(Received for publication, July 5, 1995)
From the
Sialoadhesin and CD22 are members of a recently characterized
family of sialic acid-dependent adhesion molecules belonging to the
immunoglobulin superfamily. Sialoadhesin is a macrophage-restricted
receptor containing 17 extracellular Ig-like domains which recognizes
oligosaccharides terminating in NeuAc2-3Gal in N-
and O-linked glycans. CD22 is a B cell-restricted receptor
with seven Ig-like domains which selectively recognizes
oligosaccharides terminating in NeuAc
2-6Gal in N-glycans. Sequence similarity between these proteins is
highest within their first four amino-terminal Ig-like domains. Here we
identify the domain(s) containing the binding sites of both molecules
by generating a series of extracellular domain deletion mutants fused
to the Fc portion of human IgG1. Binding activity was analyzed by solid
phase cell adhesion assays and also by surface plasmon resonance using
purified glycophorin and CD45 as ligands for sialoadhesin and CD22,
respectively. For sialoadhesin, the amino-terminal V-set Ig-like domain
was both necessary and sufficient to mediate sialic acid-dependent
adhesion of the correct specificity. In contrast, for murine CD22, only
constructs containing both the V-set domain and the adjacent C2-set
domain were able to mediate sialic acid-dependent binding. These
results are consistent with the sialic acid binding site for both
proteins residing in the membrane distal V-set domain, but for CD22 a
direct contribution in binding from the neighboring C2-set domain
cannot be excluded.
Most immunoglobulin (Ig) superfamily members mediate
intercellular recognition through protein-protein interactions (for
review see (1) ). In contrast, sialoadhesin and CD22 are
members of a new subgroup of the Ig superfamily which mediate cellular
interactions by recognizing specific sialylated glycans present in cell
surface glycoconjugates (2, 3, 4, 5) . Other members of this
subgroup (recently termed the Sialoadhesin family) include the myelin-
associated glycoprotein (MAG) ()and
CD33(6, 7) .
Members of the Sialoadhesin family
comprise a single amino-terminal V-set Ig domain (8) followed
by varying numbers of C2-set domains. The V-set domain has an unusual
arrangement of conserved cysteines that are predicted to form an
intra--sheet disulfide bond(9) . In addition, all members
of the Sialoadhesin family have conserved cysteines that are likely to
create a disulfide bond between the V- and the adjacent C2-set
domains(9, 10) . Sialoadhesin is the largest cell
surface member of the Ig superfamily identified to date with 17
extracellular Ig-like domains(2) . In comparison, the
predominant form of CD22 contains seven Ig-like
domains(11, 12, 13, 14, 15) .
The first four amino-terminal Ig-like domains of sialoadhesin and CD22
share
50% sequence similarity(2) .
A striking feature of members of the Sialoadhesin family is their highly restricted expression pattern which suggests that these receptors are likely to mediate discrete biological functions. Sialoadhesin is expressed by macrophages in hemopoietic and secondary lymphoid tissues, MAG is present on myelinating oligodendrocytes and Schwann cells, CD33 is found on myeloid cells and CD22 is B cell-restricted. In vivo and in vitro studies have shown that sialoadhesin interacts selectively with cells of the granulocytic lineage, whereas CD22 interacts preferentially with T and B lymphocytes(6, 16, 17) . In addition, CD22 has been implicated in modulating B cell signaling pathways via its association with the surface IgM-B cell receptor complex(18, 19, 20, 21) .
Although sialoadhesin and CD22 are both sialic acid-binding proteins
they have distinct preferences for sialylated glycoconjugates. Thus,
sialoadhesin recognizes oligosaccharides terminating in
NeuAc2-3Gal in N-glycans, O-glycans or
glycolipids, whereas CD22 recognizes oligosaccharides terminating in
NeuAc
2-6Gal
1-4GlcNAc, predominantly in N-glycans(3, 4, 6, 22, 23, 24) .
A key step toward understanding the molecular basis for this sialic
acid-dependent adhesion is the identification of binding regions for
members of the Sialoadhesin family. Previous studies using truncation
mutants fused to the Fc portion of human IgG1 have shown that the first
three Ig-like domains of sialoadhesin and CD22 are sufficient for
mediating sialic acid-dependent binding to a variety of
cells(3, 4, 6, 13, 17, 23, 24) .
