(Received for publication, October 10, 1995; and in revised form, February 6, 1996)
From the
CD44 alternative splicing has been implicated in the regulation of CD44 function. CD44 undergoes significant posttranslational modification in all cells, but the functional consequences of these modifications are poorly understood. In the current study, we have demonstrated that keratan sulfate modification of CD44 significantly modulates its ability to bind to hyaluronate. We observed naturally occurring differences in CD44 keratan sulfate substitution between two clonal variants of the KM12 human colon carcinoma cell line. CD44 on the highly metastatic KM12L4 clone is more heavily substituted with keratan sulfate than CD44 on the poorly metastatic KM12C6 clone. Moreover, CD44H on KM12L4 bound to hyaluronate poorly compared to CD44H on KM12C6. Removal of keratan sulfate from CD44 greatly enhanced CD44-mediated cell adhesion to hyaluronate. Removal of keratan sulfate from CD44H-immunoglobulin fusion proteins also enhanced their adhesion to hyaluronate. The influence of glycosaminoglycan substitution on CD44 function was specific to keratan sulfate substitution; treatment to remove chondroitin sulfate, heparan sulfate, or hyaluronate did not affect CD44-mediated cell adhesion to hyaluronate. Use of site-directed CD44H cDNA mutants with arginine changed to alanine at position 41 indicated that keratan sulfate modification of CD44 modulates hyaluronate adhesion through its B loop domain. These findings suggest that keratan sulfate modification of CD44 may play an important regulatory role in the broad spectrum of biological processes attributed to CD44, including normal development, tumor progression, and lymphocyte function.
CD44 is the principal cell surface receptor for hyaluronate(1, 2, 3) and has been implicated in a wide variety of processes, including cell motility(4, 5) , growth control(6) , tumor metastasis(5, 7, 8, 9) , and lymphocyte activation(10, 11, 12) . Much interest has been devoted to the extensive alternative splicing of CD44 mRNA. Several CD44 isoforms arise from mRNA alternative splicing of at least 10 exons encoding a portion of the extracellular domain(13, 14) . The predominant CD44 isoform detected in many normal tissues is CD44H, an isoform encoded by a transcript that does not contain any of the central alternatively spliced exons(15) . Inclusion of one or more of the alternatively spliced exons generates individual CD44 isoforms.
While differences in CD44 alternative splicing between cells may result in different cell behavior, cell type-specific post-translational modification of CD44 may also alter their phenotype. CD44 undergoes extensive post-translational modification, including N- and O-linked glycosylation and substitution with high molecular weight glycosaminoglycans(16, 17, 18, 19, 20) . We have recently demonstrated that the same CD44H isoform expressed on two clonal variants of a human colon carcinoma cell line display very different functional characteristics(21) . CD44H reintroduced by stable transfection back into the poorly metastatic KM12C6 colon carcinoma clone binds hyaluronate and mediates a reduction in both in vitro and in vivo growth. In contrast, CD44H transfected into the highly metastatic KM12L4 colon carcinoma clone does not bind hyaluronate and does not mediate reduction in either in vitro or in vivo growth. These results indicate that subtle differences exist between the KM12C6 and KM12L4 cells that alter the ability of CD44H expressed on their cell surface to bind hyaluronate and modulate cell growth.
In the report presented herein, we have examined how CD44H glycosaminoglycan substitution influences CD44H function. We report that CD44H is more heavily substituted with keratan sulfate when expressed on KM12L4 cells than on KM12C6 cells. Moreover, this difference in keratan sulfate substitution significantly modulates CD44H function. Removal of keratan sulfate from cell surface CD44 or from CD44H-immunoglobulin fusion proteins (CD44H receptorglobulins) greatly enhances their adhesion to hyaluronate. Use of site-directed CD44H mutants that are unable to bind hyaluronate because of an amino acid substitution in the B loop domain indicates that keratan sulfate substitution modulates hyaluronate binding through this domain. The dramatic impact of this regulatory mechanism on CD44 function indicates that it is an additional mechanism, which, together with alternative splicing, regulates the function of CD44.
