(Received for publication, November 30, 1994; and in revised form, January 13, 1995)
From the
CD22, a B cell-specific receptor of the immunoglobulin
superfamily, has been demonstrated to bind to oligosaccharides
containing 2-6-linked sialic acid (Sia) residues.
Previously, we demonstrated that the minimal structure recognized by
this lectin is the trisaccharide
Sia
2-6Gal
1-4GlcNAc, as found on N-linked, O-linked, or glycolipid structures (Powell,
L., and Varki, A.(1994) J. Biol. Chem. 269,
10628-10636). Here we utilize a soluble immunoglobulin fusion
construct (CD22Rg) to determine directly by equilibrium dialysis the
stoichiometry (2:1) and dissociation constant (32 µM) for
Neu5Ac
2-6Gal
1-4Glc binding. Inhibition assays
performed with over 30 different natural and synthetic sialylated
and/or sulfated compounds are utilized to define in greater detail
specific structural features involved in oligosaccharide-protein
binding. Specifically, the critical features required for binding
include the exocyclic hydroxylated side chain of the Sia residue and
the
2-6 linkage position to the underlying Gal unit.
Surprisingly, alterations of the 2-, 3-, and 4-positions of the latter
residue have limited effect on the binding. The nature of the residue
to which the Gal is attached may affect binding.
Bi(
2-6)-sialylated biantennary oligosaccharides are capable
of simultaneously interacting with both lectin sites present on the
dimeric CD22Rg fusion construct, giving a marked improvement in binding
over monosialylated compounds. Furthermore, data are presented
indicating that full-length native CD22, expressed on the surface of
Chinese hamster ovary cells, is structurally and functionally a
multimeric protein, demonstrating a higher apparent affinity for
multiply sialylated compounds over monosialylated compounds. These
observations provide a mechanism for strong CD22-dependent cell
adhesion despite the relatively low K
for
protein-sugar binding.
CD22 is a sialic acid (Sia) ()binding glycoprotein
found predominantly on resting IgM
IgD
B
cells(1, 2, 3, 4, 5) . It
binds to oligosaccharides containing the sequence
Sia
2-6Gal
1-4Glc/GlcNAc, and shows no affinity for
oligosaccharides containing
2-3-linked Sia residues. By
sequence analysis, it is a member of the immunoglobulin superfamily,
with an N-terminal V-type domain followed by six Ig C2-type domains, a
membrane spanning region, and a 160-amino acid cytoplasmic
tail(6, 7, 8, 9) . Two isoforms of
human CD22 have been identified by cDNA cloning, a seven domain
CD22
form and a shorter CD22
form, which lacks the third and
fourth domains present in CD22
. The extent of tissue expression of
these two isoforms is at present unexplored, although most cells
examined appear to express the larger isoform(10) . Murine
CD22
shows a 62% sequence homology to the human form and,
likewise, a lectin activity directed toward
2-6-linked
sialyloligosaccharides (9, 11) .
In vitro assays have demonstrated that CD22 (hereafter referred to as
CD22) functions in a dual capacity, both as an adhesion molecule and as
an activation molecule. Cells induced to express CD22 by cDNA
transfection acquire the ability to adhere to a variety of different
cell types, including erythrocytes, lymphocytes (both T and B cells),
and a variety of transformed cell
lines(7, 8, 9, 12, 13) . In
certain cases, a higher level of binding has been demonstrated with
cell activation, which seems to correlate with increased expression of
-galactoside
2,6-sialyltransferase(8, 9) ,
the enzyme that synthesizes the
Sia
2-6Gal
1-4GlcNAc
sequence(14, 15) . A role in activation is indicated
by the observations that CD22 defines the subset of IgM
B cells which show increased levels of intracellular
Ca
in response to stimulation with anti-µ, and
that anti-CD22 augments this response(16) . Moreover,
anti-µ stimulation of B cells rapidly induces the phosphorylation
of cytoplasmic Tyr residue(s) on CD22, and a small percentage of
surface CD22 (
2%) may be found in association with the B cell-sIg
complex, including both IgM or IgG of naive or memory B
cells(17, 18, 19) . In addition to playing a
role with B cell activation, CD22 participates in T cell activation.