In this report, we have extended this analysis and shown that for
sialoadhesin, the amino-terminal V-set Ig domain is both necessary and
sufficient for sialic acid-dependent binding to cellular and molecular
ligands. In contrast, murine CD22 appears to require both the V-set Ig
domain and the adjacent C2-set domain as a ``minimal unit''
for mediating sialic acid-dependent adhesion. These results are
consistent with the sialic acid binding site for both proteins residing
in the membrane distal V-set domain, but for CD22 a direct contribution
in binding from the neighboring C2-set domain cannot be excluded.
Sn(d1): ACAGATCTACTTACCTGTATCCGTTGTCACAGTGACCGTGGT.
Sn(d2): ACAAGCTTACTTACCTGTGGCATGTGGCACTTGCAGGTAAAC.
Sn(d3): ACAAGCTTACTTACCTGTTGCGGGGTTCATTTTGACTTCAGC.
Sn(d17): ACGAATTCCACTTACCTGTCCTCTGGAACAGCTGCAGTT.
CD22(d1): ACAGATCTACTTACCTGTCGAGACATTGAGGTGAATGGGCTC.
CD22(d2): ACAGATCTACTTACCTGTGGTATACTTAACATCCAGATGCAC.
CD22(d3): ACGAATTCACTTACCTGTGTGTGCACCGTGAGTTCCACTTCTTC.
CD22(d7): GATCGGATCCACTTACCTGTCTTGCCGATGGTCTCTGGACTGTA.
The amino-terminally truncated constructs, Sn(d1-3L)Fc, Sn(d2-3)Fc, and CD22(d2-3)Fc were obtained by PCR using cDNA templates with the appropriate reverse primers (above) and the following forward primers (5`-3`):
Sn(d1): ACAGATCTGTGGGGTGTCTCCAGTCCCAAGAATGTG.
Sn(d2): ACAGATCTGACCATTCCTGAGGAGCTGCGTGAA.
CD22(d2): ACGGATCCGTTCCAACCTTACATCCAGATG.
The PCR products were cut with BglII (Sn(d1) and Sn(d2)) or BamHI (CD22(d2)), ligated to a 66-base pair HindIII-BamHI fragment encoding the leader sequence of human CD33(33) , and cloned into the pIG vector.
The NCAM-Fc consisted of the entire extracellular domain (five Ig-like domains and two type III fibronectin-related domains) of neural cell adhesion molecule fused to the Fc portion of human IgG1. This protein was used as a negative control in solid phase binding assays. cDNA encoding this construct was kindly provided by Dr. D. Simmons (Imperial Cancer Research Fund, University of Oxford).
The Sn(d1) construct was made by PCR amplification as described above, using a reverse primer (5`-3`), GTCTAATCCGTTGTCACAGTGACCGTGGT, which resulted in the insertion of a stop codon at the end of domain 1, corresponding to Pro-120 of the mature polypeptide(2) . The PCR product was sequenced and cloned into the mammalian expression vector pEF-BOS (34) .
Figure 1: Domain deletion series of Sn-Fc and CD22-Fc chimeric proteins. Panel A, schematic representation of truncated Sn- and CD22-Fc chimeras. Although drawn as monomers, the Fc chimeras are secreted and purified as dimers as a result of disulfide bond formation between the Fc regions. Wild type sialoadhesin and CD22 have 17 and 7 extracellular Ig-like domains, respectively. Asterisks indicate the amino-terminally truncated constructs that were expressed using the leader peptide of CD33. Panel B, 5 µg of each protein was resolved by SDS-polyacrylamide gel electrophoresis under reducing conditions and visualized with Coomassie Blue. Approximate molecular masses deduced from electrophoretic mobilities (following reduction) for sialoadhesin constructs are: Sn(d2-3)Fc, 50 kDa (lane 1); Sn(d1-3L)Fc, 80 kDa (lane 2); Sn(d1-3)Fc, 80 kDa (lane 3); Sn(d1-2)Fc, 50 kDa (lane 4); Sn(d1)Fc, 45 kDa (lane 5). For CD22 constructs, the approximate molecular masses are: CD22(d2-3)Fc, 60 kDa (lane 1); CD22(d1-7)Fc, 130 kDa (lane 2); CD22(d1-3)Fc, 90 kDa (lane 3); CD22(d1-2)Fc, 66 kDa (lane 4); CD22(d1)Fc, 50 kDa (lane 5).
