(Received for publication, March 13, 1995; and in revised form, January 9, 1996)
From the
CD44 is a widely expressed integral membrane protein that acts
as a receptor for hyaluronan (HA) and is proposed to be important to
cell-extracellular matrix interaction. The Indian (In) blood group
antigens reside on CD44, and most individuals express the In antigen. Homozygosity for the In
allele
occurs as a rare event and is associated with production of
alloantibody to the common In
antigen after transfusion or
pregnancy.
The present study demonstrates that a single point
mutation (G
C) causes an Arg
Pro substitution, which is responsible for the In
/In
polymorphism. Additional mutations were found in
In(a+b-) cDNA but were not necessary to the antigenic
phenotype as determined in site-directed mutagenesis studies. In
studies using CD44 chimeric constructs, Arg
has previously
been shown to be crucial for maintenance of HA-binding ability to a
CD44 peptide. However, the present study demonstrates that the
Arg
Pro substitution does not reduce HA binding to
the intact CD44 protein, which contains two proposed extracellular
HA-binding motifs. Down-regulation of HA binding to In(a+b-)
CD44 by anti-CD44 monoclonal antibody (mAb) ligands, however, was
weakened, although all mAbs tested bound In(a+b-) and
In(a-b+) CD44 equally well. Competitive inhibition studies
using human anti-In
also showed that some mAbs that inhibit
HA binding to CD44 may do so by interacting with a domain separate
from, but affecting the structure of, the In
epitope.
Investigation of the biochemical and molecular basis of human erythrocyte blood group antigens has begun to contribute to our understanding of structure/function relationships of some biologically active red cell membrane proteins(1) . CD44, also previously identified as In(Lu)-related p80(2, 3) , the Hermes antigen(4, 5) , Pgp-1(6, 7, 8) , and ECMRIII(9, 10) , is a widely expressed integral membrane protein that bears Indian (In) blood group antigens(11, 12) . CD44 has been shown capable of binding a number of extracellular matrix components, including hyaluronan (HA), fibronectin, heparan sulfate, and collagen types I and VI(10, 13) . In addition, studies using monoclonal antibodies to this protein have suggested that CD44 plays a role in lymphocyte homing to mucosal lymphoid tissue(14) , in leukocyte activation(15) , and in tumor metastasis(16) . CD44 also appears important to lymphopoiesis (17) and to progenitor cell-ECM interactions during erythropoiesis(18) .
A large number of CD44 isoforms have been described(16, 19, 20, 21) . Nonactivated hematopoietic cells, including erythrocytes, express predominantly an 80-90-kDa ``hematopoietic'' isoform (CD44H) encoded by exons 1-5, 15-17, and 19 of the CD44 gene(22, 23) . Exons 6-14 have been shown to be expressed in various combinations under a variety of circumstances, including leukocyte activation and malignant transformation. One of the most commonly identified CD44 isoforms, CD44E, contains exons 12, 13, and 14(24) . Exon 18 encodes an alternate, shortened cytoplasmic tail. Expression of some spliceoforms has been associated with increased metastatic potential or induction of an adhesive phenotype(23) . CD44 isoforms can also vary in molecular weight due to differing glycosylation patterns. CD44H bears a number of consensus sites for covalent attachment of chondroitin sulfate, and a 180-kDa CD44 isoform has been shown to bear several chondroitin sulfate moieties(25) .
Several studies have attempted to identify
the sites at which CD44H binds hyaluronan. Resting lymphocytes express
CD44H but only bind hyaluronan poorly. Jurkat cells transfected with
CD44H cDNA will bind HA when stimulated with phorbol 12-myristate
13-acetate (PMA)()(26) . However, PMA-treated cells
transfected with CD44E cDNA did not bind HA unless first incubated with
CD44 monoclonal antibodies known to enhance HA binding(26) .
Different monoclonal antibodies to CD44 may enhance, block, or not
affect HA binding to CD44H(26, 27) ; the mechanisms
that effect alteration of HA-binding ability have not been defined. The
cytoplasmic domain of CD44H is also known to be necessary for
activation of HA-binding ability(26) .
