(Received for publication, February 19, 1997, and in revised form, April 9, 1997)
From the Departments of Pharmacology and
Neuroscience, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205, the ¶ Department of Applied Bioorganic
Chemistry, Gifu University, Gifu 501-11, Japan, the
Samuel
Lunenfeld Research Institute, Mount Sinai Hospital, Toronto M5G 1X5,
Canada, and the ** Imperial Cancer Research Fund Laboratories, John
Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
The carbohydrate binding specificities of three
sialoadhesins, a subgroup of I-type lectins (immunoglobulin superfamily
lectins), were compared by measuring lectin-transfected COS cell
adhesion to natural and synthetic gangliosides. The neural
sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell
myelin protein (SMP), had similar and stringent binding specificities.
Each required an 2,3-linked sialic acid on the terminal galactose of
a neutral saccharide core, and they shared the following rank-order
potency of binding: GQ1b
GD1a = GT1b
GM3 = GM4
GM1, GD1b, GD3, GQ1b
(nonbinders). In contrast, sialoadhesin had less exacting specificity,
binding to gangliosides that bear either terminal
2,3- or
2,8-linked sialic acids with the following rank-order potency of
binding: GQ1b
> GD1a = GD1b = GT1b = GM3 = GM4 > GD3 = GQ1b
GM1 (nonbinder). CD22 did not bind
to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was
abrogated by chemical modification of either the sialic acid carboxylic
acid group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin
binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of
MAG, as did replacement of the sialic acid acetamido group with a
hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with
sialoadhesin binding to one face of the sialic acid moiety, whereas MAG
(and SMP) may have more complex binding sites or may bind sialic acids
only in the context of more restricted oligosaccharide conformations.
Sialoadhesins (1) are a structurally and functionally related family consisting of five immunoglobulin superfamily lectins (I-type lectins) (2) including myelin-associated glycoprotein (MAG),1 Schwann cell myelin protein (SMP), CD22, CD33, and sialoadhesin. MAG and SMP are found on oligodendroglia and Schwann cells in the nervous system (3, 4), CD22 is expressed on a subset of B lymphocytes, sialoadhesin on a subset of macrophages, and CD33 on cells of myelomonocytic lineage. Sialoadhesins have been proposed to mediate cell-cell recognition, perhaps via their carbohydrate binding activities (5-7). Each sialoadhesin family member has two or more Ig-like domains: an amino-terminal V-set domain followed by one or more (up to 16) C2-set domains (8). Domain deletion and site-directed mutagenesis of sialoadhesin and CD22 localize their carbohydrate-binding sites to the amino-terminal V-set domain, with contributions (for CD22) from the adjoining C2-set domain. These first two domains share very high amino acid sequence similarity between MAG and SMP (>70%) and significant similarity across all I-type lectins (>30% in pairwise comparisons) (2, 8, 9).
Each I-type lectin binds to carbohydrate structures bearing a nonreducing terminal sialic acid (1, 6, 10). Sialic acids are a common nonreducing terminus of vertebrate glycoconjugates and appear to play uniquely important roles in recognition phenomena. Because sialic acids may be linked to Gal, GalNAc, or other sialic acid residues at various positions and because they may carry different substituents on their 9-carbon base structure, the sialic acids represent a diverse family of carbohydrate determinants (11). In certain sialic acid-dependent recognition systems, determinant stringency is low. For example, selectins bind to oligosaccharides bearing truncated sialic acids (12) or appropriately placed anionic groups (sulfates, carboxylic acids) otherwise unrelated to the sialic acid structure (13-16). In contrast, sialoadhesins appear to have more stringent sialic acid specificities (see "Discussion") (9). In this study, we used cells expressing different sialoadhesins to explore and compare the fine structural preferences of their binding to target sialylated glycoconjugates.
The ganglioside structures used in this study
are shown schematically in Fig. 3. Purified bovine brain
GM1, GD1a, GD1b, GD3, and GT1b were from EY Laboratories (San Mateo, CA) or
Matreya, Inc. (Pleasant Gap, PA), and GQ1b was from
Accurate Chemical & Scientific Corp. (Westbury, NY). GM3
(NeuAc form) was from Sigma. GQ1b, GT1
,
GM1
, GM4 and its derivatives, and
GM3 derivatives were synthesized de novo using
previously described methods (17-19). GD1a gangliosides
bearing sialic acids with truncated glycerol side chains
(7/8-aldehydes) were prepared by mild periodate oxidation followed (as
indicated) by sodium borohydride reduction to form the 7/8-alcohols
(20). GD1a gangliosides bearing sialic acid ethyl esters,
1-amides, and 1-alcohols were prepared as described (20). Products were
analyzed by thin-layer chromatography and fast atom bombardment mass
spectrometry at the Middle Atlantic Mass Spectrometry Laboratory
(21).