CD22 binds to several T cell glycoproteins, including CD45, and binding
of soluble CD22 to CD45 attenuates the increase in intracellular
calcium normally seen in T cells following stimulation with
anti-CD3(8, 12) .
In a series of experiments
utilizing both the full-length CD22 molecule expressed in COS
cells or a truncated three domain construct fused to the Fc portion of
Ig (CD22Rg), we and others have demonstrated that its ability to bind
to cells or precipitate glycoproteins from cell lysates is dependent on
the presence of Sia residues on the target cells or
molecules(8, 20, 21) . A sensitive column
assay showed that CD22 has a low but detectable affinity for sialylated N-linked oligosaccharides, providing that the Sia residues are
2-6-linked. In contrast,
2-3-linked residues are
not bound. Using purified
-galactoside
2,6-sialyltransferase
to sialylate a variety of complex oligosaccharide structures, we
further demonstrated that CD22Rg recognizes only the trisaccharide
Sia
2-6Gal
1-4Glc/NAc. Other structural features in
complex N-linked oligosaccharides, including branching,
fucosylation, and/or other core region sugars, were not recognized.
However, a higher apparent affinity was observed for multisialylated
structures, implying that the CD22Rg construct was capable of
interacting with adjacent
2-6-Sia residues present on the
same molecule. However, this column assay could not exclude higher
apparent binding merely due to a higher density of
2-6-Sia
residues on a single oligosaccharide.
To further understand the lectin properties of this molecule, several assay systems including equilibrium dialysis, ELISA capture, column binding, and cell adhesion, are utilized here to examine the binding of CD22Rg to a number of different mono- and bisialylated compounds. Additionally, cross-linking experiments have been performed to explore the possibility that cell-surface CD22 might be present in a multimeric form. These experiments, together with additional work in the accompanying papers (22, 23) examining the role of CD22 in cell-adhesion events, offer new insights into how this Sia-specific lectin may function in complex biological systems.
Figure 2:
Comparison of 2-6-sLac and FGP
as inhibitors of [
H]FGP binding to
CD22Rg. A 30-µl sample containing bisialylated
[
H]FGP (less than 0.1 nmol), combined with
[
C]Glc and buffer alone (
), 10
µM (
), 30 µM (
), or 90 µM (
) of unlabeled FGP (a mixture of mono and bisialylated
structures), was applied to CD22Rg-PAS and eluted at 4 °C (Panel A). The elution profile of
[
C]Glc for only one of the four runs is also
shown (
). From these data, a running sum on a percentage basis of
the eluted radioactivity was calculated, and is presented in Panel
B. A similar series of experiments was done with 0, 10, 30, and 90
µM
2-6-sLac, and these data are presented only
as a running sum in Panel C (symbols as in Panel
A).
Figure 7:
Inhibition of Daudi cell binding to CD22
expressing CHO cells by sialylated oligosaccharides. Radiolabeled Daudi
cells were allowed to bind to confluent cultures of CD22-CHO cells at 4
°C, and bound cells quantitated by scintillation counting. During
the initial binding step, either 2-6-sLac (
) or FGP
(mixture of mono- and bisialylated structures;
) was included at
the indicated concentrations. Each point represents the average of
triplicate assays.
Figure 4: Inhibition of CD22Rg-IgM binding by bisialylated compounds. Several synthetic or naturally occurring bisialylated compounds were examined in the ELISA assay. The different compounds tested are identified by number corresponding to the listing in Table 1.
Similarly prepared radioactive lysates
were subjected to immunoprecipitation with CD22Rg in the absence or
presence of 2-6-sLac, utilizing the same washing procedure.
Total amount of precipitable radioactivity was determined, and the
samples analyzed by SDS-PAGE/fluorography.