In the extracellular
portion of CD44H, two proposed HA-binding domains containing positively
charged amino acids (amino acids 29-46 and 150-162) have
been shown to be important for maximal HA binding, using deletion and
site-directed mutants(28, 29) . However, the first
domain is the only one that resides within the portion of CD44
homologous to cartilage link protein. Peach et al.(28) have shown that mutation of Arg
Ala in a construct containing amino acids 1-220 of CD44H almost
abolished HA binding altogether. Yang and colleagues(29) ,
using different chimeric constructs containing only small CD44
peptides, showed that an Arg
Gly substitution
abolished binding of HA to a chimeric protein containing CD44 amino
acids 38-46 and proposed that HA binds to a minimal consensus
motif BX
B, where B represents a basically charged
amino acid (Arg or Lys) and X
represents seven
nonacidic residues. HA binding was enhanced if additional basic
residues were clustered around the ends of the motif or if a basic
residue occurred within the X
domain(29) .
To date, no studies showing the effect of mutations within the first
HA-binding motif (amino acids 38-46) on HA binding by intact
CD44H have been reported.
The In blood group system comprises two
antigens presumed to be encoded by CD44
alleles(11, 30, 32) . The In antigen is of extremely high frequency, but the low incidence
antigen In
is somewhat more common among peoples of the
Middle East and Indian subcontinent. This study identifies the
molecular basis of the In
/In
polymorphism and
demonstrates that although this polymorphism abolishes the
BX
B motif within the part of CD44 homologous to
cartilage link protein, it does not inhibit HA binding by CD44H and
reduces the effect of monoclonal antibody ligands on HA binding to
CD44.
All monoclonal antibodies (mAbs) used in this study have
been previously described or have been evaluated by the Fifth
International Workshop on Leucocyte Differentiation
Antigens(2, 35) . Human anti-In were
identified using routine blood bank methodology (agglutination).
Anti-In
-Dh is from the patient previously described by
Ferguson and Gaal(36) . Anti-In
-Ra was supplied by
the International Blood Group Reference Laboratory (Drs. F. Spring, J.
Smythe, and D. Anstee, Bristol, United Kingdom). The specificity of
both antisera has been confirmed by several laboratories.
PCR of genomic DNA sequence from In(a+b-) propositus Bi was accomplished as described above, using the primers MT11 and MT12 (Table 1), 1 µg of genomic DNA, and an annealing temperature of 55 °C.
In some experiments, PMA-treated nontransfected, and CD44-transfected Jurkat cells were incubated in a final concentration of 0.005 M dithiothreitol for 1 h at 4 °C to denature cell surface disulfide bonds. Cells were then washed and resuspended in PBS-BSA. Viability of treated and sham-treated cells was determined by trypan blue exclusion and was uniformly >95% for both dithiothreitol-treated and sham-treated cells.
Radioimmunoassays were performed as described
previously(33, 34) . For competitive inhibition
assays, red cells were incubated for 1 h at room temperature with
blocking antibody, washed twice with PBS-BSA, and incubated with second
antibody for 1 h at room temperature. After repeated washing steps,
cells were resuspended in a fixed volume, incubated with radiolabeled
goat anti-mouse or anti-human IgG, and centrifuged through phthalate
oils. Centrifuge tube tips containing cell pellets were analyzed in a
scintillation counter (LKB/Wallac Inc., Gaithersburg, MD) to
quantitate radiolabel bound to cells.
Figure 1:
Sequence analysis of CD44 cDNA from
normal In(a-b+) and Dh In(a+b-) donors. Subcloned
cDNA obtained by RT-PCR was sequenced by the dideoxy nucleotide chain
termination method. Lanes represent A, T, G, and C, from left to right. Results demonstrate a G C
substitution at nucleotide 252, which causes an Arg
Pro substitution in CD44 protein.
During the course of
later studies, a third unrelated In(a+b-) EBV-transformed
lymphocyte cell line became available. PCR amplification and sequencing
of genomic DNA from this cell line once again demonstrated a G
C substitution.
Figure 2:
Demonstration that CD44 with an Arg
Pro substitution fails to react with human
anti-In
. CD44-negative human Jurkat cells were transfected
with wild-type CD44 cDNA (CD44WT), CD44 cDNA derived from the
In(b-) propositus Dh (In(b-)), or CD44 cDNA with a
G
C mutation (R46P), which causes an Arg
Pro substitution. mAb A3D8 to CD44 reacted with all
transfected cells as well as with In(b+) red cell membrane protein
(RBC). However, human anti-In
reacted only with the CD44WT
and RBC.