I-type Lectin Transfection of COS Cells
Full-length I-type
lectin cDNAs were cloned into the eukaryotic expression vector
pcDNA1/Amp (sialoadhesin only) or pCDM8. The lectins used in this
study included mouse sialoadhesin (8), both the long (L-MAG) (22) and
short (S-MAG) splice variants of rat MAG, quail SMP (4), and human CD22
(seven-Ig-like domain variant, CD22) (23, 24).
Plasmids were propagated in Escherichia coli MC1061/p3 and purified by polyethylene glycol precipitation. COS-1 cells, routinely maintained in 10% fetal calf serum in Dulbecco's modified Eagle's medium at 37 °C in a humidified atmosphere of 90% air and 10% CO2, were transiently transfected with lectin-expressing plasmids via a high efficiency procedure (using 40 µg/ml DEAE-dextran) (25). Transfected cells were returned to culture for 40-50 h to allow lectin expression to proceed and then were detached from plates for adhesion experiments (see below). Lectin expression was confirmed by flow cytometry and/or immunocytochemistry using the following monoclonal antibodies: mAb 513 (MAG/SMP cross-reactive) (4, 7), SER-4 (sialoadhesin) (26), and Chemicon 2112 (CD22; Chemicon International, Inc., Temecula, CA).
Microplate Cell Adhesion to Adsorbed GlycolipidsAdhesion was performed as reported previously (20, 22, 27). Aliquots (50 µl) of ethanol/water (1:1) containing phosphatidylcholine (0.5 µM), cholesterol (2.0 µM), and gangliosides (concentrations as indicated) were added to microwells (96-well Serocluster, Costar Corp., Cambridge, MA). Plates were incubated for 90 min uncovered at ambient temperature to allow partial evaporation and lipid adsorption (28, 29), after which the wells were washed with water. Wells were preblocked by addition of 100 µl/well Hepes-buffered Dulbecco's modified Eagle's medium containing 1.5 mg/ml bovine serum albumin. Plates were covered and incubated for 10 min at 37 °C prior to cell addition (see below).
Transfected COS cells were harvested using hypertonic Ca2+/Mg2+-free phosphate-buffered saline containing 1 mM EDTA as described (22), collected by centrifugation, and resuspended at 107 cells/ml in Dulbecco's phosphate-buffered saline containing 2 mg/ml bovine serum albumin. Transfected cells were pretreated with neuraminidase, which enhances cell adhesion without changing carbohydrate binding specificity (20), as follows. Aliquots of cells (500 µl) were placed in 1.5-ml microcentrifuge tubes, and 10 milliunits of Vibrio cholerae neuraminidase (Calbiochem) were added. Suspensions were incubated for 1.5-2 h at 37 °C with end-over-end mixing. Cells were collected by centrifugation, washed twice with Dulbecco's phosphate-buffered saline containing 2 mg/ml bovine serum albumin, and resuspended at 250,000 cells/ml in Hepes-buffered Dulbecco's modified Eagle's medium containing 1.5 mg/ml bovine serum albumin. Cell viability was determined by trypan blue exclusion on representative transfected cells. Prior to pretreatment, cells were 84% viable. After neuraminidase or control pretreatment, viability ranged from 81 to 85%, essentially unchanged from the freshly collected cells. Quantitation of cell adhesion was via an enzyme assay (see below) that measured only viable cells.
