(Received for publication, November 13, 1995; and in revised form, January 24, 1996)
From the
The sialoadhesins are a distinct subgroup of the immunoglobulin
superfamily, comprising sialoadhesin, CD22, the myelin-associated
glycoprotein, and CD33. They can all mediate sialic acid-dependent
binding to cells with distinct specificities. Sialoadhesin is a murine
macrophage-restricted cell-surface molecule with 17 extracellular
immunoglobulin-like domains that recognizes NeuAc2-3Gal in N- and O-glycans and interacts preferentially with
cells of the granulocytic lineage. Its sialic acid-binding site is
located within the NH
-terminal (membrane-distal) V-set
domain. Here we have carried out site-directed mutagenesis in an
attempt to identify the binding site of sialoadhesin. A subset of
nonconservative mutations disrupted sialic acid-dependent binding
without affecting binding of three monoclonal antibodies directed to
two distinct epitopes of sialoadhesin. A CD8
-based molecular model
predicts that these residues form a contiguous binding site on the
GFCC`C"
-sheet of the V-set domain centered around an arginine in
the F strand. A conservative mutation of this arginine to lysine also
abolished binding. This amino acid is conserved among all members of
the sialoadhesin family and is therefore likely to be a key residue in
mediating sialic acid-dependent binding of sialoadhesins to cells.
Carbohydrate-binding proteins mediate diverse biological functions in animals (reviewed in (1) ). The mammalian lectins so far identified can be divided into groups according to their structural similarities. These include the C-type lectins, the galectins, the P-type lectins, and the recently characterized immunoglobulin-type or I-type lectins (reviewed in (2) ). The best characterized I-type lectins are the sialoadhesins, a homologous group of cell-surface glycoproteins that recognize distinct sialylated glycans(3, 4) . Members of the sialoadhesin family include the eponymous member, sialoadhesin, which is expressed only on macrophage subpopulations(5) ; CD22, a B-cell-restricted antigen(6, 7) ; the myelin-associated glycoprotein expressed by oligodendrocytes and Schwann cells(8) ; and CD33, an antigen restricted to myelomonocytic cells(9) .
Each
member of the sialoadhesin family contains a single
NH-terminal (membrane-distal) V-set domain followed by
differing numbers of C2-set domains, ranging from 1 in CD33 to 16 in
sialoadhesin. The greatest sequence similarity between sialoadhesins is
found in the NH
-terminal two to four domains(5) .
In contrast, their cytoplasmic regions show little homology, indicating
distinct intracellular functions of these proteins.
Sialoadhesin
specifically recognizes NeuAc2-3Gal as a minimal
oligosaccharide in O- and N-linked glycans and
glycolipids(10, 11) . In bone marrow, sialoadhesin is
selectively concentrated at regions of membrane contact between
macrophages and developing granulocytes(12) , and the isolated
molecule binds preferentially to granulocytes(13) . These
observations indicate that sialoadhesin is involved in the specific
recognition of neutrophils by macrophages during hemopoiesis.
Previous work using a series of domain deletion constructs has shown
that the sialic acid-binding site on sialoadhesin is located within the
V-set domain(14) . V-set domains consist of nine -strands,
designated A-G, making up two
-sheets, the GFCC`C" sheet,
and the ABED sheet (reviewed in (15) ). All members of the
sialoadhesin family have unusual structural characteristics within
their V-set domains that are unique within the Ig superfamily. Instead
of the canonical intersheet disulfide bridge between the B and F
-strands, they possess an unusual intrasheet disulfide between the
B and E strands. Furthermore, an interdomain disulfide bond is thought
to bridge the first two NH
-terminal
domains(16, 17) . (
)
In this study, we
have undertaken a site-directed mutagenesis screen of the V-set domain
of sialoadhesin in an attempt to locate and characterize the sialic
acid-binding site. Drastic changes to a subset of amino acids predicted
to be on the surface of the V-set domain resulted in complete
abrogation of sialic acid-dependent binding without affecting binding
to anti-sialoadhesin monoclonal antibodies (mAbs). ()When
superimposed onto a CD8
-based model of the V-set domain of
sialoadhesin, these residues are seen to constitute a contiguous
binding site clustered around an arginine on the F strand. This residue
is conserved in all members of the sialoadhesin family and appears to
be essential for sialic acid-dependent binding.