These cell lines were also
transfected with three constructs, and the resulting transfectants have
been characterized previously(21) . Briefly, cells designated
with the suffix H were transfected with CD44H full-length
cDNA in the pRC/CMV vector (Invitrogen, San Diego, CA), and these cells
express CD44H in addition to high molecular weight CD44 isoforms. A
mutant form of CD44H with arginine 41 changed to alanine, thereby
destroying its affinity for hyaluronate, is expressed on the cell
surface of transfectants designated with the suffix
41R/A. Control
transfectants were transfected with the vector only (no insert) and are
designated with the suffix
neo. Transfectants were grown in
Dulbecco's modified Eagle's medium/Ham's F-12 with
glutamine (Life Technologies, Inc.) and G418 (Sigma) added to a final
concentration of 500 µg/ml for KM12 and HT29 transfectants and 1500
µg/ml for SW620 transfectants.
The CD44H receptorglobulin was prepared as described previously(1) . Briefly, oligonucleotide-primed amplification of cDNA sequences of CD44H was performed by polymerase chain reaction. The oligonucleotide primers were designed to encode endonuclease restriction sites to facilitate subsequent cloning into Ig vectors digested with the same restriction enzymes. CD44-Ig constructs were introduced into COS cells by the DEAE-dextran method, and supernatants were harvested 5-8 days post-transfection. Receptorglobulins purified on protein A-Sepharose beads (Repligen, Cambridge, MA) were eluted with 0.1 M citric acid, pH = 3.0, dialyzed overnight, and purified protein concentration was determined using the BCA assay (Pierce).
An
enzyme-linked immunosorbent assay was used to measure the adhesion of
receptorglobulins to hyaluronate. 96-well flat-bottomed plates were
coated with hyaluronate or BSA in PBS overnight at 4 °C. The plates
were washed, and nonspecific sites were blocked with BSA.
Receptorglobulin (5 µg/ml) was added to the hyaluronate- or
BSA-coated plates and incubated at room temperature for 2 h. For some
experiments, receptorglobulin was treated with keratanase (1 unit/ml)
for 30 min. The plates were washed with PBS containing Tween 20 (0.1%)
four times. Horseradish peroxidase-conjugated anti-human immunoglobulin
Fc (Sigma) was added to the plates and incubated at room temperature
for 1 h. The plates were washed with PBS containing Tween 20 four times
and incubated with o-phenylenediamine (0.4 mg/ml) (Sigma) in
citrate phosphate buffer containing HO
at 37
°C. The reaction was stopped by addition of 2 N H
SO
, and the optical density was measured
at wavelength 492 nm.
Figure 1:
Analysis of sulfated glycosaminoglycan
substitutions on CD44. A, CD44 immunoprecipitates derived from SO
-labeled KM12L4
H and KM12C6
H
transfectants were treated with 1 unit/ml keratanase, 0.5 unit/ml
chondroitin ABC lyase (chondroitinase), or 0.2 unit/ml heparitinase in
PBS (pH = 7.4) at 37 °C for 1 h. Proteins were then
separated by SDS-PAGE and subjected to autoradiography. B,
CD44 immunoprecipitates derived from KM12L4
H, KM12C6
H, and
SW620
H transfectants that had been cell surface-labeled with
biotin were treated with either medium or 1 unit/ml keratanase at 37
°C for 1 h. SW620
H cells do not substitute CD44H with keratan
sulfate and therefore serve as a negative control for keratanase
treatment. Molecular size standards are shown at the left in
kDa.
However, these two cell lines
differ in their degree of keratan sulfate substitution of CD44. This
was determined by labeling cell surface proteins with biotin.
Keratanase-treated CD44H immunoprecipitates from the KM12L4H cells
demonstrated a greater shift in molecular mass than keratanase-treated
CD44H immunoprecipitates from the KM12C6
H cells (Fig. 1B). SW620
H is derived from separate colon
carcinoma cell line and does not substitute CD44H with keratan sulfate.
Therefore, this cell line serves as a negative control for keratanase
treatment. The results indicate that KM12L4 cells more heavily
substitute CD44H with keratan sulfate than do the KM12C6 cells. Because
the CD44H core protein backbones are identical in the two cell lines,
CD44H on KM12C6 presumably has more of its molecular mass made up by
other glycosaminoglycan substitutions or N- or O-linked oligosaccharides.