Figure 2: Solid phase adhesion assays of truncated Sn-Fc chimeras to human erythrocytes and CD22-Fc chimeras to mouse mesenteric lymphocytes. Sn- and CD22-Fc chimeras were adsorbed at varying concentrations onto microtiter wells precoated with goat anti-human IgG. For sialoadhesin (panel A), binding assays were carried out with human erythrocytes and bound cells quantified by measuring the pseudoperoxidase activity of hemoglobin. For CD22 (panel B), binding assays were performed with mouse mesenteric lymphocytes that had been labeled with the fluorescent esterase substrate BCECF-AM. Data shown are means of duplicate wells from a single experiment representative of at least three performed.
For CD22, mesenteric lymphocytes bound at high levels to CD22(d1-7)Fc and CD22(d1-3)Fc with intermediate binding to CD22(d1-2)Fc, but no binding was observed with CD22(d1)Fc or CD22(d2-3)Fc (Fig. 2B). To exclude the possibility that the failure of CD22(d1)Fc to mediate binding was due to denaturation during purification (see BIAcore experiments below), binding assays were also carried out with unpurified Fc chimeras in the form of tissue culture supernatants. Under these conditions, no binding of lymphocytes to CD22(d1)Fc was observed, whereas high and equivalent levels of binding were seen with CD22(d1-2)Fc and CD22(d1-3)Fc (data not shown). Binding of lymphocytes to CD22 was sialic acid-dependent as pretreatment of cells with sialidase abolished binding with all constructs (data not shown). Since the CD22(d1)Fc construct may be incorrectly folded, these results are not inconsistent with the possibility that, like sialoadhesin, the binding site of CD22 lies in domain 1.
Figure 3: Solid phase binding assays of Sn(d1)Fc and to Sn(d1-3)Fc to derivatized human erythrocytes. Human erythrocytes were either untreated (native) or treated with sialidase (asialo). Sialylated glycans on asialo erythrocytes were then reconstituted using purified sialyltransferases to give exclusively 3-O, 3-N, or 6-N. Binding assays were performed as described in the legend to Fig. 2.
To show unequivocally
that domain 1 of sialoadhesin contains the sialic acid binding site we
engineered a soluble form of domain 1, Sn(d1), lacking the Fc portion.
The purified recombinant protein migrated on SDS-polyacrylamide gel
electrophoresis with an apparent M of 12,500 (Fig. 4), close to the calculated M
of
13,302. Solid phase binding assays were carried out by immobilizing
Sn(d1) onto microtiter plates coated with (Fab`)
fragments
of 3D6 mAb. Sn(d1) was able to bind human erythrocytes at high levels (Fig. 4). Binding was sialic acid-dependent as sialidase
treatment completely abolished binding (data not shown).
Figure 4:
Solid phase binding assay of Sn(d1) with
human erythrocytes. Sn(d1) protein at varying concentrations was
adsorbed onto microtiter plates that had been coated with 40 µg/ml
F(ab`) fragments of 3D6 mAb. Binding assays were carried
out with human erythrocytes as described in the legend to Fig. 2. Data show the mean values of duplicate well at each
point; similar results were obtained in three independent experiments.
The inset shows SDS-polyacrylamide gel electrophoresis of 5
µg of purified Sn(d1) protein under reducing conditions, stained
with Coomassie Blue.
For sialoadhesin, we used BSA and asialoglycophorin to control for nonspecific and non-sialic acid-dependent binding to glycophorin, respectively. As shown in Fig. 5, A, C, and E, when the protein samples were injected before the binding of Sn(d1-3)Fc to the surface, the response rose quickly to a plateau level and remained at that level until the injection ended, after which it dropped quickly to the same level as before the injection. In contrast, when glycophorin was injected following the immobilization of Sn(d1-3)Fc, the response did not rise to a plateau level but instead continued to increase during the injection, indicating that glycophorin was binding to the Sn(d1-3)Fc protein. Following the injection, the response initially dropped quickly as the injected sample was washed out of the flow cell. However, the response remained elevated after the injection, indicating that glycophorin remained bound to the Sn(d1-3)Fc and slowly dissociated (see Fig. 5A, inset). In comparison, following immobilization of the Sn(d1-3)Fc protein, no changes in response were seen with the control proteins, asialoglycophorin and BSA.
