From the
Carbohydrate-binding proteins (lectins) are widespread among
prokaryotes and eucaryotes. Among the latter, lectins mediate many
specific biological functions including cell-cell interactions, protein
trafficking, and primitive defense
reactions(1, 2, 3, 4, 5, 6) .
We describe here a newly recognized family of mammalian lectins
belonging to the immunoglobulin superfamily
(IgSF)
CD22 is a cell surface phosphoglycoprotein detected on the
majority of resting mature B cells. It appears to facilitate
antigen-dependent B cell triggering by association with the B cell
antigen receptor and with cytoplasmic tyrosine
kinases(10, 17, 20, 21, 22) . It
can also induce intercellular adhesion, recognizing ligands on
activated lymphocytes, monocytes, and endothelial
cells(10, 12, 23, 24, 25) .
Sialic acids (Sias) are known to repel cell-cell interactions because
of their negative charge(26) , and sialidase treatments usually
enhance such interactions. In contrast, treatment of target cells with
sialidase abolished CD22-mediated adhesion, and de novo expression of a sialyltransferase induced binding(23) . A
positive role for Sias in CD22 recognition was confirmed by loss of
binding upon treating target cells with mild sodium periodate, under
conditions that selectively oxidize the C
Sn was first identified on populations of bone marrow and
tissue macrophages, mediating adhesion to various lymphohematopoietic
cells(34, 35, 36, 37, 38, 39) in a
Sia-dependent manner. Elucidation of the primary sequence demonstrated
it to be a member of the IgSF with 17 extracellular Ig-like
domains(14) . The first four NH
Sias are a family of 9-carbon carboxylic acids usually found
in the terminal position on vertebrate
glycoconjugates(26, 45) , attached by different
Each of the
many types of sialic acid linkage is generated by one or more unique
sialyltransferases, many of which have been characterized and/or
cloned(45) . With
The most common Sia is N-acetylneuraminic acid (see Fig. 3), believed to be the
biosynthetic precursor for more than 25 others in the
family(26) . A common natural modification, the
9-O-acetylation of the polyhydroxy side chain, abrogates CD22
recognition(31) . This fits with the earlier finding that
chemical cleavage of this side chain abolishes
binding(29, 30) . Indeed, a major fraction of CD22
ligands on some murine lymphoid cells appears to be
``masked'' by 9-O-acetylation of the
The five I-type lectins described above share close homology
as well as the ability to recognize sialic acid. Many classical
immunoglobulins (antibodies) can of course recognize specific
carbohydrate structures. However, the Ig superfamily itself is of
ancient evolutionary origin, predating the establishment of the
antibody response(7, 8, 55) . Thus, lectin-like
properties might be present in other IgSF members with more distant
homologies. Indeed, the neural cell-adhesion molecule (NCAM) is
reported to recognize high mannose oligosaccharides on other proteins,
evidently via domain 4(56, 57) . This property is
independent of its homotypic protein-based interactions. ICAM-1,
another IgSF member with well known protein-based interactions, may
also function as a receptor for the anionic oligosaccharide
hyaluronan(58) , as well as recognizing a heavily sialylated
glycoprotein leukosialin(59) . Indirect evidence suggests that
the single anionic complex-type oligosaccharide present on P0 may be
involved in modulating some of its binding
functions(60, 61, 62) . Furthermore NCAM and MAG
interact with the anionic polysaccharide heparin(63) . These
findings suggest that carbohydrate recognition by IgSF members may be
more widespread.
IgSF members are known to mediate homotypic or
heterotypic binding via protein-protein
interactions(7, 8) . There is no reason why such roles
would be completely supplanted in the I-type lectins by their
carbohydrate recognition properties (NCAM and ICAM-1 may be examples).
An interesting possibility is that the two types of recognition might
even be related, e.g. the occupancy of the lectin sites on
CD22 on activated B cells might unmask alternative cell-cell
interactions mediated by this protein. In this regard, several internal
C2-type domains of the Sia-binding I-type molecules share homology with
the carcinoembryonic antigen-related family of IgSF proteins known to
function as homotypic peptide binding partners(64) .