Figure 1:
Equilibrium dialysis binding of
2-6-sLac to CD22Rg. CD22Rg (5 µM protein
concentration based on a M
of 210,000) was
incubated with increasing amounts of unlabeled
2-6-sLac in
the presence of a fixed amount (2-4 pmol) of
[
H]2-6-sLac, in a total volume of 50
µl. Dialysis was against 50 µl of buffer, across a M
cut-off 100,000 dialysis membrane, for 18 h at 4 °C. Aliquots
were taken from either side of the membrane and counted, the
concentration of bound and free
2-6-sLac determined, and the
data plotted according to Scatchard. The data were fit to a single line
by a linear least squares program. The slope corresponds to a K
of 32 µM, and the x intercept indicates a stoichiometry of 2.2. R
, total concentration of receptor in
(µM).
Previously, we utilized a
column elution assay to determine CD22Rg-oligosaccharide
binding(21, 37) . By this approach, sialylated
oligosaccharides with two or more 2-6-linked Sia residues
were found to be retained longer as compared to monosialylated
structures. This observation suggested tighter binding of bisialylated
structures with the two possible binding sites on the dimeric CD22Rg
chimera. To more quantitatively study interactions with a bisialylated
biantennary oligosaccharide, Pronase glycopeptides were generated from
bovine fibrinogen, which contains exclusively biantennary N-linked structures containing only
2-6-linked Sia
residues(38) . On several batches of commercial fibrinogen
examined, sialylation was incomplete, and large quantities of pure
bisialylated material could not be generated. However, as described
under ``Experimental Procedures,'' a small quantity of
radioactive bisialylated material could be generated using
glycosyltransferases and ion exchange chromatography. This material was
employed in a ``single point'' dialysis experiment utilizing
radiotracer amounts of either
[
H]
2-6-sLac or
H-bisialylated FGP. Under conditions of large excess of
lectin over ligand, the free receptor concentration closely
approximates the total receptor concentration (R
).
Thus, provided the stoichiometry is known or can be estimated, the K
can be calculated directly from the ratio of
bound to free ligand and the R
. By this approach,
a bisialylated glycopeptide, prepared from fibrinogen, exhibited a
17-fold higher apparent affinity to CD22Rg than
2-6-sLac.
This ratio would correspond to an apparent K
of
1-2 µM.
The enhanced binding affinity of the
bisialylated FGP over that of 2-6-sLac was further
demonstrated by examining their ability to inhibit the binding of a
radiolabeled oligosaccharide to immobilized CD22Rg. The CD22Rg columns
initially utilized to demonstrate oligosaccharide binding contained
200 µg of protein, and the elution of most multisialylated
compounds required warming the column to 22-24
°C(21, 37) . A new column was constructed which
contained
25-50 µg of protein on 0.15 ml of protein
A-Sepharose. With a column of this small scale, the
[
H]FGP, applied in a total volume of 30 µl,
eluted significantly slower than the nonbinding
[
C]Glc marker (Fig. 2), and warming of
the column was not required for successful elution. When this same
30-µl sample, containing 10-90 µM unlabeled FGP
(with concentrations based on Sia groups, not peptide), is applied to
the column, the radioactivity elutes significantly earlier (Fig. 2A). These column profiles can be presented with
greater clarity by calculating a running sum, on a percentage basis, of
the eluted radioactivity (Fig. 2B). In contrast to this
result, when 10-90 µM
2-6-sLac is
included, significantly less inhibition of the binding of the
[
H]FGP is observed (Fig. 2C). As
a greater level of inhibition is seen with FGP than
2-6-sLac
for identical concentrations of competing
2-6-Sia groups,
then the FGP must have an intrinsically higher affinity for CD22Rg than
with
2-6-sLac. These results are consistent with those of
the single point dialysis experiment. The most likely mechanism for
this increased affinity would be its ability to simultaneously interact
with more than one sialic acid binding site. Alternatively, other
segments of the N-linked glycopeptides may interact with
CD22Rg (e.g. Man residues, chitobiose core sugars). However,
in prior investigations we have found no evidence to suggest that other
structural features on an N-linked oligosaccharide are
recognized by CD22Rg(37) .