Figure 2:
Influence of keratan sulfate modification
on CD44 adhesion to hyaluronate. Adhesion of CD44H cDNA
transfectants (designated with the suffix H) and control
transfectants (designated with the suffix
neo) to hyaluronate was
measured before and after keratanase treatment (1 unit/ml) in the
absence or presence of anti-CD44 mAb BRIC205 (5 µg/ml). Data are
presented as the mean ± S.D. of triplicate experiments.
, medium; &cjs2108;, BRIC205; &cjs2113;, keratanase;
,
keratanase + BRIC 205.
Similarly, keratanase treatment of KM12L4H cells also
dramatically enhanced their binding to hyaluronate. The degree of
enhancement suggests that keratanase treatment enhanced both CD44H and
high molecular weight CD44 isoform adhesion to hyaluronate. Again,
blocking experiments with mAb BRIC 205 indicated that the
keratanase-mediated enhancement was attributable to hyaluronate
adhesion by CD44, and not another cell surface protein. Untreated
KM12C6
H cells bind to hyaluronate extremely well, and treatment of
these cells with keratanase did not enhance their binding. These data
reveal that the differences detected in the ability of CD44H to bind
hyaluronate when expressed on KM12C6 cells compared to KM12L4 cells are
partially a result of the inherent differences in CD44H keratan sulfate
substitution noted between the cells. Nonetheless, the persistent
difference in hyaluronate binding between KM12C6 and KM12L4
transfectants even after keratanase treatment suggests that additional
factors which influence CD44 function differ between these two KM12
clones. The significant inhibition of adhesion by mAb BRIC 205 measured
in all of the keratanase-treated cells suggests that keratanase
treatment enhances hyaluronate binding of at least two different CD44
isoforms through a similar mechanism.
To examine the possibility that keratanase treatment enhanced cell adhesion to hyaluronate through an increase in cell surface CD44, we performed fluorescence-activated cell sorting analysis with mAb BU52 to measure cell surface CD44 before and after keratanase treatment (Fig. 3). No immediate changes in cell surface CD44 were detected, indicating that keratanase treatment modified pre-existing cell surface CD44 and did not induce an up-regulation of CD44 expression.
Figure 3: Cell surface CD44 expression with and without keratanase treatment. Cells were stained with CD44 mAb BU52 with (solid lines) or without (dotted lines) keratanase treatment. No changes in cell surface CD44 were detected.
Figure 4:
Influence of glycosaminoglycanase
treatment on CD44 adhesion to hyaluronate. Adhesion of CD44H
cDNA transfectants (H) and control transfectants (
neo) to
hyaluronate before and after treatment with chondroitin ABC lyase (0.5
unit/ml), hyaluronidase (2 units/ml), heparitinase (0.2 unit/ml), or
keratanase (1 unit/ml). Data are presented as the mean ± S.D. of
triplicate experiments.
, medium; &cjs2108;, chondroitinase;
&cjs2113;, hyaluronidase; &cjs2110;, heparitinase;
,
keratanase.
Figure 5:
Influence of keratan sulfate modification
on CD44H receptorglobulin adhesion to hyaluronate. CD44H
receptorglobulin before and after keratanase treatment was separated by
SDS-PAGE, transferred to nitrocellulose, and detected with mAb
F10-44-2. The molecular weight of CD44H receptorglobulin decreased
after keratanase treatment (A). CD44H receptorglobulin
adhesion to hyaluronate or BSA before and after keratanase treatment
was measured and found to be significantly greater after removal of
keratan sulfate (B). &cjs2113;, hyaluronate; &cjs2108;, BSA;
, hyaluronate + keratanase; &cjs2110;, BSA +
keratanase. Binding activity is expressed as the value of
OD
. Data are presented as the mean ± S.D. of
triplicate experiments.