Activation of B cells increases expression of ST6N, the
enzyme that generates
Equilibrium dialysis studies with the soluble dimeric CD22Rg
and the monomeric ligand
The relatively weak affinity
of the monovalent CD22 interaction indicates that, as with most
lectins, functional avidity may be attained primarily by multivalent
binding(3) . Indeed, N-linked oligosaccharides with
multiple
Many cell surface proteins of CD22-positive B cells can bear
The restricted expression of the I-type lectins () implies that they mediate specific biological functions.
The selective occurrence of Sn at the contact sites between bone marrow
macrophages and myeloid precursors (35) strongly suggests an
adhesive role. This is supported by the selective interactions of Sn
with cells of the myeloid lineage in vitro(39) .
However, the biological significance of these interactions remains
obscure. The privileged location of MAG within the nervous system has
suggested a specific role in axonal myelination. However, the results
of homozygous MAG gene disruption in mice (69) indicate that MAG
is not critical for myelin formation but is necessary for maintenance
of the cytoplasmic collar and periaxonal space of myelinated fibers.
Additionally, MAG has recently been shown to be a potent inhibitor of
neurite outgrowth in vitro(41) . CD22 is in a position
to mediate interactions of B cells with T cells, other B cells,
activated endothelial cells, or accessory
cells(12, 24, 32) . However, the in vivo occurrence and significance of such interactions have not been
demonstrated. The same is true of CD33. CD22, MAG, and CD33 have
cytosolic domains with potential phosphorylation sites that, at least
in the case of CD22, are known to be utilized upon B cell
activation(17) . Thus, it is possible that the major role of
these lectins is not in primary adhesive events but rather in secondary
activation events caused by engaging either soluble ligands or cell
surface ligands in cis.
Classical immunoglobulins can
recognize oligosaccharides with a high degree of structural
specificity. Indeed, many germline V regions appear to have native
carbohydrate binding properties(55) , suggesting the importance
of carbohydrate recognition in the immune response(55) .
However, the IgSF is of more ancient evolutionary origin than the
immunoglobulins of the vertebrate immune
system(7, 8, 55) . Thus, while I-type lectins
may have evolved from carbohydrate-binding immunoglobulins, it is
equally possible that they are products of a parallel evolutionary
process, driven by the need to generate carbohydrate binding
specificities in complex multicellular systems. If so, all I-type
lectins might not be closely homologous to one another. Thus, the
molecules closely related to CD22 (Sn, CD33, MAG, and SMP) may be only
one subfamily of the larger group of I-type lectins. Indeed, the less
clear-cut examples of potential I-type lectins presented in (NCAM, P0, and ICAM-1) may give some hint to the true
diversity of this family.
Investigators who have made the most contributions toward
defining I-type lectins that recognize Sias (Sn, CD22, CD33, and MAG)
have proposed that these should be collectively called the
``sialoadhesins''(40, 43) . While this is a
very reasonable suggestion, each of these proteins has one name that is
already well established in the literature, adhesive functions have not
been conclusively shown for some, and at least some are thought to have
other non-lectin-based functions as well. Furthermore, if more I-type
lectins are discovered that do not recognize sialic acids, these could
not be properly called sialoadhesins. Perhaps some more time should
elapse before definitive nomenclature changes are adopted.
These
recent advancements have established the principle that IgSF members
other than immunoglobulins can specifically recognize and bind
carbohydrates. It is reasonable to predict that there will be more
members of this family, including some not closely homologous to those
that bind Sias. Indeed, some as yet unidentified I-type lectins might
be well known IgSF members whose carbohydrate binding properties have
never been tested. It is important to know how the Sia-binding lectins
mediate specific biological functions (presumably mediated by high
affinity ligands) in the midst of a large excess of low affinity
ligands. In this regard, it would be useful to know if the
homomultimeric state discovered for CD22 is a feature of the other
lectins. Specific focus upon the ligands with the highest apparent
affinity (e.g. CD45 and IgM for CD22) also seems warranted.
Ultimately, genetic manipulation of these lectins and of the
glycosyltransferases that generate their ligands must be performed in
transgenic animals.
We thank Stephen Hedrick, Rod Langman, and Jamey Marth
for the careful review of this manuscript and several authors for
providing manuscripts in press.