Figure 5: Analysis of glycophorin binding to truncated Sn-Fc chimeras. Purified forms of the indicated Fc chimeras were injected into a flow cell in which an anti-human IgG mAb had been immobilized onto the sensor surface. At the end of the injection the response is higher than the base line, which represents bound Fc chimera. BSA (B, 0.5 mg/ml), asialoglycophorin (aG, 0.5 mg/ml), and glycophorin (G, 0.5 mg/ml) were all injected for 3 min (short bars) before and after the Fc chimera was bound. An increase in the response both during and following the injection of glycophorin reflects binding to that particular Fc chimera. The inset in panel A shows a magnified view of glycophorin binding before (dotted lines) and after (solid lines) injection of Sn(d1-3)Fc. Results of three experiments are shown. In each experiment binding of Sn(d1-3)Fc is compared with CD22(d1-3)Fc (panels A and B), Sn(d1-17)Fc (panels C and D), and Sn(d1)Fc (panels E and F). Similar results were obtained for each construct in at least three independent experiments.
Because of the variability in absolute responses between experiments, binding to glycophorin of CD22(d1-3)Fc, Sn(d1-17)Fc, and Sn(d1)Fc was compared with Sn(d1-3)Fc in separate experiments (Fig. 5). Sn(d1)Fc and Sn(d1-17)Fc bound glycophorin as indicated by a small but clearly increased response during and immediately after glycophorin injection, whereas the response after BSA and asialoglycophorin injection remained unchanged. Binding of Sn(d1)Fc and Sn(d1-17)Fc to glycophorin occurred at levels similar to that of Sn(d1-3)Fc. As might be expected, no binding was observed with CD22(d1-3)Fc.
Similar binding experiments were performed with the truncated CD22-Fc chimeras to CD45 purified from rat thymus (Fig. 6). BSA and CD45 were injected before and after injection of the CD22-Fc chimeras. CD22(d1-3)Fc bound as well as CD22(d1-7)Fc. In comparison, CD22(d1-2)Fc bound poorly to CD45 (Fig. 6A). No binding of CD45 was observed with Sn(d1-3)Fc.
Figure 6: Analysis of CD45 binding to truncated CD22-Fc chimeras. Panel A, purified forms of the indicated Fc chimeras were injected (long bars) into a flow cell in which an anti-human IgG mAb had been immobilized onto the sensor surface. BSA (0.5 mg/ml) and purified rat thymus CD45 (30 µg/ml) were injected before and after (short bars) the Fc chimera was bound. Panel B, tissue culture supernatants (TCS) containing the indicated Fc chimeras were injected (long bars) into a flow cell in which an anti-human IgG mAb had been immobilized onto the sensor surface. BSA (10 µg/ml) and CD45 (10 µg/ml) were injected before and after (short bars) the Fc chimera was bound. An increase in the response both during and following the injection of CD45 reflects binding to that particular Fc chimera. Similar results were obtained for each construct in at least three independent experiments.
One explanation for why CD22(d1-2)Fc bound weakly to CD45 might be that a large fraction of the protein had been denatured during affinity purification of the protein which involves elution at pH 3.0. To test this possibility, we compared binding of unpurified CD22(d1-2)Fc and CD22(d1-3)Fc present in COS cell supernatants. As shown in Fig. 6B, unpurified CD22(d1-2)Fc and CD22(d1-3)Fc bound at equivalent levels to CD45, suggesting that CD22(d1-2)Fc had undergone selective denaturation during low pH elution. Binding of the CD22 constructs to CD45 was sialic acid-dependent because sialidase treatment of immobilized CD45 prevented subsequent binding (data not shown).
Figure 7: Epitope mapping of mAbs directed against sialoadhesin. Purified forms of the indicated Fc chimeras were injected into a flow cell (long bars) in which an anti-human IgG mAb had been immobilized onto the sensor surface. The anti-sialoadhesin mAbs 3D6 (panels A, D and G), SER-4 (panels B, E, and H), or 1C2 (panels C, F, and I) were then injected at a concentration of 10 µg/ml (short bars). An increase in response both during and after the injection of mAbs reflects binding to that particular Fc chimera.
