(Received for publication, November 13, 1995; and in revised form, January 24, 1996)
From the
B-lymphocyte antigen CD22 is a member of the recently described
sialoadhesin family of immunoglobulin-like cell-surface glycoproteins
that bind glycoconjugates terminating in sialic acid. One prominent
ligand for CD22 is the highly glycosylated leukocyte surface protein
CD45. Using surface plasmon resonance spectroscopy, we characterized
the interaction of recombinant mouse CD22 with native CD45 purified
from rat thymus (CD45-thy). By in situ desialylation and
resialylation of immobilized CD45-thy, we show that mouse CD22 binds to
the sialoglycoconjugate NeuGc2-6Gal
1-4GlcNAc
carried on CD45-thy N-glycans. Previous studies have shown
that the sialic acid-binding site lies within the two membrane-distal
domains of CD22 (domains 1 and 2), which are V-set and C2-set
immunoglobulin superfamily domains, respectively. To further localize
the binding site, we have made 42 single amino acid substitutions
throughout both domains. All 12 mutations that abrogated binding to
CD45-thy without disrupting antibody binding were of residues within
the GFCC`C"
-sheet of domain 1. These residues are predicted to
form a contiguous binding site centered around an arginine residue in
the F strand that is conserved in all members of the sialoadhesin
family. Our results provide further evidence that immunoglobulin
superfamily cell adhesion molecules use the GFCC`C"
-sheet of
membrane-distal V-set domains to bind structurally diverse ligands,
suggesting that this surface is favored for cell-cell recognition.
Immunoglobulin superfamily (IgSF) ()domains are
probably the commonest domain type involved in cell-surface
recognition, being present in
40% of all proteins identified on
the surface of leukocytes(1) . One possible reason for this is
that IgSF domains provide a stable, but versatile, recognition
platform, capable of binding to structurally diverse
ligands(2) . Typically, IgSF cell adhesion molecules bind
either to other IgSF molecules or to integrins (3, 4) , but recent reports indicate that some IgSF
cell adhesion molecules bind carbohydrate ligands (reviewed in (5) ). The best characterized of these lectin-like IgSF
proteins are a group of homologous proteins (termed the sialoadhesin
family) that bind carbohydrate structures terminating in sialic
acid(5, 6, 7) . Members of this family
include the leukocyte proteins CD22, sialoadhesin, and CD33 as well as
myelin-associated glycoprotein and Schwann cell myelin
protein(5, 6, 7) .
CD22 is expressed on a subpopulation of mature B-cells and has been implicated in cell adhesion as well as in modulating signaling through the B-cell antigen receptor (BCR) (reviewed in (8) ). CD22 associates loosely with the BCR (9, 10) and is tyrosine-phosphorylated following BCR ligation(11) . This leads to association with and activation of the tyrosine phosphatase SHP(12) , which can inhibit signaling through the BCR(13, 14, 15) . The binding of anti-CD22 antibody-coated beads to B-cells decreases the activation threshold of the BCR, presumably by removing CD22 (and associated SHP) from the vicinity of the BCR(12) . Together, these findings suggest that physiological interactions between CD22 and natural cell-surface ligands may function to modulate signaling through the BCR(12) .
The extracellular region of mouse CD22 (18) consists of a single membrane-distal V-set IgSF domain
(domain 1), followed by six C2-set IgSF domains (domains 2-7).
However, two cDNA clones of human CD22 have been identified (CD22 (16) and CD22
(17) ), one of which (CD22
)
lacks the sequence encoding IgSF domains 3 and 4. Human CD22
(which is equivalent to mouse CD22 and is henceforth called CD22) binds
with a low affinity (K
30
µM at 4 °C(18) ) to the sialylated
glycoconjugate
NeuAc
2-6Gal
1-4Glc(NAc)(19, 20) .
Although this structure is very common on N-glycans,
recombinant CD22 appears to bind only to a limited number of lymphocyte
cell-surface (19, 21, 22) and plasma (23) glycoproteins, suggesting that some of these molecules are
preferred ligands. One prominent ligand is the large, abundant, and
highly glycosylated leukocyte cell-surface glycoprotein
CD45(22, 24, 25) , which carries multiple N-glycans terminating in
2-6-linked sialic
acid(26) .