Figure 3:
Structural parameters influencing the
interaction of sialosides with CD22Rg. The relative affinities of
several different sialylated oligosaccharides for CD22Rg was inferred
by determining their ability to inhibit the binding of CD22Rg to
immobilized IgM in an ELISA assay. Binding was performed in the absence
or presence of the indicated inhibitor at 4 °C for 12-15 h,
and then bound CD22Rg determined as described under ``Experimental
Procedures.'' The different compounds tested are identified by
number corresponding to the listing in Table 1. The data
presented are representative experiments, and each data point
represents duplicates ± S.D. For each series of experiments
performed on a given day, 2-6-sLac was included as a
reference compound. The data presented in Panels A, B, and C are from one series of experiments, and the inhibition
profile of
2-6-sLac is shown only in Panel A for
simplicity.
Figure 5:
Demonstration of relative binding of
bisialylated structures to CD22Rg. Compounds 26-29 were screened
for their ability to inhibit [H]FGP-CD22Rg
binding in the column assay as described in Fig. 2, using a
single concentration of 30 µM (based on Sia groups). The
elution profiles are presented as a running sum. The elution profiles
of [
H]FGP in the absence of any inhibitor (no
inhibitor) and of one typical profile of
[
C]Glc (Glc) are also
shown.
These experiments indicate that CD22Rg
is capable of binding a broad range of 2-6-sialosides and,
moreover, that structural features away from the Sia residue may
significantly influence CD22Rg binding. In confirmation of prior
studies,
2-3-linked Sia residues are not recognized (5, Fig. 3). Several molecules containing a 6-thio
derivative of Gal (2, 3, 7, 8, 9,
and 11) were examined. These compounds were originally developed
as (potentially) nonhydrolyzable inhibitors for different bacterial and
viral sialidases(31) . All these compounds bound to CD22Rg
although, for some, with a significantly poorer affinity as indicated
by a higher RIC. Compound 8 had a RIC 3-fold higher than its
non-thio derivative (6), while 9 had a lower RIC than its
non-thio derivative (10). However, this chemical modification
did not produce dramatic changes in the apparent binding affinity of
these compounds for CD22Rg.
Several C-methyl-Gal
sialosides were similarly examined. This chemical modification limits
the rotamer conformations possible with the Sia-Gal disaccharide. In
aqueous solution, two rotamer orientations (tg and gt) are commonly
found, formed by a 120° rotation around the C
-C
bond in Gal. In one (gt), the two saccharide residues are bent
back over themselves, and in the other (tg), the two residues are in an
extended conformation(31) . The (6S)- or
(6R)-C
-methyl group sterically limits this
rotation and shifts the equilibrium in favor of one or the other of
these two rotamers. These compounds have been useful in determining the
orientation of the Sia-Gal disaccharide preferred by different
sialidases, most of which show a marked preference for tg over the
gt(31) . For CD22Rg binding, the C
-methyl
derivatives all showed poorer RICs relative to non-methyl derivatives (Fig. 3B and Table 1). However, the
(6S)-C
-methyl modification appeared to be more
detrimental to sialoside binding (6versus10),
indicating a preference for the tg rotamer. This preference is lost in
the methyl-thio derivatives (8, 9, and 11), which
are poorer inhibitors of CD22Rg-IgM binding.
A large number of
different Sia 2-6-Hex(NAc) sialosides (with Hex(NAc) being
GlcNAc, GalNAc, or Gal) are recognized by CD22Rg (Fig. 3, C and D, and Table 1). A preference for GlcNAc over
GalNAc is suggested by the different RICs of compounds 12versus13 and 15versus16,
although a difference in the linkage (
versus
) of
the blocking groups may also explain the differences seen. CD22Rg
binding was significantly influenced by the structure of the group
attached to the Hex(NAc) residue. For example, the RIC of 18,
which has a ONP group attached to a GlcNAc residue, is 40-fold better
than that of 15, which contains a benzyl alcohol group instead.