Figure 1:
Alignment
of sialoadhesins with CD8, a member of the Ig superfamily of known
structure. The predicted protein sequence of the V-set Ig domain of
sialoadhesin (Sn) was manually aligned with the V-set domains
of mouse CD22(42) , mouse myelin-associated glycoprotein (MAG)(43) , human CD33(9) , and human
CD8
(44) . Numbering of amino acids corresponds to the
mature protein sequence of sialoadhesin(5) . The
-strand
assignments (solid bars) were based on the structure of
CD8
. Broken lines instead of bars are shown where there
are no grounds for making precise assignments to
-strands. It
should be noted that for the assignment of residues that are identical
between members of the sialoadhesin family but are not characteristic
of other V-set domains within the Ig superfamily, the other species
homologues are taken into account, namely rat and human
myelin-associated glycoproteins(45, 46) , human
CD22(7) , and mouse CD33(47) . Thus, Trp-2 in
sialoadhesin is replaced by Gln in mouse CD33, and Gly-66 in
sialoadhesin is replaced by Lys in human
CD22.
The approach we used to localize the binding site was to make drastic changes to residues predicted to lie on the surface of the V-set domain and to examine the effects on sialic acid-dependent binding. We made drastic changes because it has been shown that more conventional changes to alanine may only identify a fraction (25-40%) of the residues in the structural binding site(23, 24) . Those residues implicated by drastic mutations were subsequently mutated to alanine to determine whether they are important for sialic acid recognition.
The drastic
mutations fell into two classes regarding their effects on sialic acid
binding. Eleven mutants bound human erythrocytes to the same level as
the wild-type Sn(d1-3)Fc protein and maintained specificity for
NeuAc2-3Gal
1-3GalNAc/NeuAc
2-3Gal
1-3(4)GlcNAc
over NeuAc
2-6Gal
1-4GlcNAc (Table 1). Fig. 2A and Fig. 3A show the results of
erythrocyte binding to two mutants of this class, D29K and T93D. Six
mutants were unable to mediate sialic acid-dependent binding, but bound
both mAbs 3D6 and SER-4 (Table 1). Fig. 4shows binding
data for R97D as an example of this class of mutant.
Figure 2:
Binding assays of the sialoadhesin mutant
D29K, typical of mutants that bind normally to erythrocytes and mAbs.
Wells were precoated with anti-human IgG and then incubated with
varying concentrations of wild-type or mutant Sn(d1-3)Fc or with
NCAM-Fc protein as a control. A, for erythrocyte binding
assays, human erythrocytes were allowed to adhere for 30 min to the
coated wells, and unbound cells were removed by washing. ,
wild-type protein;
, D29K;
, NCAM-Fc. B, for mAb
binding assays, wells were incubated with anti-sialoadhesin mAbs 3D6
and SER-4, followed by peroxidase-conjugated goat anti-rat IgG. SER-4:
-
, wild-type protein;
-
, D29K;
-
, NCAM-Fc.
3D6:
- - -
, wild-type protein;
- - -
, D29K;
- -
-
, NCAM-Fc. Binding for both assays was quantified using o-phenylenediamine dihydrochloride as substrate, followed by
optical density measurement of the reaction product at 450 nm. Results
are expressed as mean values of four wells from single experiments. The
standard deviations were consistently within 10% of the mean values and
are not shown for clarity. Similar results were obtained in at least
three independent experiments.
Figure 3:
Binding assays of the sialoadhesin mutant
T93D, typical of mutants that bind erythrocytes normally but that are
selectively unable to bind mAb 3D6. Binding assays were carried out as
described in the legend to Fig. 2. A, erythrocyte
binding. , wild-type protein;
, T93D;
, NCAM-Fc. B, mAb binding. SER-4:
-
, wild-type
protein;
-
, T93D;
-
,
NCAM-Fc. 3D6:
- - -
, wild-type protein;
- - -
, T93D;
- -
-
, NCAM-Fc.