With the exception of antibody-carbohydrate
interactions, little is known about carbohydrate recognition by IgSF
molecules(5) . As a first step toward understanding the
structural basis of sialic acid recognition, we undertook to identify
the sialic acid-binding site on CD22. Previous studies on human (27) and mouse (22, 28) CD22 have shown that
the sialic acid-binding site lies within domains 1 and 2. In the
present study, we extend this work by making single amino acid
substitutions of surface residues throughout domains 1 and 2 of mouse
CD22. Our results suggest that the CD22 sialic acid-binding site is
situated on the GFCC`C" -sheet of domain 1 centered on an arginine
residue in the F strand that appears to be essential for sialic acid
recognition.
Figure 1:
Mouse CD22Fc binds
NeuGc2-6Gal
1-4GlcNAc carried on CD45 N-glycans. A-D, CD45-thy was covalently coupled
to the BIAcore sensor surface. CD22Fc and the sialic acid-binding
lectins MAA and SNA were then injected at 0.5 mg/ml for 4 min each (bars) over unmodified thymic CD45 (A),
sialidase-treated CD45 (B), sialidase-treated CD45
resialylated with NeuAc using Gal
1-4GlcNAc
2-6-sialyltransferase (C), or sialidase-treated
CD45 resialylated with NeuGc using Gal
1-4GlcNAc
2-6-sialyltransferase (D). Following each
injection, bound protein was eluted with a 4-min injection of 100
mM HCl (arrows mark the beginning of these
injections). E, sCD45ABC-CHO was coupled to the sensor
surface. Mouse CD22Fc, MAA, and SNA were injected (0.5 mg/ml for 4 min)
first over unmodified sCD45ABC-CHO and then after the indicated
desialylation and resialylation steps. The binding response during each
injection was measured 20 s after the injection (to eliminate the bulk
phase effect) and is expressed as a percentage of the maximal response
seen for each ligand during the experiment, which was 1380, 3640, and
7300 response units for CD22Fc, MAA, and SNA, respectively. ST6N, Gal
1-4GlcNAc
2-6-sialyltransferase; ST3N,
Gal
1-3(4)GlcNAc
2-3-sialyltransferase.
When CD22Fc was injected
over a sensor surface to which CD45-thy had been covalently
immobilized, there was an increase in the response (measured in
response units), which indicates binding of CD22Fc to CD45-thy (Fig. 1A). Following completion of the injection, the
response decreased slowly, reflecting dissociation of bound CD22Fc (Fig. 1A). The remaining CD22Fc was eluted rapidly by
the injection of 100 mM HCl (Fig. 1A, arrow). The lectins MAA and SNA (which are specific for
2-3- and
2-6-linked sialic acids, respectively)
were also bound (Fig. 1A), indicating that CD45-thy
carries both
2-3- and
2-6-linked sialic acids.
Treatment of the immobilized CD45-thy with sialidase abolished CD22Fc
binding and substantially decreased both MAA and SNA binding (Fig. 1B). When desialylated CD45 was
2-6-resialylated with Gal
1-4GlcNAc
2-6-sialyltransferase using NeuAc as substrate, SNA binding
increased substantially, indicating successful
2-6-resialylation, but CD22Fc was still unable to bind (Fig. 1C). In contrast, CD22Fc binding was fully
restored when CD45-thy was
2-6-resialylated using NeuGc as
substrate (Fig. 1D). The specificity of the
2-6-resialylation is indicated by the increase in SNA (but
not MAA) binding following resialylation (Fig. 1, C and D).
These results indicate that mouse CD22 binds to
2-6-linked NeuGc carried on CD45-thy N-glycans.