INTRODUCTION
CD22 Is a Sialic Acid-binding Lectin
IgSF Members Homologous to CD22 Are Also Lectins
The Role of Sialic Acids in Recognition
Other IgSF Members That May Have Lectin-like Properties
Occupancy of the Lectin Site by Ligands in Cis
Further Studies of Carbohydrate Binding
Low Affinity Ligands and Soluble Inhibitors as Modulators
of Biologically Relevant Recognition
Biological Roles and Evolutionary Considerations
Future Directions
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(7, 8) . Relative emphasis
is placed on a prototypic member, CD22. Previously known mammalian
lectins can be classified into distinct families based on protein
sequence homologies (1) (). Recently, another family
has emerged from independent investigations of the proteins CD22 and
sialoadhesin (Sn) (see ). Unlike previously recognized
animal lectins, these belong to the IgSF, and several have a
characteristic V
-C2
domain structure (Fig. 1)(9, 10, 11, 12, 13, 14, 15, 16) ,
suggesting the name ``I-type'' lectins. All are integral
membrane proteins, preferentially expressed on the plasma membrane, and
some (CD33, CD22, and MAG) have large cytosolic domains with multiple
potential and established phosphorylation sites (both Ser/Thr and
Tyr)(17, 18, 19) . Here we focus primarily on
the lectin properties of the extracellular domains.
Figure 1:
Sequence homologies among some I-type
lectin. Comparisons are made between the N-terminal two Ig-type domains
(V and C2). All sequences are from the mouse. Predicted strand
assignments for each are indicated in the toprows by
by a, b, c, c`, etc. Shared residues indicated in blue are homologous to previously established motifs of Ig V and C2
type domains (see bottomrows, where a is
acidic, f is aliphatic, h is hydrophobic, o is aromatic, p is polar, and s is small (8)).
Shared residues indicated in red appear to be common
(identical or conserved) to these I-type lectins
only.
-C
exocyclic side chain of
Sias(27, 28, 29, 30, 31) . The
predominant isoform of human CD22 has 7 extracellular domains, and
isoforms lacking domains 3 and/or 4 have been
reported(10, 12, 24) . A soluble chimeric form
containing the three amino-terminal domains fused to the hinge and two
Fc domains of IgG (CD22Rg) was employed to study its interactions with
lymphoid cells (23). From radiolabeled T and B cell lines, CD22Rg
specifically precipitated several glycoproteins including CD45, the
leukocyte-specific phosphotyrosine
phosphatase(27, 28, 32) . CD45 was also
identified as a specific target for CD22 in cell adhesion assays (23,
27, 32). Blocking experiments with a panel of monoclonal antibodies
mapped the binding region to the first two domains of
CD22(24, 33) . In all of these studies, CD22 binding was
Sia-dependent.
-terminal domains
showed considerable homology with CD22 (14) ( Fig. 2and Fig. 3), and soluble immunoglobulin fusion proteins containing
these domains preserved the sialic acid-dependent
binding(14, 40) .
Figure 2:
Biosynthetic pathways generating terminal
sialylated oligosaccharides recognized by some I-type lectins. GlcNAc
or GalNAc residues of glycoproteins and/or glycolipids can be extended
by several enzymes. Note that some theoretically possible structures, e.g. Sia2-6Gal1-3GlcNAc, have not been
reported in nature. The sialylated sequences shown are the minimal
structural motifs necessary for binding. Natural high affinity ligands
may be more complex.
Figure 3:
Proposed model of CD22-sialoside binding.
A disaccharide containing a 9-carbon sialic acid (N-acetylneuraminic acid) in 2-6 linkage to a Gal
residue is presented interacting with the binding pocket of CD22 (based
on the data in Ref. 46). In that study, R
could be GlcNAc,
GalNAc, H, or a synthetic group. R
, R
, and
R
could be substituted in various ways without
substantially affecting binding.