Figure 4:
Binding assays of the sialoadhesin mutant
R97D, typical of mutants that are unable to bind erythrocytes but bind
normally to mAbs. Binding assays were carried out as described in the
legend to Fig. 2. A, erythrocyte binding. ,
wild-type protein;
, R97D;
, NCAM-Fc. B, mAb
binding. SER-4:
-
, wild-type protein;
-
, R97D;
-
, NCAM-Fc.
3D6:
- - -
, wild-type protein;
- - -
, R97D;
- -
-
, NCAM-Fc.
Interestingly, the mutants K8E, T93D, and K110E showed greatly reduced binding to mAb 3D6, with little or no effect on binding to mAb SER-4 or to erythrocytes (Table 1). These observations suggest that Lys-8, Thr-93, and Lys-110 form part of the epitope recognized by mAb 3D6. Results obtained using T93D, which is typical of these mutants, are shown in Fig. 3.
Four of the six residues implicated in sialic acid recognition by drastic changes were studied further by mutating them to alanine. All alanine mutants bound to both mAbs (Table 1), and the majority had no effect on erythrocyte binding. R97A was the only alanine mutation that abolished erythrocyte binding. To investigate the effect of a much more conservative substitution, Arg-97 was also changed to lysine. Similar to the R97D and R97A mutants, the R97K mutant showed greatly reduced binding to erythrocytes, but normal binding to both mAbs (Table 1). Arg-97 therefore appears to be a key residue in mediating sialic acid-dependent binding of sialoadhesin.
Figure 5:
Ribbon diagram of the CD8-based model
of the sialoadhesin V-set domain. Residues that abolished sialic acid
binding when mutated are shown in black. Those that had no
effect are shown in white. Those that resulted in a reduction
in binding to mAb 3D6 are shown in gray. Residues that occur
in regions of the model where sequence alignment is uncertain are shown
only as C-
spheres, whereas those that occur in regions of greater
certainty are represented with side chains. This figure was prepared
using MOLSCRIPT Version 1.4 (48) with modifications by R.
Esnouf (see Footnote 3). Minimal modifications were made to the
CD8
framework in the C`-D region. Sequence considerations
(see Fig. 1) would imply greater structural differences between
sialoadhesin and CD8
in this region, and therefore, the model only
represents a general guide for amino acid
positions.
We have used site-directed mutagenesis in an attempt to
locate the binding site for sialic acid within the V-set domain of
sialoadhesin. Drastic mutations of six residues abolished erythrocyte
binding with little or no effect on protein folding, as assessed by mAb
binding. A model of this domain predicts that these residues form a
discrete cluster on the G, F, and C strands, surrounded by residues
that when mutated have no effect on erythrocyte binding. Of the alanine
mutants, only R97A led to complete abolition of binding. A more
conservative mutation of Arg-97 to lysine also disrupted sialic
acid-dependent binding without affecting mAb binding. Arg-97 is
conserved in all sialoadhesins, and mutation of the equivalent residue
to alanine or lysine in CD22 (49) and to alanine in CD33 ()abolishes the binding activity of these proteins,
suggesting that this residue is critical in sialic acid recognition by
sialoadhesins.
The molecular model of sialoadhesin based on the
CD8 structure (Fig. 5) is used primarily to depict the
approximate positions of the mutants, and some aspects are likely to be
inaccurate, particularly in the loop regions and in the C` and C"
-strands. However, the B, C, E, and F
-strands of Ig
superfamily domains are structurally highly conserved. These strands
also have characteristic sequence patterns that allow accurate
alignments of superfamily proteins of unknown structure with those of
known structure. For example, the central portions of the C and F
strands have the patterns X
XCX and
DXGXYX, respectively. The X residues will invariably be surface residues and will always be in
well defined positions within the
-sheets. Thus, the positions of
the F and C strand mutants are likely to be accurate in the model. The
other mutations affecting binding were predicted to lie on the G
strand, but their precise positions cannot be determined with
confidence because of the poor alignment with CD8
in this region.