KM12 clones
transfected with a site-directed mutant CD44H cDNA to express
CD44H with arginine at position 41 changed to alanine have been
described previously. As expected, KM12L441R/A and
KM12C6
41R/A cells demonstrate the same hyaluronate binding
characteristics as do KM12L4
neo and KM12C6
neo cells (Fig. 6). These results confirm that the mutated CD44H expressed
by the KM12L4
41R/A and KM12C6
41R/A cells bind to hyaluronate
poorly. Treatment of these cells with keratanase did not enhance their
binding to hyaluronate to any greater extent than observed in the
control cells. In other words, the keratanase treatment enhanced
hyaluronate binding of only the high molecular weight CD44 isoforms,
and not of the site-directed mutant CD44H. These results strongly
suggest that keratan substitution on CD44H modulates its interaction
with hyaluronate through its B loop domain.
Figure 6:
Effect of keratan sulfate modification of
mutant CD44 adhesion to hyaluronate. Mutation of arginine 41 to alanine
in the B loop domain abolishes hyaluronate adhesion. Transfectants
expressing this mutant CD44 (designated with the suffix 41R/A) and
control transfectants (designated with the suffix
neo) were tested
for adhesion to hyaluronate or BSA before and after keratanase
treatment (1 unit/ml). Data are presented as the mean ± S.D. of
triplicate experiments.
, hyaluronate; &cjs2108;, BSA;
&cjs2113;, hyaluronate + keratanase;
, BSA +
keratanase. Keratanase treatment did not enhance adhesion of the mutant
CD44 transfectants any more than it enhanced adhesion of the control
transfectants.
Figure 7:
Influence of CD44 glycosaminoglycan
substitution on adhesion to hyaluronate in HT29 human colon carcinoma
cells. Adhesion of CD44H cDNA transfectants (HT29H) and
control transfectants (HT29
neo) to hyaluronate was measured after
treatment with medium, 0.5 unit/ml chondroitin ABC lyase
(chondroitinase), 2 units/ml hyaluronidase, 0.2 unit/ml heparitinase,
or 1 unit/ml keratanase at 37 °C for 1 h. Data are presented as the
mean ± S.D. of triplicate experiments. Only treatment with
keratanase enhanced adhesion to hyaluronate.
, medium;
&cjs2108;, chondroitinase; &cjs2113;, hyaluronidase; &cjs2110;,
heparitinase;
, keratanase.
The diversity of biological functions attributed to CD44 may result from its role as a cell surface adhesion molecule that binds to hyaluronate. Given the broad tissue distribution of CD44, it is reasonable to assume that CD44 interaction with extracellular matrix is tightly regulated. This hypothesis is supported by our finding that CD44H expressed on two individual clonal variants of a single colon carcinoma cell line drastically differ in their ability to bind hyaluronate. It has also been reported previously that hyaluronate binding by CD44 expressed on murine T cells is transiently activated during an in vivo immune response(28) . Potential mechanisms that regulate CD44 interaction with extracellular matrix include: 1) alternative splicing, 2) phosphorylation of residues in the cytoplasmic domain, 3) interaction of the cytoplasmic domain with intracellular proteins, 4) posttranslational modification by glycosylation or glycosaminoglycan substitution, 5) interaction with other cell surface proteins, 6) interaction with extracellular ligands, and 7) masking or shedding of cell surface CD44 (for review, see (29) ).
Several tumor types express different CD44 alternative splice isoforms compared to their normal tissue counterparts(7, 30, 31, 32, 33, 34) ; therefore, CD44 alternative splicing has received the most attention in studies of CD44 function. Our studies have focused on a distinct and separate mechanism that influences CD44 function, namely keratan sulfate substitution on the CD44 protein. CD44 is known to undergo extensive posttranslational modification, including N- and O-linked glycosylation, and substitution with several glycosaminoglycans(16, 17, 19, 20) . Recently it was reported by Jackson and colleagues that only CD44 isoforms that contain exon v3 are modified with heparan sulfate or chondroitin sulfate on Namalwa lymphoma cells(18) . These authors reported that CD44H and other isoforms lacking exon v3 were not modified with either heparan sulfate or chondroitin sulfate. In contrast, we have detected keratan sulfate substitution on CD44H, which does not contain exon v3, in three different colon carcinoma cell lines, and demonstrated its influence on CD44 function. Furthermore, the degree of keratan sulfate modification of CD44 differs between different tumor cell lines.