Additionally, Glc is favored over trimethylsilane (compare 1 and 4), and we previously demonstrated that Glc was favored over
glucitol(37) . Other pairs of compounds differing only in their
reducing group were not available. Several structures based on the
2-6-sialylated Gal
1-3GalNAc/GlcNAc trisaccharide (12 and 13), which is not found in nature, also were
reasonable inhibitors of CD22Rg-IgM binding. Moreover, significant
modifications of the Hex(NAc) residue are permissible, including
3-O-galactosylation (compounds 15 and 16) as
well as capping O
-O
with alkyl groups, as found
in the diisopropylidene derivatives. Taken together, these results
indicate that Sia
2-6Hex(NAc) sialosides are recognized by
CD22Rg. These observations are significant as they indicate that a wide
range of
2-6-sialylated structures, found on both N- and O-linked structures and glycolipids, may be
potential ligands for CD22Rg.
Previously, we demonstrated that
glycopeptides from bovine submaxillary mucin, a rich source of
Sia2-6GalNAc
-(peptide) residues, was not recognized
with high affinity by CD22 even after de-O-acetylation of
sialic acids(37) . When this same bovine submaxillary mucin
preparation was screened by the column assay as described in Fig. 2, a level of inhibition of [
H]FGP
binding corresponding to 30 µM
2-6-sLac was
seen with 900 µM bovine submaxillary mucin (based on Sia
concentration; data not shown), confirming our earlier
result(37) . Thus, the low RIC of compound 17 (Table 1), which is comparable to the mucin structure must be
explained either by a positive effect of the ONP aglycone, or by a
negative effect of the polypeptide in the case of bovine submaxillary
mucin.
Several modifications were without effect on sialoside binding. Sulfation did not affect the binding of sialylated compounds, and sulfate did not substitute for sialic acid (Fig. 3E). The sulfated derivative of 16 did exhibit a 4.5-fold improvement in its RIC (16versus14, Table 1), yet these compounds also differ in their reducing terminus blocking group. As with 17, 18, and 19, the ONP group improves the compound's RIC considerably.
Given this
limitation with the ELISA assay, compounds 26-29 were
examined with the column retention assay, using each at a concentration
of 30 µM. These results indicate measurable differences in
the ability of these compounds to compete with the binding of the
[H]FGP sample to CD22Rg, with 26 being the
most potent inhibitor (Fig. 5). Although it would appear to be
more potent than FGP at 30 µM, the FGP preparation
contained a mixture of mono- and bisialylated structures. While these
four sialosides are structurally very similar, they are all branching
isomers and, consequently, the terminal
2-6-Sia residues
will be oriented differently(29, 30) . Molecular
modeling predicts that the C
-C
distance between
the two Sia molecules is 17-19 Å for 26 and 29, and 9-10 Å for 27 and 28(29) , thus, these measurements do not correlate with the
different RICs seen for these four compounds (Table 1). Other
structural features, such as the relative orientation of the
Sia
2-6Gal units, must be involved. Earlier we presented
evidence indicating that CD22Rg was capable of discriminating between
two different bi(Sia
2-6)-tetraantennary isomers, although we
were not able to directly prove which antennae were
sialylated(37) . The observations with these four synthetic
bisialylated compounds confirms our earlier hypothesis that CD22Rg
binding is influenced by the relative positioning of multiple Sia
groups on a single oligosaccharide.
Figure 6:
Inhibition of CD22Rg binding to Daudi cell
glycoproteins by 2-6-sLac. Equal aliquots of
[
S]Met-labeled Daudi cell lysate were precleared
with protein A-Sepharose and then precipitated with CD22Rg and
additional protein A-Sepharose in the presence of buffer alone (lane 2), or 30 µM (lane 3), 90
µM (lane 4), or 1 mM (lane 5)
2-6-sLac. Material nonspecifically adsorbed to an equal
volume of protein A-Sepharose in the absence of CD22Rg is shown in lane 1. After boiling the beads with sample buffer, equal
aliquots of the solubilized radioactivity were examined by SDS-PAGE
analysis. Other aliquots were used to determine the amount loaded (cpm)
of 800 (lane 1), 12,620 (lane 2), 6,860 (lane
3), 3,620 (lane 4), and 2,500 (lane 5); and to
show that all of this radioactivity is trichloroacetic
acid-precipitable (data not shown). TD, tracking
dye.