Although it could be argued that the effect of the mutations in disrupting sialic acid-dependent binding is due to changes in the overall folded structure of sialoadhesin, several arguments suggest that this is unlikely. First, all mutants that were unable to bind erythrocytes were able to bind three anti-sialoadhesin mAbs that recognize two distinct epitopes. It is known that the majority of mAbs bind discontinuous, conformationally sensitive epitopes(26) , and our results demonstrate that this is the case for 1C2 and 3D6, mAbs directed to the V-set domain of sialoadhesin. Second, of 23 mutations made, only 8 disrupted erythrocyte binding, and these lie in a single contiguous region. Finally, mutations in the equivalent region of the V-set domain of CD22 also disrupt sialic acid-dependent binding of CD45 ligand without disrupting the binding of a conformationally sensitive mAb directed to this domain(49) .
A combination of x-ray
crystallography, molecular modeling, and mutagenesis has resulted in
detailed descriptions of the binding sites of other sialic acid-binding
proteins. Those studied so far include influenza virus
hemagglutinin(27, 28, 29) , viral and
bacterial sialidases (30, 31, 32, 33) , the polyoma virus
VP1 protein(34) , wheat germ agglutinin (35) , and the
mammalian cell adhesion molecule
E-selectin(36, 37, 38) . Thus, sialic
acid-binding sites can occur in a variety of distinct protein folds,
including -propellor (sialidases), ``jelly roll''
(polyoma VP1), and carbohydrate recognition domains typical of C-type
Ca
-dependent lectins (selectins). The present study,
together with the accompanying report on CD22(49) , is the
first description of the putative sialic acid-binding site within
adhesion molecules that belong to the Ig superfamily. Interestingly,
the GFCC`C" face that appears to be used by sialoadhesin and CD22 to
interact with sialic acid is commonly used by other members of the Ig
superfamily to bind protein ligands, as discussed in the accompanying
paper(49) .
For both influenza hemagglutinin and wheat germ agglutinin, interactions with sialic acid require diverse uncharged side chains, such as tryptophan and tyrosine(27, 35) . For several other sialic acid-binding proteins, positively charged amino acids (usually arginine) are also required. In a low resolution crystal structure of the polyoma virus VP1 protein, the guanidinium group of an arginine is thought to form a salt bridge with the carboxylate of sialic acid(34) . Viral and bacterial sialidases have three conserved arginine residues thought to stabilize the carboxylate group of sialic acid(33) . The E-selectin-sialic acid interaction is very sensitive to substitution of an arginine, and binding is abolished even when changed to lysine, indicating the critical role of this residue(36, 37, 38) . Similarly, for sialoadhesin and CD22, we demonstrated that substitution of a conserved arginine to alanine or lysine abolishes sialic acid-dependent binding.
In common with many sialic acid-binding
proteins, members of the sialoadhesin family exhibit distinct
preferences for sialic acid linkage to galactose. Sialoadhesin, CD33,
and myelin-associated glycoprotein prefer NeuAc2-3Gal,
whereas CD22 shows a strict requirement for
NeuAc
2-6Gal(3, 4, 10, 39, 40, 41) .
In the present study, none of the sialoadhesin mutants showed altered
sialic acid linkage preference, so no insight was gained into how
sialoadhesin discriminates between
2-3-linked and
2-6-linked sialic acids. Structural studies with the polyoma
virus VP1 protein have shown that the specificity for particular sialic
acid linkage is determined by direct interactions of amino acid side
chains with the sialic acid and galactose(34) . A similar
situation may occur with members of the sialoadhesin family since in
recent studies with CD22, recognition of oligosaccharides terminating
in NeuAc
2-6Gal appeared to require an interaction of the
CD22 binding site with both the sialic acid and galactose
moieties(41) . Further studies will be required to understand
the molecular basis of sialic acid linkage discrimination between
members of the sialoadhesin family.