We studied HA binding by cells transfected with CD44WT, CD44Dh (cDNA from the In(a+b-) propositus Dh), and CD44(R46P). As shown in Fig. 3, when compared with PMA-treated CD44WT-transfected cells, PMA-treated CD44Dh transfectants showed equal ability to bind HA, as measured both by percentage of positive cells as well as by degree of binding per cell, indicated by mean fluorescence channel. When the ability of CD44(R46P) to bind HA was measured, it also was shown to bind HA as well (Fig. 4).
Figure 3: HA binding to In(b+) CD44WT and In(b-) CD44Dh expressed on intact cells. Jurkat cells that had been stably transfected with either CD44WT cDNA derived from an In(b+) individual or CD44Dh derived from an In(b-) individual were stained with mAb A3D8 and fluorescein-conjugated goat anti-mouse Ig (black bars) or with directly fluoresceinated hyaluronan (shaded bars). Cells were tested with and without preincubation with PMA, which has previously been shown to induce HA binding by CD44. A, between 85 and 95% of cells in all aliquots were reactive with mAb A3D8, directed against a nonpolymorphic region of CD44. Only 1.1% of untreated Jurkat/CD44WT cells bound hyaluronan, while 36.2% of PMA-treated cells bound hyaluronan. Jurkat/CD44Dh cells showed a similar number of A3D8+ cells; again, PMA treatment caused marked increase (4.0-45.1%) in the fraction of cells that bound HA. B, when linear mean fluorescence channels of all cells were compared, Jurkat/CD44WT and Jurkat/CD44Dh cells showed similar increases in immunofluorescence with HA after PMA treatment (MFC = 142 and 159 cells, respectively). The level of CD44 expression, as measured by fluorescence with A3D8, appeared higher in Jurkat/CD44Dh cells, probably accounting for the increased number of Jurkat/CD44Dh HA-binding cells demonstrated in panel A.
Figure 4:
Effect of Pro substitution on
HA binding. Nontransfected Jurkat cells and cells transfected with
CD44(R46P), which encodes the Arg
Pro substitution
responsible for the In
/In
polymorphism, were
cultured with PMA and then incubated with either FITC-labeled HA or
with anti-CD44 mAb A3D8 or control myeloma protein P3, followed by
FITC-labeled anti-mouse Ig. Fluorescence is graphed on a log scale (horizontal axis), and mean fluorescence channel is calculated
on a linear scale. As shown in the lower middle panel, mAb
A3D8 bound 87% of CD44(R46P)-transfected cells (MFC = 187).
HA-FITC bound 49% of CD44(R46P) with MFC = 107.8 (lower
right panel) and did so as strongly as did CD44WT-transfected
cells (data not shown) as compared with 1.6% untransfected cells (MFC
= 64, upper right panel).
Therefore, despite
alteration of the first BXB motif of CD44, intact
In(b-) CD44Dh and CD44(R46P) maintained their ability to bind HA.
In experiments in which PMA-treated transfected Jurkat cells were pretreated with dithiothreitol, neither CD44WT nor CD44Dh bound HA (data not shown).
All antibodies were shown by Western blotting to bind
equally well to In(a+b-) and In(a-b+) CD44 (data
not shown). mAbs BRIC235, HP2/9, BU75, 5F12, and 3F12 had been
previously shown to partially or completely inhibit HA binding to
CD44-transfected Jurkat cells; mAb F10.44.2 had been shown to enhance
HA binding 2-fold(42) . mAbs BU75 and 3F12 had further been
shown to inhibit 5F12 binding to red cells by 84 and 71%,
respectively(27) . Saturating dilutions of all CD44 monoclonal
antibodies tested produced >10% inhibition of binding of
subsaturating dilutions of human anti-In. However, only
three antibodies (Bric235, Bu75, and HP2/9) produced more than 25%
competitive inhibition of anti-In
binding (Fig. 5).
In reverse experiments, human anti-In
was unable to inhibit
or enhance binding of subsaturating dilutions of anti-CD44 mAbs by
greater than 25% (data not shown).