Further evidence that the sequence
NeuGc
2-6Gal
1-4GlcNAc was both necessary and
sufficient for CD22Fc binding was obtained in an experiment using
sCD45ABC-CHO. The lectin MAA bound unmodified sCD45ABC-CHO, whereas SNA
did not, indicating that sCD45ABC-CHO contains no detectable
2-6-linked sialic acid (Fig. 1E, Untreated). Therefore, it is not surprising that CD22Fc did
not bind unmodified sCD45ABC-CHO (Fig. 1E, Untreated). However, CD22Fc did bind sCD45ABC-CHO following
2-6-resialylation with NeuGc (Fig. 1E). In
contrast, neither
2-6-resialylation with NeuAc nor
2-3-resialylation with NeuGc could restore CD22Fc binding (Fig. 1E). Taken together, these results establish that
mouse CD22Fc binds to CD45-thy through an interaction with the
structure NeuGc
2-6Gal
1-4GlcNAc carried on
CD45-thy N-glycans. This is consistent with a recent analysis
of the specificity of mouse CD22Fc using resialylated
erythrocytes(44) .
Figure 2:
Alignment of CD22 with IgSF molecules of
known structure. The predicted protein sequences of domains 1 and 2 of
the mouse CD22 alleles C57Bl (this study), BALB/c
(GenBank/EMBL accession number L02844), and DBA/2J
(GenBank
/EMBL accession number L16928) were manually
aligned with human CD22 (GenBank
/EMBL accession number
X59350), mouse sialoadhesin (GenBank
/EMBL accession number
Z36293), and either rat CD2 (domain 1 (d1)) (SwissProt
accession number P08921) or VCAM-1 (domain 2 (d2)) (SwissProt
accession number P19320). The
-strand assignments (solid
bars) were based on the structures of CD2 (57, 64) and VCAM-1 (59) as well as on
structural data from other IgSF
domains(2, 60, 61) . The division between
domains 1 and 2 of mouse CD22 is made at the junction of exons 4 and
5(55) . Dashed lines instead of bars are shown where
there are no grounds for making precise assignments to
-strands. Boxed mouse CD22 residues were mutated in the present study,
whereas boxed sialoadhesin residues were mutated in the
accompanying study(65) .
To aid in the selection of residues to mutate,
domains 1 and 2 of CD22 were aligned with IgSF domain sequences for
which there are structural data available (Fig. 2). Domain 1 of
CD22 was aligned with the V-set domain (domain 1) of rat CD2 (57, 58) (Fig. 2), whereas domain 2 was aligned
with domain 2 of VCAM-1(59) . CD22 residues in domains 1 and 2
could be assigned accurately to the structurally conserved B, C, E, and
F -strands (Fig. 2) by aligning residues characteristic of
IgSF domains(2, 60, 61) . In a similar
manner, residues in CD22 domain 1 could be assigned to the beginning of
the D strand and to the end of the G strand, and residues in domain 2
could be assigned to the A strand and to the end of the G strand (Fig. 2). In contrast, CD22 residues could not reliably be
assigned to the C` and C" strands of domain 1 or to the C`/D strand of
domain 2 (Fig. 2). The assignment of residues to the loop
regions was tentative except for the E-F loop, which is
structurally conserved in V-set and C2-set IgSF
domains(2, 60, 61) .
The sialic
acid-binding site on sialoadhesin has been definitively localized to
its V-set domain (domain 1(28) ), but in the case of CD22, a
contribution from domain 2 has not been ruled
out(27, 28) . To further localize the sialic
acid-binding site on CD22, we mutated residues predicted to lie on the
surface of domain 1 or 2. We introduced drastic changes rather than
mutating to alanine because our primary aim was to delineate the
structural binding site. It has been shown that alanine mutagenesis may
only identify a fraction (25-40%) of the residues within the
binding site(62, 63) . We have previously used this
approach of making drastic mutations to identify the interacting
surfaces of the cell adhesion molecules CD2 and CD48 (42) (
)and obtained results that agree well with
structural studies(58, 64) .