Two mAbs, CY34 (30) and NIMR6(15) , have been shown to recognize mouse CD22. Using the BIAcore with unpurified forms of the CD22-Fc constructs, CY34 mAb bound to CD22(d1-2)Fc, CD22(d1-3)Fc, and CD22(d1-7)Fc but not to CD22(d1)Fc (Fig. 8). Since it is possible that the CD22(d1)Fc construct was incorrectly folded, these results suggest that the epitope recognized by CY34 mAb may lie in either domain 1 or domain 2 or in a region shared between both of these domains. In contrast, NIMR6 mAb bound CD22(d1-3)Fc and CD22(d1-7)Fc but not CD22(d1-2)Fc (Fig. 8). This shows that the epitope recognized by this antibody is either in domain 3 or is in a region shared between domains 2 and 3.
Figure 8: Epitope mapping of mAbs directed against CD22. Unpurified forms of the indicated Fc chimeras were injected into a flow cell (long bars) in which an anti-human IgG mAb had been immobilized onto the sensor surface. Anti-mouse CD22 mAbs NIMR6 and CY34 in the form of tissue culture supernatants were injected (short bars). An increase in response both during and after the injection of mAbs reflects binding to that particular Fc chimera.
By using a series of domain deletion constructs we have
mapped the sialic acid binding sites of sialoadhesin and CD22 as well
as epitopes recognized by mAbs specific to each molecule. We
demonstrate that for sialoadhesin, the amino-terminal V-set Ig-like
domain (domain 1) is both necessary and sufficient for binding in all
assays. Domains 2 and 3 do not appear to contribute significantly to
the adhesive functions since binding of Sn(d1)Fc was at levels
comparable to that of Sn(d1-3)Fc. However, a recombinant
construct that contained the first two domains displayed reduced
binding as compared with the construct containing domain 1 on its own
or domains 1+2+3. Since mAbs 3D6 and 1C2 bound to
Sn(d1-2)Fc at reduced levels compared with Sn(d1)Fc and
Sn(d1-3)Fc, this suggests that a fraction of Sn(d1-2)Fc was
partially denatured, which could therefore explain its reduced binding
activity. Similar observations were made with CD22(d1-2)Fc, and
in this case it was found that full activity could be restored if
binding assays and epitope mapping were carried out with unpurified
forms of the Fc proteins. Since the routine purification of Fc proteins
involves an elution step at pH 3.0, these results indicate that the Sn-
and CD22(d1-2)Fc constructs are unusually susceptible to acid
denaturation. In this respect it is interesting that our attempts to
generate purified MAG(d1-2)Fc have been unsuccessful so far. ()
In contrast to sialoadhesin, our results show that
murine CD22 requires at least the first two domains for binding to
cellular and molecular ligands. These observations are consistent with
a recent report (38) demonstrating that for human CD22
expressed in COS cells the presence of the first two amino-terminal
domains together is essential for sialic acid-dependent adhesion. In
view of the similarities in sequence and domain organization between
the amino-terminal domains of sialoadhesin and CD22 it might be
expected that, similar to sialoadhesin, the binding site of CD22 would
reside in the first amino-terminal Ig-like domain. Indeed, recent
mutagenesis screens of sialoadhesin and CD22 have shown that domain 1
of both molecules contains residues that are critical for sialic
acid-dependent binding. ()We therefore favor the idea that
the sialic acid binding site of CD22 lies in domain 1 and that domain 2
is required for correct folding of domain 1. Indirect support for this
interpretation is provided by the observation that human CD22
constructs lacking domain 2 show reduced reactivity with mAbs that are
directed against domain 1 and/or domain 2(13, 38) ,
thereby suggesting that domain 2 is critical for the correct
conformation of domain 1. Likewise, in the present study it is possible
that the failure of the CY34 mAb to bind CD22(d1)Fc is a result of
incorrect folding. Generation of more mAbs and/or production of
chimeric constructs between appropriate mouse strains (12) will
be required to resolve this issue. Unpublished observations in our
laboratory with MAG and CD33 show that, like CD22, domain 1 on its own
is unable to mediate sialic-acid dependent adhesion. (
)Within the Sialoadhesin family, therefore, it appears that
sialoadhesin is unusual in that domain 1 can function independently of
domain 2. It is possible that formation of a disulfide bond between
domains 1 and 2 (9, 10) could be important for the
correct folding of domain 1 in the cases of CD22, MAG, and CD33, but
not in the case of sialoadhesin.