Figure 8:
Cross-linking of CD22 on CD22 expressing
CHO cells. Cells metabolically labeled with
[S]Met were cross-linked with the thio-cleavable
agent, DTSSP, at the indicated concentration, and immunoprecipitated
with mAb To15, directed against human CD22. After purification,
one-half of each sample was reduced with 2-mercaptoethanol (2-ME), as indicated, and both reduced and nonreduced samples
were analyzed on a 6% SDS-PAGE. The molecular weight standards are
indicated on the left. TD, tracking
dye.
Previously, we established that the B cell sialic
acid-binding protein, CD22, specifically recognized the trisaccharide
Sia2-6Gal
1-4GlcNAc as commonly found on N-linked oligosaccharides, but also on some O-linked
oligosaccharides and glycolipids(21, 37) . These
studies were all accomplished with a column-binding assay, employing
the bivalent immunoglobulin fusion construct CD22Rg bound to protein
A-Sepharose, in which the elution of appropriately sialylated
structures was significantly retarded beyond that of a nonbinding
monosaccharide. Structures with more
2-6-Sia residues were
separated from those with less Sia residues. This observation suggested
the possibility of multivalent interaction between a multiply
sialylated oligosaccharide and the bivalent CD22Rg construct.
Alternatively, the better retention could be merely due to a
statistical effect, i.e. increased probability of interactions
with the column during the elution. The results described herein
demonstrate that CD22Rg chimera has two binding sites for
2-6-sLac, each with a K
of
30
µM. Although equilibrium dialysis could not be performed
on a bi(Sia
2-6)-biantennary oligosaccharide, additional
studies indicated that this compound bound to CD22Rg with approximately
17-fold lower K
. Additionally, a comparison of the
inhibitory potency of several bisialylated oligosaccharides against
2-6-sLac in the column retention assay further supports the
likelihood that simultaneous interaction is occurring. In these assays,
the bisialylated compounds are clearly more potent inhibitors than
2-6-sLac when compared on an equimolar basis relative to
2-6-Sia groups, indicating that they have a higher affinity
than
2-6-sLac alone. The work presented here and previously (21) has indicated that no other structural features in a
multiantennary oligosaccharide are recognized by CD22Rg. Thus, the
enhanced affinity of the bisialylated oligosaccharides is most likely
due to the binding of CD22Rg to the two
Sia
2-6Gal
1-4GlcNAc structures terminating the
antennae. Extensive studies on antibody-hapten binding (reviewed in (26) ) would argue that this enhanced affinity is due to the
simultaneous binding of the two
Sia
2-6Gal
1-4GlcNAc moieties to CD22Rg.
Unambiguous proof of this point, of course, would require direct
equilibrium binding studies with mono- and bivalent forms of CD22 with
mono- and bivalent sialylated oligosaccharides.
Even if a single
multisialylated oligosaccharide does simultaneously interact with the
two lectin binding sites of CD22, the latter is in fact an artificially
created chimeric protein. It is more important to know if a similar
phenomenon can occur with native full-length CD22 which is
initially synthesized as a monomer. Indeed, two lines of investigation
(inhibition of binding by oligosaccharides and cell surface
cross-linking) suggest that the native molecule expressed on the
surface of CHO cells, associates to form noncovalent oligomers. This
multimeric association offers a mechanism of producing a higher
apparent binding affinity than would be observed with single chain
CD22.
Collectively, the results obtained with the different
monosialylated compounds examined in Table 1indicate that the
minimum structure required for CD22Rg binding is
Sia2-6-Hex(NAc), where Hex(NAc) is Gal, GalNAc, or GlcNAc.