Figure 3: Analysis of CD45 binding to CD22 mutants. An outline of the approach used in these experiments is shown in A (upper left). TCS containing the indicated CD22Fc mutant was injected (long bars) for 20-40 min over a sensor surface to which an anti-Fc mAb had been covalently coupled. BSA and purified rat CD45 (both at 26 µg/ml) were injected for 3 min each (short bars) before and after CD22Fc was bound to the sensor surface. An increase in the response with the second injection of CD45 reflects binding to that particular CD22 mutant. The mutants in A and B were analyzed in different experiments and should be compared with wild-type CD22Fc in A and B, respectively. The results with R130A (solid lines) and R130K (dotted line) are superimposed (B, lower left)
Initially, nine
mutations were made in each of domains 1 and 2 (Table 2). Of
these, only two mutations, both in domain 1, led to a decrease in CD45
binding (R130E and E140K) (Fig. 3A and Table 2).
Both mutants bound normally to mAb CY34 (Fig. 4A and Table 2). The sequence alignment (Fig. 2) places Arg-130
and Glu-140 on adjacent F and G -strands in domain 1 (Fig. 5). Interestingly, Arg-130 is one of only five residues in
domain 1 (apart from residues characteristic of IgSF domains) that are
completely conserved within the sialoadhesin family (indicated by &cjs3622; in Fig. 2)(2, 56, 60, 61) ,
suggesting that it may play an important role in sialic acid
recognition. To provide stronger evidence for this, we made the
substitutions R130A and R130K, which are less likely to abrogate
binding by introducing unfavorable effects. Both mutations abolished
CD45 binding (Fig. 3B) without affecting the binding of
mAb CY34 (Table 2), suggesting that Arg-130 is critical for
sialic acid recognition.
Figure 4: A, analysis of CY34 binding to CD22 mutants. TCS containing the indicated CD22Fc mutants was injected for 12 or 40 min over a sensor surface to which an anti-Fc mAb had been covalently coupled (see Fig. 3). The anti-CD22 mAb CY34 (in TCS) was then injected over the immobilized CD22 mutants for 4-6 min. An elevated base line following the injection of CY34 TCS indicates binding. B, CY34 and CD45 bind to different regions of CD22. In this experiment, an anti-Fc mAb was immobilized to the sensor surface. Left, BSA and CD45 (65 µg/ml each) were injected for 4 min before and after the binding of wild-type CD22Fc (30 µg/ml, 10 min) to the sensor surface. Middle, purified CY34 (30 µg/ml, 6 min) was injected before and immediately after the binding of CD22Fc to the sensor surface, followed by injection of BSA and CD45. Right, after the binding of CD22Fc, BSA and CD45 are injected, followed by CY34.
Figure 5:
Approximate positions of mutations in
domain 1 of mouse CD22 that disrupt CD45 and CY34 binding. This ribbon
drawing is intended to represent a typical V-set IgSF domain and is
drawn with MOLSCRIPT (73) using the coordinates of domain 1 of
human CD2(58) . The positioning of the mutated CD22 residues is
based on the alignment of CD22 with rat CD2 in Fig. 2. Because
the alignment is poor over the entire C`-C" region, the precise
positions of Lys-74, Thr-76, Lys-85, and Lys-88 cannot be predicted,
and so they are depicted with broken circles. For clarity, the
residues mutated in -strand B (Arg-43 and Lys-47) and the
B-C loop (Lys-49 and Asp-58) are not labeled. Lys-149 is included
in this figure, although it lies at the junction of domains 1 and
2.
The binding site was further defined with
22 additional mutations in and around the GFCC`C" -sheet of domain
1 ( Fig. 2and Table 2). Of a total of 42 mutations made (Table 2), 30 had little or no effect on CD45 binding (examples
include K74E, R120D, K149D, and K185E (Fig. 3)), 10 completely
abolished CD45 binding (examples include R130E, R130K, R130A, and W138R (Fig. 3)), and 2 substantially decreased, but did not abolish,
CD45 binding (E140K and K73E (Fig. 3)). The partial effect of
the latter mutants suggests that they lie on the periphery of the
binding site. According to the alignment shown in Fig. 2, the
mutations that abrogate CD45 binding fall within the GFCC`C"
-sheet and are predicted to form a well defined contiguous region
centered around Arg-130 in the F strand ( Fig. 2and Fig. 5). The positioning of the F and C strand mutations is
likely to be correct because the alignment of CD22 with CD2 in both
these regions is excellent (Fig. 2), and these strands form part
of the structurally conserved core of IgSF
domains(60, 61) . Because of a poor alignment with
CD2, the positioning of the G and C` strand mutants is more tentative (Fig. 2). However, it is clear that residues in the F-G
loop and/or the beginning of the G strand contribute to the
CD45-binding site.