For the majority of Ig superfamily adhesion molecules involved in heterophilic interactions, structure-function studies have shown that the dominant binding sites are localized to the first amino-terminal Ig-like domain (reviewed in (1) ). In general, it can be expected that the amino-terminal domain is more accessible for mediating interactions with corresponding counterreceptors. The binding activities of the membrane proximal domains are less defined, but they may be important in making the binding site more accessible. Thus, the binding site on ICAM-1 for lymphocyte function-associated molecule-1, rhinovirus, and malaria-infected erythrocytes is localized in the first amino-terminal domain, but removal of the membrane proximal domains results in decreased ligand binding when ICAM-1 is expressed in monkey COS cells(39, 40) . In certain cases where domain 1 has been shown to contain the binding site, it has also been shown that there is a conformational dependence on domain 2. With ICAM-1 and CD4, it has not been possible to express domain 1 in the absence of domain 2(39, 40) . This conformational dependence is thought to be a result of the close packing of the first two domains as shown in the crystal structure of CD4(41, 42) . Thus, it appears that for several adhesion molecules belonging to the Ig superfamily, the ligand binding site is located in the first amino-terminal domain, but an adjacent domain is required for correct conformation.
Several cell adhesion molecules of the Ig superfamily exhibit a dual homophilic/heterophilic adhesion activity, including NCAM, CD31, and CEA family proteins(1) . In these molecules, the homophilic and heterophilic binding sites appear to be distinct. For example, homophilic NCAM-NCAM binding interactions in trans appear to be mediated by a 10-amino acid sequence within the third Ig-like domain (43) . This is distinct from its heterophilic glycosaminoglycan binding region in the second amino-terminal domain. NCAM can also mediate carbohydrate-dependent cis interactions with L1 via a region located in its fourth Ig-like domain(44) . CD31-CD31 homophilic interactions involve a more extensive area over multiple domains than NCAM homophilic interactions. It is proposed that domains 2 and 3 on one CD31 molecule interact in an antiparallel fashion with domains 5 and 6 on an apposing CD31 molecule(45) . A similar mechanism has been proposed for CEA homotypic interactions, which involve reciprocal interactions between antiparallel CEA molecules aligned in trans(46) . It is therefore evident that cell adhesion molecules containing several Ig-like domains can bind multiple ligands using distinct domains.
The Sialoadhesin family represents a new class of carbohydrate-binding proteins in which an Ig fold has acquired the capacity to mediate sialic acid-dependent adhesion. The overall molecular organization of sialoadhesins bears several similarities to selectins, arguably the best characterized mammalian lectins involved in cell-cell interactions (reviewed in (47) ). Selectins contain an amino-terminal C-type lectin domain that functions in carbohydrate binding. Our results show that for sialoadhesin, the lectin region is within the amino-terminal V-set domain. We anticipate that the same region is used by CD22, MAG, and CD33. The localization of the carbohydrate binding sites at the amino terminus of selectins and sialoadhesins could be important in allowing their respective lectin domains to extend out of the cell glycocalyx to interact with carbohydrate ligands on apposing cells(7) . Selectins use varying numbers of complement control protein domains to extend the lectin domain from the cell surface, whereas sialoadhesins use variable numbers of C2-set Ig domains. In selectins, correct folding of the lectin domain may require an adjacent epidermal growth factor-like domain(48, 49, 50, 51) . This is analogous to the apparent requirement of the second Ig domain in CD22 (and possibly MAG and CD33) for correct folding of the V-set domain.
The V-set and adjacent C2-set domains of sialoadhesins
contain structural features that are not present within other adhesion
molecules of the Ig superfamily and could therefore be involved in
sialic acid-dependent binding. These include an inter--sheet
disulfide between
-strands B and E of the V-set domain (9) and a predicted disulfide between the V- and C2-set
domains
(10) . Since the V-set domain of
sialoadhesin can function independently of the C2-set domain, a direct
role of the interdomain disulfide for binding of sialoadhesin to sialic
acid is unlikely, but it may be important for correct folding of the
V-set domain in the cases of CD22, MAG, and CD33. The role in sialic
acid-dependent binding of the conserved inter-
-sheet disulfide
within the V-set domain of sialoadhesins awaits further investigation.