An examination of the different RICs of the compounds listed in Table 1, together with our earlier studies offers considerable
insight into the regions of the sialoside which are recognized by the
protein. Fig. 9shows Neu5Ac
2-6Gal
1-R in the tg
rotamer (the gt rotamer is formed by a 120 °C rotation around the
Gal C
-C
bond), and we propose a model for
CD22-sialoside binding based on these data and earlier
studies(13, 21, 39) . The
C
-C
-C
side chain of Sia is
essential for binding, as either its removal or its
9-O-acetylation abolishes
binding(13, 20, 21) . The N
substituent on Sia (glycolyl versus acetyl) may be in
close proximity to the binding site, as the murine form of CD22 shows a
partial preference for Neu5Gc over Neu5Ac (a preference not seen with
human CD22)(11) . The Gal O
-C
face of
this disaccharide must also be involved in CD22 binding as these
positions are the key structural features which distinguish
2-6-sLac from
2-3-sLac. The generally unfavorable
effect of C
methylation of Gal likewise indicates that this
region of the disaccharide faces the binding site. In contrast, the
other hydroxyl groups of the Gal residue are not involved in CD22
binding, as (a)
2-6-sialylated Gal, GalNAc, and
GlcNAc are all recognized; (b) capping some or all of the
hydroxyl groups with alkyl groups (as in the case of the
di-O-isopropylidene derivative) or with an additional Gal
residue was without effect on binding; (c) the configuration
at Gal C
is not significant (as both
2-6-sialylated GalNAc and GlcNAc are recognized). The group
at the reducing terminus of Gal (R
in Fig. 9) must
also face against the protein (as opposed to being oriented out into
the solvent) as different blocking groups at this position
(trimethylsilane, benzyl, ONP, H, Glc, Glc-OH, or peptide) markedly
influence binding. Thus, we would predict that the CD22 binding site
would be an area sufficiently large to accommodate these multiple
points of contact.
Figure 9:
Proposed model of CD22-sialoside binding.
The disaccharide Sia2-6Gal, in the tg rotamer conformation,
is presented interacting with the binding pocket of CD22. This model is
designed to incorporate the data presented in Table 1, and under
``Discussion.'' R
: Gal, GlcNAc, GalNAc,
trimethylsilane, benzyl, ONP, allyl, isopropylidene or H; R
: isopropylidene, OH, or NAc; R
: Gal, isopropylidene, or OH; R
: isopropylidene or OH; and R
: methyl or OH.
The binding specificity of CD22 is somewhat
comparable to that of the lectin SNA. As studied by precipitation
assays, this lectin is specific for the sequence
Sia2-6Gal/GalNAc, with a K
(by
equilibrium dialysis) of 2.5 µM(25, 40) .
As with CD22, the C
-C
-C
side chain
of Sia is required for binding(40) . Unlike CD22, however, SNA
recognizes additional structural determinants in an oligosaccharide
structure, as evidenced by the 100-fold greater inhibitory potency of
Sia
2-6Gal
1-4GlcNAc
1-3Gal
1-4Glc
over
2-6-sLac(40) . Moreover, lactose at
concentrations of 0.1 M are capable of inhibiting
SNA-oligosaccharide binding(25, 40) , an effect which
is not found with CD22Rg(37) . Mucins containing the
Sia
2-6GalNAc sequence bind well to SNA (judged by a
precipitin assay)(40) , while preparations of such mucins bind
very poorly to CD22(37) . While 9-O-acetylated
2-6-Sia groups are not recognized by CD22, it is not known
if SNA binding can be affected by this substitutions. 6-Thio
derivatives are not recognized by SNA, nor does it show preferential
binding to any of the synthetic disialosides examined here (26-29)(29, 30) .
The CD22Rg chimera
was constructed to be bivalent by inclusion of the hinge region of IgG.
