger
receptor binds to acetylated low density lipoproteins(25) , 90K
binds to MAC-2(26) , and cyclophilin-C (27) ,
complement factor I, binds to the activated complement proteins C3b and
C4b(28) , the SPERACT receptor binds to the peptide SPERACT (29) , and MARCO binds Escherichia coli and other
bacteria (30) . In some cases, these receptor-ligand
interactions do not involve the SRCR domain(s). This is exemplified by
the interaction between the type I macrophage scavenger receptor and
acetylated low density lipoprotein. In this case, receptor-ligand
binding is not mediated through the SRCR domain(25) . In other
cases, indirect evidence for the role of the SRCR domain in
receptor-ligand binding has been reported. As an example, in studies of
the E. coli binding function of MARCO, polyclonal antiserum
raised against sequences found in the SRCR domain of MARCO was found to
inhibit binding to E. coli(30) . Presently it is not
known if this effect is due to direct blocking of the ligand binding
site or steric hindrance. However, in most cases, the role of the SRCR
domain(s) in receptor-ligand interactions has not been examined. Our
finding that CD6 SRCR-D3 binds ALCAM provides the first direct
demonstration that an SRCR domain can participate in binding
interactions. Future work defining the role of other SRCR domains in
receptor-ligand binding will be required to determine the range of
molecular interactions mediated by this well conserved protein domain.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)family of proteins(1) . The extracellular domain
of the mature CD6 protein is composed of three SRCR domains followed by
a short 33-amino-acid stalk region. This extracellular domain is
anchored to the cell membrane via a short hydrophobic domain followed
by a cytoplasmic domain of variable length (2) . Cloning of
cDNAs encoding the murine homologue of CD6 (mCD6) and comparison of its
predicted amino sequence with that of the human clone revealed the
possible existence of multiple CD6 isoforms which result from the
variable splicing of exons encoding the cytoplasmic domain (3) . Within the SRCR family of proteins, CD6 is most closely
related to CD5(1) . These two proteins share a similar
extracellular domain organization and are both expressed by thymocytes,
T cells, B cell CLLs, and a subset of normal B cells.
(4) . On T
cells, ligation of CD6 with the anti-CD6 mAb G3-5 causes an increase in
CD3-induced cytoplasmic calcium concentration (5) while CD6
cross-linking with the anti-CD6 mAb T12 has been shown to enhance the
activation mediated by suboptimal doses of anti-CD3 mAbs(6) .
Additional evidence for the role of CD6 in T cell activation comes from
studies showing that CD6 becomes hyperphosphorylated on Ser and Thr
residues (7, 8, 9) and phosphorylated on Tyr
residues (10) following T cell activation. This last
observation suggests that the cytoplasmic domain of CD6 may provide a
membrane anchor for interaction with SH2-containing proteins involved
in intracellular signaling.
constant domains, resulted in the identification
of cells which express a CD6 ligand(11) . The identification of
these cells allowed the characterization (11, 12) and
cloning (13) of a human CD6 ligand. This CD6 ligand, which is
recognized by mAb J4-81(14) , was named ALCAM. ALCAM is a
member of the immunoglobulin supergene family and appears to be the
human homologue of the chicken neural adhesion molecule
BEN/SC-1/DM-GRASP (15, 16, 17) and the rat
protein KG-CAM(18) . ALCAM is a type I membrane protein
composed of two amino-terminal V-set domains followed by three C2-set
domains, a hydrophobic transmembrane domain, and a short cytoplasmic
anchor sequence. ALCAM is expressed by thymic epithelial (TE) cells (11, 12) and transiently on activated leukocytes (13) . In addition, ALCAM is expressed by neurons in the
brain(12) . Cell adhesion assays demonstrated that CD6-ALCAM
interactions are in part responsible for mediating thymocyte binding to
TE cells(13) .
Cell Lines, Tissue Culture, and Antibodies
The
human T cell line HPB-ALL was obtained from the American Type Culture
Collection (Rockville, MD) and maintained in culture in Iscove's
modified Dulbecco's medium + 10% fetal bovine serum. The
preparation and characterization of the rat anti-mCD6 mAbs M6-1A.1,
M6-3A.1, and M6-3B.1 mAbs will be described elsewhere. ()All
three mAbs bound to murine thymocytes as determined by flow cytometry
analysis. Analysis of which domains contained the epitope recognized by
each mAb was determined by examining the binding pattern of the mAbs on
the mCD6 Rg fusion proteins by ELISA. M6-1A.1 bound to mCD6 Rg,
mCD6D1-2 Rg, and mCD6D1 Rg but did not bind mCD6D2-S Rg, mCD6D2 Rg, or
mCD6D3 Rg. M6-3A.1 and M6-3B.1 each bound mCD6 Rg, mCD6D2-3 Rg, and
mCD6D3 Rg but not mCD6D1-2 Rg, mCD6D1 Rg, or mCD6D2 Rg. We therefore
concluded that M6-1A.1 recognizes mCD6 SRCR-D1 and that both M6-3A.1
and M6-3B.1 recognize mCD6 SRCR-D3.