Little is understood about the functional consequences of CD44 glycosaminoglycan substitution. Chondroitin sulfate substitution on CD44 enhances melanoma motility and invasive properties in vitro(35, 36) . It has recently been reported that heparan sulfate modification of exon v3 containing CD44 isoforms allows presentation of heparin-binding growth factors such as basic fibroblast growth factor and epidermal growth factor(19) . However little else is known about the functional consequences of CD44 glycosaminoglycan substitution, especially in epithelial cells. Our finding that two clonal variants of a colon carcinoma cell line, KM12L4 and KM12C6, differ in their CD44H function (21) led us to the current series of investigations in which we report that naturally occurring differences in keratan sulfate modification of CD44 between KM12L4 and KM12C6 cells account for their differences in hyaluronate adhesion.
The mechanism by which keratan sulfate modification of CD44 alters its adhesion to hyaluronate remains unclear. CD44 contains a cluster of basic residues in the B loop region that is responsible for the majority of its adhesion to hyaluronate(27) , and mutation of arginine 41 drastically reduces its adhesion to hyaluronate(26) . Our data using mutant CD44H indicate that keratan sulfate substitution modulates the interaction of this specific region with hyaluronate. CD44 binding to hyaluronate can also be influenced by treatment with mAb IRAWB 14 (37) or by inclusion of alternatively spliced exons in the membrane proximal region(18, 38) . In concert, these data suggest that CD44 keratan sulfate substitution may modulate adhesion to hyaluronate via changes in protein conformation.
Although we have demonstrated that naturally occurring differences between cells in their addition of keratan sulfate to CD44H modulates its binding to hyaluronate, the actual sites on CD44H containing the keratan sulfate substitutions are not known.
Serine residues as part of a S-G-X-G sequence preceded by several acidic residues appear to serve as sites for chondroitin sulfate and heparan sulfate substitution(39) ; however, no clear consensus sequence exists for keratan sulfate substitution. A hexapeptide motif repeated in the keratan sulfate-enriched region of bovine cartilage proteoglycan has been suggested as a possible consensus sequence for keratan sulfate substitution(40) , but CD44H does not contain this sequence. The amino acid sequence A-P-S-P-G, which was deduced to contain the keratan sulfate linkage site in pig nucleus pulposus proteoglycan(41) , is also not contained in CD44H. Keratan sulfate is linked to bovine fibromodulin through asparagine-glycosidic linkages(42) . These linkages do not appear to be the major site of keratan sulfate substitution on CD44H, because growth of KM12 and HT29 cells in tunicamycin does not reduce keratan sulfate substitution (data not shown). Consequently, we are unable to use site-directed mutagenesis of CD44H cDNA to inhibit keratan sulfate substitution on CD44H in colon carcinoma cells.
The enhancement in hyaluronate adhesion by KM12 and HT29 cells occurred nearly immediately after treatment with keratanase. This rapid increase in hyaluronate adhesion suggests that keratanase treatment modified preexisting CD44 molecules, rather than induced new CD44 expression. This conclusion is supported by fluorescence-activated cell sorting data that demonstrates no change in cell surface CD44 after keratanase treatment. This conclusion is also supported by the receptorglobulin adhesion data that demonstrate enhanced CD44H receptorglobulin adhesion to hyaluronate after treatment with keratanase. The rapid influence of keratanase treatment combined with the receptorglobulin data also suggest that new protein synthesis is not required for the enhanced hyaluronate adhesion. This regulatory mechanism is distinct from that of phorbol 12-myristate 13-acetate-inducible binding of lymphocytes to hyaluronate, in which new protein synthesis is essential(43) .
In conclusion, we have demonstrated that CD44 function on colon carcinoma cells is modulated by posttranslational modification. Specifically, we have identified differences in keratan sulfate substitution on CD44 that markedly modulate its interaction with hyaluronate through its B loop domain. The impact of keratan sulfate substitution on CD44 function is significant and may be functionally as important as CD44 alternative splicing. Closer examination of this CD44 regulatory mechanism in investigations of development, tumor metastasis, and lymphocyte function will likely reveal it to be an important regulator for these biological processes.