The ability of a bi(Sia2-6)-biantennary oligosaccharide to
simultaneously interact with both binding sites is indicated by the
higher levels of inhibition of the bisialylated compounds over
2-6-sLac, both in the cell binding assay and the column
assay. The different levels of inhibition achieved by synthetic
bisialylated compounds 26-29 indicate that the construct
shows a preference for certain branching orientations. The ability of
multiantennary oligosaccharides to simultaneously interact with
multiple binding sites on multisubunit lectins has been demonstrated
with several
lectins(29, 41, 42, 43, 44) .
One well studied example is the hepatic Gal/GalNAc lectin. This protein
is a trimer, with each monomer containing two Gal/GalNAc binding sites.
An oligosaccharide with a single Gal residue on its nonreducing
terminus binds with a K
of 1 mM, and
branched structures containing two and three terminal Gal residues bind
with affinities approximately 10
and 10
tighter, respectively (41, 43, 45) .
Likewise, the affinity of the cation-independent Man-6-phosphate
receptor, which contains two binding sites, for ligands containing two
Man-6-P groups is 300-fold higher than for ligands containing just
one(44) . From basic thermodynamic principles, the apparent K
for dimeric receptor-ligand binding should be
the product of each individual binding interaction; the extent that
this theoretical limit is not met is reflective of strain or steric
factors which limit the simultaneous independent interaction of the two
receptor-ligand pairs(26) . The differences in apparent binding
affinity of the different bisialylated compounds (26-29; Table 1) is indicative that the two Sia binding sites on CD22Rg
are not optimally oriented for all bisialylated structures, i.e. that different levels of intramolecular strain are induced in
these compounds when bound to CD22Rg.
The inability of the ELISA assay to detect the bivalent nature of oligosaccharide recognition is surprising and without obvious explanation. However, the ELISA capture assay, involving a primary binding and then a secondary reagent, with multiple washing steps in between, is very different from a solution phase equilibrium or column assay. This discrepancy indicates that ELISA capture assays may not always be the best assay for describing interactions which may potentially be multivalent.
The binding
affinity for a monovalent ligand seen here, 30 µM, is
in line with that observed for other lectin-oligosaccharide
interactions(45) . For a single receptor-ligand interaction,
this affinity is comparatively low and would suggest that in nature,
CD22's function as an adhesion molecule is dependent upon
multiple interactions. Conversely, it is possible that higher affinity
interactions will be found with naturally occurring ligands. The
CD22-containing oligomers we have reported here may be part of a
mechanism to create a receptor structure capable of distinguishing
between different
2-6-sialyloligosaccharides. Such a
mechanism(s) could be very important in the role(s) CD22 plays in
cell-cell adhesion and signaling.
The cross-linking studies
presented here indicate that native CD22 may also utilize multivalency
to achieve functional interactions. No dimers were seen in these
studies, indicating that at least a portion of cell-surface CD22 exists
in a multimeric state. Also, no association was seen with any other
metabolically-labeled proteins, implying that these complexes are
homomultimers. In support of this, the intensity of the CD22 monomer
band decreased in the presence of the cross-linker to an extent
qualitatively consistent with the appearance of the multimer bands.
Furthermore, the anti-CD22 antibody adsorbed less than 0.2% of the
total macromolecular [S]Met material, and in the
presence of the cross-linker, the total amount of counts/min adsorbed
to To15/protein A-Sepharose was
75% of that in its absence. If
CD22 were being cross-linked to a heterogeneous array of different
proteins (and thus not visible as a discrete band after reduction) then
the amount of counts/min would be expected to increase in the presence
of the cross-linking agent. Of course, we cannot rule out the presence
of non-labeled proteins in these multimers (either long-lived or poor
in Met/Cys residues). Regardless, since CHO cells (a fibroblastic cell
line) are capable of supporting the formation of these multimers, we
can be certain that no B lymphocyte-specific proteins are required. It
is possible that the CD22 molecules self-associate in the membrane
following biosynthesis. In this regard, CD22 might be similar to the
cation-dependent Man-6-P receptor, which although it has a
stoichiometry of 1:1 for the cognate oligosaccharide(46) , is
able to generate high-affinity binding via the reversible formation of
dimers and multimers(47, 48, 49) .