Aliquots of the cell suspension (200 µl) were added to preblocked,
lipid-adsorbed microwells and incubated at 4 °C for 10 min to allow
the cells to settle and then at 37 °C for 45 min. To gently remove
nonadherent cells after the incubations, plates were immersed in
phosphate-buffered saline, inverted, and placed in an immersed
Plexiglas box that was sealed with a gasket to exclude air (27). The
inverted plate in its fluid-filled chamber was placed in a centrifuge
carrier and centrifuged at 110 × g. The box was again
immersed in phosphate-buffered saline; the plate was removed and
righted (while immersed); and excess surface buffer was removed by
aspiration, leaving 300 µl/well. Adherent cells were lysed by
addition of 20 µl of 10% Triton X-100 to each well, and 80 µl were
removed to a fresh 96-well plate for quantitation. Cell adhesion was
quantitated by measuring lactate dehydrogenase activity in the cell
lysate after addition of 120 µl of phosphate-buffered saline
containing 0.7 mM NADH and 4.7 mM pyruvate. The
decrease in absorbance at 340 nm as a function of time was measured
simultaneously in each well using a Molecular Devices UV multiwell
kinetic plate reader. This method is amenable to testing large numbers
of samples. The data presented are compiled from 4000 individual
data points and are presented as the mean ± S.E. of the mean for
3-103 replicate determinations. Where indicated, the statistical
significance of adhesion to ganglioside-adsorbed surfaces compared with
control surfaces (adsorbed with phosphatidylcholine and cholesterol,
but no ganglioside) was determined using a two-tailed Student's
t test.
MAG-,
SMP-, and sialoadhesin-transfected COS cells bound specifically to
ganglioside-adsorbed surfaces (Figs. 1, 2, 3). Adhesion to
the most potent target gangliosides was typically very high (>80% of
the cells added), whereas background adhesion to surfaces adsorbed with
phosphatidylcholine and cholesterol without ganglioside was low. COS
cells transfected with CD22 failed to adhere to any ganglioside tested
(GD1a, GD1b, GD3, GT1b,
GQ1b, and GQ1b). COS cells transfected with
either of the two splice variants of MAG (L-MAG and S-MAG) demonstrated
the same extent and specificity of adhesion to a representative set of
ganglioside-adsorbed surfaces (GM1, GD1a,
GD1b, GT1b, and GQ1b
) (data not
shown). Therefore, L-MAG-transfected COS cells were used in subsequent
experiments, and all data presented on MAG-mediated adhesion refer to
the long splice variant.
The two neural sialoadhesins, MAG and SMP, had similar ganglioside
binding specificities (Figs. 1, 2, 3). The abundant brain gangliosides
GD1a (at 12.5 pmol/well) and GT1b (at
25
pmol/well) supported highly significant adhesion (p < 0.0002) of both MAG- and SMP-transfected COS cells (Fig. 1,
A and B). Other gangliosides including
GM3 and GM4 also supported significant adhesion
of both lectins, although only at
10-fold higher ganglioside
concentrations compared with GD1a. In contrast, neither MAG
nor SMP bound to GM1, GD1b, or GD3,
indicating that both lectins require a terminal
2,3-linked sialic
acid. All gangliosides that supported statistically significant
adhesion of SMP contained the NeuAc
2,3Gal terminal structure (see
Fig. 3), whereas all nonsupportive gangliosides lacked this terminal
structure. MAG supported adhesion to the same gangliosides, although
typically with higher efficiency (greater number of adherent cells).
This may be due to more efficient transfection with the MAG plasmid,
higher expression of the transfected MAG, and/or more effective
ganglioside binding by MAG. Flow cytometry using a MAG/SMP
cross-reactive antibody (mAb 513) indicated that more MAG-transfected
cells (48.2%) expressed the highest level of lectin compared with
SMP-transfected cells (28.3%). Within these highest expressing
populations, the mean fluorescence intensities were similar (496 and
441 relative units for MAG and SMP, respectively).
In addition to gangliosides bearing the NeuAc2,3Gal terminus,
GQ1b (which bears only
2,8-linked sialic acid termini)
supported a low amount of adhesion by MAG-transfected cells. This
preparation of GQ1b, however, was contaminated with a small
amount of GT1b (20). We conclude that MAG and SMP bind with
similar rank-order potency to gangliosides terminated with
NeuAc
2,3Gal (see Fig. 3).
In contrast to MAG and SMP, sialoadhesin had a distinctly broader
binding specificity. Several gangliosides with terminal NeuAc2,3Gal
structures (GD1a, GT1b, GM3, and
GM4) as well as GD1b (which bears only a
terminal NeuAc
2,8NeuAc structure) supported nearly equivalent
sialoadhesin-mediated adhesion (Fig. 1C). GD3 and GQ1b, which also bear only NeuAc
2,8NeuAc termini,
supported sialoadhesin binding with moderate potency. Binding was
structurally specific in that GM1 did not support
sialoadhesin-mediated adhesion.
Prior studies indicated that MAG bound with markedly high affinity to
one of the "Chol-1" gangliosides (22). These minor brain
gangliosides bear a sialic acid linked 2,6 to the
GalNAc(III) of the gangliotetraose core (structures in Fig.