Murine CD6 Rg Constructs
Complementary DNA
fragments encoding individual or groups of mCD6 extracellular domains
were obtained by polymerase chain reaction. The mCD6 fusion protein
contains bases 119-1181 of the mCD6 cDNA (3) fused to a cDNA
fragment encoding the hinge, CH2, and CH3 domains of human
IgG1(19) . The mCD6D1, mCD6D2, mCD6D3, mCD6D1-2, mCD6D2-3,
mCD6D1-3, mCD6D2-S, and mCD6D3-S fusions contain mCD6 bases
119-468, 469-775, 776-1081, 119-775, 469-1081,
119-1081, 469-1181, and 776-1181, respectively. To ensure that no
mutations were introduced by the procedure, all chimeric genes were
sequenced. In all cases, the amino-terminal secretory signal sequence
used in these chimeric constructs was derived from the human CD5 gene.
The fusion proteins were produced by transient expression in COS cells
and were purified by adsorption and elution from a protein A-Sepharose
column. Protein concentrations were determined using a Bradford
dye-binding procedure (Bio-Rad) and verified by SDS-polyacrylamide gel
electrophoresis.Binding Assays
Goat anti-mouse IgG (4 µg/ml,
Jackson Laboratories, West Grove, PA) was coated on 96-well plastic
dishes overnight at room temperature. After washing to remove excess
antibody, ALCAM Rg (2 µg/ml) was added. The ALCAM Rg fusion protein
used for these studies contains a murine IgG domain. Dilutions of mCD6
Rg fusion proteins (7.5 ng/ml-1.0 µg/ml) and control CD7 Rg
were added and allowed to bind to the immobilized ALCAM Rg. After
washing, the bound mCD6 Rg and CD7 Rg fusion proteins were detected
with horseradish peroxidase-conjugated donkey anti-human IgG (Jackson
Laboratories). Absorbance readings were taken at 450 nm and 630 nm.
Standard deviations were calculated on the basis of three independent
binding measurements. 10
) were
labeled with 20 µg/ml purified fusion protein for 1 h at 4 °C.
Cells were washed and subsequently labeled with fluorescein
isothiocyanate-goat anti-human IgG (Tago, Burlingame, CA) for 30 min at
4 °C and washed. The level of fusion protein binding was determined
by flow cytometry (FACScan, Becton-Dickinson).
Antibody Blocking Experiments
The ability of the
anti-mCD6 mAbs to inhibit the binding of ALCAM Rg to mCD6 was examined
using murine thymocytes obtained from C57BL/6 mice. In each assay, 5
10
cells were incubated with 50 µl of
conditioned media obtained from each of the hybridomas producing the
anti-mCD6 mAbs or from a hybridoma producing a rat anti-human gp39 mAb
as a negative control for 30 min at 4 °C. Cells were washed and
stained with 20 µg/ml human ALCAM Rg for 30 min at 4 °C. Cells
were then washed and labeled with fluorescein isothiocyanate-donkey
anti-human IgG (Jackson Laboratories) with minimal cross-reactivity to
rat and bovine IgG. The cells were then washed and subsequently
analyzed by flow cytometry using a FACScan.
Mapping of the CD6 ALCAM Binding Site
The
extracellular region of mature CD6 can be divided into four domains,
three consecutive SRCR domains (D1-D3) followed by a
33-amino-acid membrane proximal ``stalk'' (S) domain. To
determine which of these domains contains the ALCAM binding site, we
prepared six different mCD6 immunoglobulin fusion proteins (Rg,
recombinant globulins) which contain different mCD6 domains alone or in
combinations: mCD6 Rg which contains the complete extracellular domain
of CD6 or mCD6D1 Rg, mCD6D2 Rg, mCD6D3-S Rg, CD6D1-2 Rg, and CD6D2-S Rg (Fig. 1A). These mCD6 Rg fusion proteins were prepared
by transient expression in COS cells and purified by adsorption to and
elution from a protein A-Sepharose column (Fig. 1B).
constant domains including hinge, CH2, and CH3 are represented by
a bold line. The name of each of the mCD6 Rg fusion proteins
is shown to the right. B, radiolabeled fusion proteins were
purified as described under ``Materials and Methods'' and
electrophoresed under reducing conditions. The electrophoretic mobility
of molecular mass standards in kDa is shown to the left.
Blocking of CD6-ALCAM Interactions by Domain-specific
Anti-CD6 Antibodies
Recently we prepared a number of rat
anti-mCD6 mAbs. The ability of these mAbs to bind to the different CD6
Rg domain constructs was examined. These studies allowed
the identification of domain-specific anti-mCD6 mAbs. Three anti-mCD6
mAbs were selected for blocking studies. One of these antibodies
recognizes the CD6 SRCR-D1 (M6-1A.1), the other two (M6-3A.1 and
M6-3B.1) recognize CD6 SRCR-D3. As shown in Fig. 3, mAb M6-3A.1
completely blocked the ability of ALCAM Rg to bind murine thymocytes
expressing CD6. MAb M6-3B.1 was able to partially block while M6-1A.1
blocked only very weakly or not at all the binding of ALCAM Rg to
murine thymocytes. As expected, an isotype-matched rat anti-human gp39
mAb used as a control was unable to block the binding of ALCAM Rg to
murine thymocytes (Fig. 3). Inhibition of ALCAM binding to mCD6
by domain 3 but not domain 1 antibodies was confirmed by ELISA (data
not shown). These results provide further evidence that the third SRCR
domain of CD6 contains the ALCAM binding site.
We thank Vicki McDonald, Marcia Gordon, Gary Carlton,
and Nicola Tinari for their assistance, Jeff Ledbetter for critical
review of this manuscript, and Debby Baxter for help in its
preparation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.