Our finding that none of the nine mutations in
domain 2 affect CD45 binding ( Fig. 3and Table 2) suggests
that domain 2 does not contribute directly to sialic acid recognition.
This is consistent with the observation that domain 1 of sialoadhesin
is sufficient for sialic acid binding(28) . Furthermore,
mutagenesis of sialoadhesin (65) suggests that its sialic
acid-binding site is also localized to the GFCC`C" -sheet of
domain 1, centered around the same conserved F strand arginine ( Fig. 2and Fig. 6). Taken together, these data suggest
that sialic acid recognition by CD22 and sialoadhesin involves only
domain 1. Prior observations that domain 1 of CD22 binds poorly (22) or not at all (27, 28) to ligand when
expressed in the absence of domain 2 may be explained by an inability
of domain 1 to fold correctly in the absence of domain 2. Support for
this is provided by two lines of evidence that suggest that domains 1
and 2 of CD22 are intimately associated. First, the conserved cysteines
present in domains 1 (A-B loop) and 2 (B-C loop) of
sialoadhesin family members appear to form an interdomain disulfide
bridge (66) . (
)Second, residues in both domains 1
and 2 contribute to the CY34 epitope (see below and Table 2).
Figure 6:
Ligand-binding sites on the cell-cell
recognition molecules CD22, sialoadhesin, VCAM-1, CD80, and CD2. Shown
are the positions (filled circles) of mutations reported to
disrupt the following interactions: CD22 (this study) and sialoadhesin (65) with sialoglycoconjugates, VCAM-1 with VLA-4(59) ,
CD2 with CD58(58, 69, 70, 71) , and
CD80 (72) with CD28 or CTLA-4. All the mutations lie within
membrane-distal V-set IgSF domains of these molecules. The ribbon
drawings of human CD2 and VCAM-1 are based on their crystal
structures(58, 59) . A ribbon drawing of domain 1 of
CD2 is used as a template to display the CD22, sialoadhesin, and CD80
mutants. The positioning of the residues was guided by the -strand
assignments in Fig. 2and (72) . This figure was drawn
using MOLSCRIPT(73) .
A potential source of artifact in the present study is the possibility that some or all of the mutants do not lie within the sialic acid-binding site, but instead disrupt the overall folded structure of CD22. While this possibility cannot be eliminated, several considerations suggest that this is unlikely. First, all mutants that did not bind CD45 still bound mAb CY34. Our mutagenesis studies suggest that CY34 binds to a ``discontinuous'' or ``conformational'' epitope on CD22 (see below), which requires the correct folding of domain 1 and 2. This is supported by our observation that several, widely spaced mutants that were expressed only at very low levels bound neither CD45 not CY34 (see Footnote a to Table 2). Second, the 12 mutations that decrease CD45 binding (without affecting CY34 binding) lie within a single contiguous area, with the mutations that have a partial effect (K73E and E140K) situated on the edge of this area. And finally, mutations in the equivalent region of sialoadhesin also disrupt sialic acid binding without disrupting the binding of mAbs directed to this domain 1(65) .
The
variable IgSF domains in B- and T-cell antigen receptors are capable of
binding to an enormous variety of structures. However, antigen
recognition involves loop regions between the -sheets, which are
known to display considerable structural diversity. In contrast, the
-sheets show far less structural diversity. Indeed, the central
portion of each
-sheet (comprising the B, C, E, and F
-strands) forms the structurally conserved core of the IgSF
fold(60, 61) . The observation that GFC(C`C")
-sheets bind to structures as diverse as integrins, IgSF
molecules, and sialoglycoconjugates provides impressive evidence of the
versatility of IgSF domains(2) .