3) (30). Fig. 2 presents a comparison of adhesion
of MAG-, SMP-, and sialoadhesin-transfected COS cells to synthetic
Chol-1 and related gangliosides. MAG and SMP again had markedly similar
binding specificities (Fig. 2, A and B).
GT1
was equipotent to GT1b in supporting MAG
and SMP binding, whereas GQ1b
was 10-fold more potent.
GM1
, which contains a single
2,6-linked sialic acid,
failed to support adhesion of either lectin. Therefore, the terminal
NeuAc
2,3Gal structure is required for both SMP- and MAG-mediated
cell adhesion, and additional sialic acids on the internal GalNAc(III)
and Gal(II) of the gangliotetraose core enhance binding of MAG and SMP
to a similar extent. In contrast, GQ1b
was only modestly
(<3-fold) more potent than GT1b in supporting
sialoadhesin-mediated adhesion. Binding potencies for all gangliosides
tested using MAG-, SMP-, and sialoadhesin-mediated cell adhesion are
summarized in Fig. 3.
The MAG/SMP cross-reactive antibody mAb 513 (4, 7), shown previously to
block MAG binding to neurons (31) and gangliosides (22), demonstrated
the carbohydrate-binding site structural similarity between MAG and SMP
and their difference from sialoadhesin. As shown in Fig.
4, mAb 513 eliminated or markedly reduced binding of MAG
and SMP to GT1b, whereas binding of sialoadhesin was
unaffected. The anti-sialoadhesin blocking mAb 3D6 (32) inhibited
binding of sialoadhesin to GT1b (data not shown).
Sialic Acid Substructure Binding Specificities of I-type Lectins
Sialic acid is a complex monosaccharide, with a
carboxylic acid, an N-acyl group, and a glycerol side chain
within its structure (see Fig. 7). Chemically modified and synthetic
gangliosides were used to determine which sialic acid substituent
groups are required for binding by sialoadhesin family members.
Since GD1a supports highly significant adhesion of MAG,
SMP, and sialoadhesin (Fig. 1), it was used as a basis for testing sialic acid chemical modifications. GD1a was selectively
oxidized with periodate under conditions that cleave exclusively
between C-7-C-8 and C-8-C-9 on the sialic acid glycerol side chain.
Mass spectrometry indicated equal conversion of GD1a sialic
acids to their corresponding 7- and 8-carbon aldehydes (data not
shown). A portion of the resulting GD1a aldehydes was
reduced with sodium borohydride, resulting in conversion to the
corresponding 7- and 8-carbon alcohols. As shown in Fig.
5, neither the 7/8-aldehyde nor 7/8-alcohol sialic acid
derivatives of GD1a supported binding of any of the I-type
lectins tested. Similarly, modifications of the carboxylic acids on
GD1a abrogated binding. Conversion of both sialic acids on
GD1a to the corresponding 1-ethyl esters, 1-amides, or
1-alcohols completely eliminated binding of MAG-, SMP-, and
sialoadhesin-transfected COS cells (Fig. 5). The structures of all
GD1a derivatives were confirmed by thin-layer
chromatography, DEAE-Sepharose chromatography (of carboxylate
derivatives), and fast atom bombardment mass spectrometry at the
Middle Atlantic Mass Spectrometry Laboratory (21).
Since GM3 and GM4 (bearing a terminal
N-acetylneuraminic acid) supported substantial adhesion
mediated by both sialoadhesin and MAG, a series of synthetic analogs
based on these structures (19) was used to determine the role of each
sialic acid hydroxyl group and the sialic acid N-acyl group
on adhesion (binding of SMP to GM3 and GM4 was
insufficient to allow valid comparisons). Consistent with chemical
modification studies, the 8-deoxy and 9-methoxy forms of
GM3 failed to support adhesion mediated by either MAG or
sialoadhesin (Fig. 6). In contrast, the 4-deoxy and
7-deoxy forms of GM3 were comparable to GM3 in
supporting sialoadhesin-mediated adhesion, but failed to support
substantial MAG-mediated adhesion. Furthermore, the sialic acid
acetamido group appears to be involved in lectin binding.
GM4 supported sialoadhesin and MAG binding, whereas a
derivative bearing a 5-deaminated analog of neuraminic acid
(KDN-GM4) failed to support binding by either lectin (Fig.
6). These data are consistent with the prior published observations
that glycoconjugates bearing N-glycolylneuraminic acid fail to support MAG (20) or sialoadhesin (33) binding.