Figure 5:
Inhibition of anti-In reactivity with human red blood cells by anti-CD44 mAbs with
various effects on HA binding. mAbs BRIC235, 3F12, BU75, HP2/9, and
5F12 have been previously shown to inhibit HA binding to CD44, while
mAb NIH44-1 had no effect on HA binding, and mAb F10.44.2
increased HA binding 2-fold (42) . mAbs were individually
incubated with red cells, which were then washed and exposed to human
anti-In
and radiolabeled anti-human Ig. Binding was
compared with that achieved when cells were preincubated with P3
nonreactive murine myeloma protein. BRIC235 and HP2/9 showed marked but
incomplete inhibition of anti-In
binding, while mAb 5F12,
which inhibits HA binding equally well, showed little inhibition. No
antibody enhanced binding of
anti-In
.
Figure 6: Inhibition of HA binding to various CD44 proteins by mAb 5F12. HA binding to PMA-treated Jurkat cells transfected with CD44WT, CD44Dh, or CD44(R46P) was measured in the presence (shaded bars) and absence (black bars) of anti-CD44 mAb 5F12. The percentage of cells expressing CD44, as measured by monoclonal antibody A3D8, was equivalent for all three cell lines (89.9, 89.0, and 88.6% positive, respectively). CD44 copy number, as measured by linear mean fluorescence channel, varied slightly among cell lines (for CD44WT MFC = 205.4; for CD44Dh MFC = 219.6; and for CD44(R46P) MFC = 224.7). In both immunofluorescence and Western blot assays, mAb 5F12 as well as other mAbs bound equally well to all CD44 proteins expressed (data not shown). In the absence of 5F12, HA-FITC bound to CD44 transfectants in proportion to CD44 expression. However, saturating concentrations of 5F12 antibody inhibited HA binding to CD44WT by 92%, while HA binding to CD44Dh and CD44(R46P) was inhibited by only 32 and 66%, respectively.
The CD44 protein (Fig. 7) is known to be a major
receptor for HA, and it may also have physiologic importance as a
receptor for fibronectin and laminin(18, 46) . The N
terminus of CD44 is homologous to cartilage link protein, but this
domain only contains one of the two amino acid motifs (KNGRYSISR,
residues 38-46) thought to be active in cellular binding to
HA(29) . The more C-terminal portion of the extracellular
domain of CD44 encompasses two overlapping BXB
motifs (RDGTRYVQKGEYR, residues 150-162). Thus far, there has
been insufficient evidence to indicate whether one of these two amino
acid motifs is the more important in HA binding. Both motifs, expressed
either as peptides or as part of chimeric molecules, have shown
HA-binding activity(28, 29) , and Liao et al. have suggested that the two motifs can come into proximity to one
another and can cooperate in HA binding(44) .
Figure 7:
Schematic diagram of CD44 and its putative
HA-binding motifs. The N-terminal domain of CD44 comprises a
disulfide-cross-linked peptide highly homologous to cartilage link
protein. This domain contains a single BXB
putative HA-binding motif, constituted by residues 38-46. The
In
/In
polymorphism results from Pro or Arg at
position 46, respectively. Two overlapping BX
B
motifs are formed by residues 150-162, outside of the region
homologous to cartilage link protein.
We have shown
that a naturally occurring polymorphism of CD44 coding sequence is
responsible for the expression of In/In
blood
group antigens. Evidence that the basis of the In
/In
polymorphism is an Arg
Pro substitution
includes both cDNA and genomic DNA sequence analysis as well as antigen
expression by transfected cell lines expressing various CD44 cDNA
constructs. The localization of this polymorphism to the region of CD44
homologous to cartilage link protein is consistent with previously
described characteristics of In antigens, including sensitivity to
degradation by proteases and reducing reagents. Analysis of additional
propositi expressing the In
antigen would be required to
ascertain whether either the silent mutations or variable residues at
positions 109 and 239 are in linkage disequilibrium with the
In
-type sequence at position 46.
Since the
In(a+b-) phenotype has not been associated with any clinical
abnormalities, it was surprising to find that the
In-associated Arg
Pro mutation
disrupted an HA consensus binding motif. Studies of transcripts from
both the CD44Dh and CD44(R46P) constructs containing this amino acid
substitution showed that altering the C-terminal basic amino acid of
the proposed first HA-binding motif in intact CD44 did not inhibit HA
binding to CD44 expressed at the cell surface. In contrast, previously
described alteration of this residue in a model studied by Yang and
colleagues (29) abolished binding of HA. Their experimental
system, however, did not utilize the intact protein; instead, amino
acids 38-46 of CD44 were expressed as part of a chimeric
construct, and mutation of Arg
Gly abolished
binding of biotinylated HA to the CD44 peptide/RHAMM chimeric protein
immobilized on nitrocellulose.
The work of Liao and colleagues (44) has shown that mutation of Arg
His in
the second putative HA-binding motif also did not abrogate HA binding
to recombinant intact CD44 expressed in Jurkat cells, although other
studies on the second HA-binding motif have shown that altering basic
residues within this motif did reduce the ability of CD44 chimeric
constructs to bind HA(28, 29) . Clearly, conclusions
from studies of HA binding to small peptides or chimeric constructs
cannot necessarily be extrapolated to the full protein expressed by
intact cells.
The cooperation of both extracellular
BXB motifs in the binding of HA has been proposed
by Liao et al.(44) . This hypothesis is further
supported by the complete abrogation of HA binding to PMA-treated
CD44-transfected Jurkat cells pretreated with dithiothreitol. As
depicted in Fig. 7, the conformation of CD44 is likely to be
highly dependent on disulfide bonds. Interaction of the
BX
B motifs would thus be likely to be disrupted by
reduction of these bonds and subsequent alteration of CD44
conformation. However, it should be noted that other studies concerning
HA binding to proteins containing BX
B binding
motifs have shown that the motif may not be required for the ligand to
bind the molecule. Specifically, mutants of the cartilage link protein
in which the exon containing this motif had been deleted nevertheless
bound HA(47) .
Studies using monoclonal antibodies that can
affect ligand binding have helped to identify possible recognition
sites on receptors(48) . The ability of some anti-CD44 mAbs
that inhibit HA binding to also partially inhibit binding of
anti-In to red cells is in concordance with the hypothesis
that the In
epitope is closely related to an HA-binding
domain. Inability to achieve more complete inhibition may be due to the
polyclonal nature of human anti-In
sera or may be due to
the mechanism whereby these mAbs may adversely affect HA-binding
ability. Since anti-In
binding was partially inhibited by
preincubation of cells with CD44 mAbs, but anti-In
did not
reduce mAb reactivity to CD44 on erythrocytes, these monoclonal
antibodies likely inhibit HA binding via transmitted effects, i.e. alteration of conformation of the CD44 molecule. Such an
alteration of the structure of CD44 would prevent binding of
anti-In
without directly blockading the In
epitope, whereas the reverse incubations might have no effect on
the binding of the CD44 mAbs that themselves affect HA binding.
Similarly, the preincubation of anti-In
with PMA-treated
CD44WT-transfected cells did not prevent binding of HA, as the antibody
might not alter the critical configuration of the binding site(s).
Further, our data regarding the effects of CD44 mAbs on HA binding
to CD44Dh and CD44(R46P) might then be interpreted as due to alteration
of ligand effect on the interaction of the two HA-binding motifs. If
mutating Arg
Pro alters the mobility of the first
HA-binding motif of CD44, then alteration of the tertiary structure by
CD44 ligands might be hampered. It is thus likely that the substitution
of a basic residue at the carboxyl-terminal end of the first motif,
although it does not inhibit HA binding, does influence the effect
mediated by monoclonal antibodies to CD44. Studies by Zheng et al.(48) have shown that transmission of effects of CD44 mAbs
on HA binding can be hampered when residues have been altered, although
ligand binding is maintained. The conformational change of CD44 thought
to be induced by the previously described mAbs (42) may be
reduced when Pro is substituted for Arg
, as shown by
maintenance of HA-binding ability. Our results may thus be considered
consistent with the theory that various domains of CD44 interact to
form a recognition site for HA(44, 48) .
It should
be noted that, although the Tyr
Ser substitution
is common in human CD44H and conserved among species, the presence of
Tyr
does not inhibit CD44 binding to HA, as theorized by
Dougherty et al.(31) Both CD44WT and CD44(R46P)
proteins, both of which contain Tyr
, bind to HA.
In conclusion, demonstration of a blood group antigen-related polymorphism within a functional domain of a protein may no longer be considered unexpected(1) . Another recently described example is the Diego blood group antigen polymorphism of the anion exchange protein AE1(45) . One may expect that future exploration of the molecular basis of blood group antigen variants will uncover further such polymorphisms, which can then be used to extend our understanding of structure/function relationships of other membrane proteins. Such polymorphisms are especially useful because, as they occur naturally, their impact on protein function and the importance of a particular protein in various physiologic processes can be assessed from clinical evaluation of individuals with the variant phenotype.