Sialoadhesins (1, 8, 9) are a functionally and structurally related subfamily of carbohydrate-binding immunoglobulin superfamily members (I-type lectins) (2). The sialoadhesin family consists of the eponymous member (sialoadhesin), MAG, SMP, CD22, and CD33. MAG and SMP are expressed on myelinating cells in the nervous system, sialoadhesin on a subset of macrophages, CD22 on certain B lymphocytes, and CD33 on cells of myelomonocytic lineage (9). Sialoadhesins mediate cell-cell interactions by binding to target sialylated glycoconjugates (1, 6, 10, 32). They share the same general polypeptide domain structure: an amino-terminal V-set Ig-like domain followed by one or more C2-set Ig-like domains, a transmembrane domain, and a short cytoplasmic tail. The ligand recognition site has been localized to the amino-terminal V-set domain (sialoadhesin) (34) or the V-set domain with contributions from the adjacent C2-set domain (CD22) (34, 35). Additionally, sialoadhesins have extensive sequence similarity. The first two amino-terminal Ig-like domains of MAG and SMP are 56% identical (72% similar, including conservative amino acid replacements), and other sialoadhesins range from 32 to 43% sequence similarity in pairwise comparisons. Site-directed mutagenesis (36, 37) indicates that sialoadhesin and CD22 bind to sialylated glycoconjugates via amino acids on one surface of the V-set domain. This is consistent with sialoadhesin's sialic acid substituent group binding specificity (see below), whereas MAG's specificity indicates a more complex binding site.
Target ligands for the sialoadhesins are glycoconjugates in which a
terminal sialic acid is essential for binding (1, 2, 6, 9, 10, 38). In
previous studies, CD22 bound only to 2,6-linked sialic acids (1,
39), whereas MAG and CD33 bound only to
2,3-linked sialic acids (1,
10, 22). Sialoadhesin bound predominantly to terminal
2,3-linked
sialic acids (1), although weaker binding to
2,8-linked sialic
acids was demonstrated (32). Among structures with
2,3-linked sialic
acids, MAG bound preferentially to "3-O" structures
(NeuAc
2,3Gal
1,3GalNAc), which are common termini on gangliosides
(the major sialoglycoconjugates of the nervous system) (40) and
O-linked glycoproteins (1). Sialoadhesin and CD33 bound
similarly to "3-O" and "3-N"
(NeuAc
2,3Gal
1,4GlcNAc) structures (1, 10).
Our prior studies demonstrated that (i) MAG bound to gangliosides with
the specificity GQ1b > GT1b = GD1a > GM3
GM1, GD1b, GQ1b, the latter of which did not support
adhesion; and (ii) modification of the glycerol side chain, carboxylic
acid, or N-acyl group abrogated MAG-mediated adhesion (20,
22). This study confirms and extends those findings. MAG-mediated
adhesion was repeated to the above gangliosides as well as to
GM4, KDN-GM4, six synthetic derivatives of
GM3, and various gangliosides with
2,6-sialic acids
linked to the GalNAc(III) of the gangliotetraose core. These new data
were compared directly with adhesion of COS cells expressing CD22,
sialoadhesin, SMP, and the short isoform of MAG.
CD22 failed to bind to 2,3- or
2,8-linked sialic acid termini on
gangliosides (data not shown). In contrast (Figs. 1 and 2),
sialoadhesin bound to most of the gangliosides tested, including many
with terminal
2,3- or
2,8-sialic acids. Nine of 12 gangliosides tested supported sialoadhesin binding within the same
10-fold concentration range, indicating that sialoadhesin does not markedly distinguish the sialic acid linkage (
2,3 versus
2,8)
or the neutral core (e.g. compare GD1b and
GM4 in Fig. 3). A previous study of detergent-solubilized
125I-sialoadhesin binding to gangliosides using thin-layer
chromatography plate overlay (32) also reported a broad
specificity.
Consistent with their extensive sequence similarity, the two neural
sialoadhesins, SMP and MAG, were remarkably similar in their
ganglioside binding (Figs. 1, 2, 3). Both bound only to structures bearing
terminal 2,3-linked sialic acids (e.g. GT1b and GD1a) and failed to bind to those terminated with
2,8-linked structures (e.g. GD1b and
GD3). Among glycoconjugates with
2,3-linked sialic acid
termini, SMP and MAG distinguished sialic acid linkage patterns and
neutral core variations. In contrast to sialoadhesin, di- and
trisialogangliosides with the gangliotetraose core (GD1a and GT1b) supported adhesion of SMP and MAG
10-fold
better than did monosialogangliosides (GM3 and
GM4). Furthermore, the Chol-1 ganglioside,
GQ1b
, was 10-fold more potent than any other ganglioside
tested (Figs. 1, 2, 3). Chol-1 gangliosides are quantitatively minor
structures that are expressed exclusively on cholinergic neurons (30,
41). The functional significance of their preferential binding to the
neural sialoadhesins is not known. Although the terminal
tetrasaccharide on GQ1b
is also found on
O-linked glycoproteins (42, 43), polyclonal antibodies
against Chol-1 gangliosides do not cross-react with any glycoprotein
(44), suggesting that the oligosaccharide on GQ1b
adopts
a unique conformation that fits particularly well in the SMP and MAG
binding pockets. In addition to having similar carbohydrate recognition specificities, the observation that both SMP- and MAG-mediated adhesion
to gangliosides is inhibited by the same conformationally restricted
monoclonal antibody (mAb 513) (45) confirms the similarity of their
binding sites.
Sialic acids are unusual among monosaccharides in their complexity and diversity (11). They carry a carboxylic acid (C-1), an N-acyl group attached to C-5, and a glycerol side chain attached to C-6 (Fig. 7), each of which is involved in molecular recognition by certain sialoadhesins. Blocking the carboxylic acid abrogates binding (Fig. 5), as does replacement of the acetamido group with a hydroxyl (compare GM4 with KDN-GM4 in Fig. 6) or truncation of the glycerol side chain (Fig. 5). These data are consistent with prior studies on the sensitivity of sialoadhesin and CD22 binding to modifications of the sialic acid residue (6, 33, 46-48) and contrast with studies on selectins, in which extensive modifications of sialic acids have no effect (12, 14, 49). In fact, substitution of the entire sialic acid (e.g. on sialyl-LeX or sialyl-Lea) with a sulfate ester results in retention of ligand binding by all selectins (13, 14), but abrogates binding by CD22 (47).
The sialic acid substructural binding specificities of sialoadhesin and
MAG have implications for ligand docking on the proteins. For
sialoadhesin, modification of the C-8 or C-9 hydroxyl, the acetamido
nitrogen or methyl group (33), or the C-1 carboxylic acid eliminated
binding (Figs. 5 and 6), whereas removal of the C-4 or C-7 hydroxyl was
without effect. This pattern is consistent with binding primarily to a
single face of the sialic acid (top face in Fig. 7). Sialic acid
binding to sialoadhesin can be compared with x-ray crystallography of
sialic acid binding to the influenza virus hemagglutinin (50), in which
a carboxylate oxygen, the acetamido nitrogen, and the 8- and 9- hydroxyls face into a depression on the hemagglutinin surface, whereas
the 7-hydroxyl faces the solvent. This model is consistent with
Ig-domain studies and site-directed mutagenesis (34, 37), which place
the ligand-binding site of sialoadhesin on a contiguous cluster of
residues on the surface of the GFCCC"
-sheet of the V-set Ig-like
domain.
Sialic acid modifications that block sialoadhesin binding also block MAG binding. In addition, removal of either the 4- or 7-hydroxyl inhibits MAG binding (Fig. 6). Since the 7-hydroxyl and 8/9-hydroxyls extend in opposite directions (Fig. 7), a more complex model of MAG binding is implicated. One possibility is that the MAG binding site consists of a deep pocket or apposing polypeptide sheets. Alternatively, the 7-hydroxyl group may stabilize a conformation of the oligosaccharide that is preferentially bound by MAG at a single protein surface. To date, no direct evidence addresses whether one or more than one protein surface on MAG is responsible for sialic acid binding, although biophysical and electron microscopic studies suggest that MAG may have a bent rod configuration with apposed Ig-like domains (51, 52). Studies using chimeric molecules indicate that the first three Ig domains of MAG are necessary and sufficient for binding to neurons (45) and sialoglycoconjugates (1), although the sialic acid substructure specificities of truncated forms of MAG have not been reported. Further protein structural and functional studies will be needed to establish the sialoglycoconjugate-binding site on MAG (and on SMP) and to determine the precise role each sialic acid hydroxyl group plays in protein binding.
This work is dedicated to the vision and collaborative spirit of our friend and colleague, Dr. Akira Hasegawa, who passed away October 10, 1996.
We gratefully acknowledge Dr. Melitta
Schachner for mAb 513 and Dr. Ivan Stamenkovic for the plasmid carrying
CD22.