Some previously known proteins have considerable sequence homology to
Sn and CD22 ( Fig. 2and Fig. 3). MAG is involved in the
assembly of the myelin sheath and may control the growth properties of
neurons(41) . CD33 is a marker of early myeloid cells and some
monocyte populations(9, 16, 42) . Schwann cell
myelin protein (SMP) is an avian protein with close homology to
mammalian MAG(15) . The function(s) of CD33 and SMP are
currently unknown. Based on their close homology to CD22 and Sn,
MAG(40) , CD33(43) , and SMP(
)were
studied for sialic acid-dependent recognition and found to share this
property. As shown in , these molecules vary widely in
their number of Ig-like domains. However, in each case, the first two
amino-terminal Ig-like domains (V- and C2-type, respectively) appear to
be necessary and sufficient for sialic acid-dependent
binding(40, 43) . In CD33, the entire extracellular
domain consists of just these two domains. Sequence comparisons of the
corresponding regions of these proteins (Fig. 1) indicate several
regions of close homology not universally found in IgSF
members(7, 8) . Notably, some of these residues are
located in the predicted interstrand regions that form the
antigen-binding site in classical immunoglobulins. In each case, the
first V-type domain contains an unusual intradomain disulfide bond
between the predicted b and e
-strands (rather
than b and f
-strands classically found in
immunoglobulins)(7, 8) . Also, an interdomain disulfide
bond is reported in MAG between unpaired cysteines in domains 1 and
2(44) , which are conserved in CD22, SMP, Sn, and CD33. This
unpaired Cys residue is also found in some other IgSF adhesion proteins
(ICAM-1, ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1) as part of a
C-X
-C motif essential for integrin
binding. However, by deletional mutagenesis experiments, this motif is
not essential for CD22-sialic acid binding(33) .
-ketosidic linkages from the 2-position to the underlying sugar
chain (Fig. 3). CD22 interactions involve recognition of the
structural motif
Sia
2-6Gal
1-4GlcNAc
1-(29, 30) ,
known to occur in varying numbers on the N-linked
oligosaccharides of some cell surface glycoproteins(45) . The
2-6 linkage is an absolute requirement ( Fig. 2and Fig. 3), and other
2-6-linked Sia structures
(including linkage to GalNAc and GlcNAc) may also be recognized with a
lower affinity(46) . In contrast, Sn binds to
Sia
2-3-containing structures (Fig. 2)(37, 40) . The binding specificity of CD33
appears to be similar to that of Sn(43) , while MAG binding
shows a somewhat more restricted range (see Fig. 2)(40) .
Another well known family of sialic acid-binding lectins (the
selectins) recognizes a further variation on these terminal sequences
(not shown in Fig. 2), involving fucosylation of
Sia
2-(3)4Gal
1-4GlcNAc
1-(5) .
-galactoside
-2,6-sialyltransferase
(ST6N), which transfers terminal
2-6-linked Sia to
Gal
1-4GlcNAc
1-(45) , tissue-specific promotors
have been identified(47) , and activation and/or cell
cycle-dependent expression in lymphoid and endothelial cells have been
demonstrated(48, 49, 50, 51) . Regulated
expression of
2-6-specific and
2-3-specific
sialyltransferases in other tissues and organs has also been
shown(45, 52, 53) . However, expression of a
given sialyltransferase does not guarantee expression of its product,
as they must compete with other enzymes for the same acceptors in the
Golgi apparatus (Fig. 2).
2-6-linked sialic acids(31) . Similar effects of
9-O-acetylation have been reported for Sn(54) . Another
common substitution of sialic acids is the conversion of the N-acetyl group at the 5-position to an N-glycolyl
group. This modification has no major effect on recognition by human
CD22 (31) but enhances binding by murine CD22 (54). The effects
of the many other known natural modifications of sialic acids (26) remain unexplored.
2-6-sialylated CD22
ligands(47, 53) . Thus, recently activated B cells carry
both CD22 and CD22 ligands(23, 47, 65) , e.g. B cells in the mantle zone of secondary lymph node
follicles(53, 65) . Can CD22 can still mediate cell
adhesion under such circumstances? Braesch-Andersen and Stamenkovic (66) first showed that when CD22 is transiently coexpressed with
ST6N in COS cells, the lectin property is lost and can be restored by
sialidase digestion. We confirmed and extended this observation both
with Chinese hamster ovary cell lines stably expressing CD22 and/or
ST6N (67) and with cultured B lymphoma cells co-expressing CD22
and its
2-6Sia ligands. Direct probing of the lectin
function of CD22 on cell surfaces by ligand staining confirmed the loss
of CD22 lectin function on the B lymphoma cells(67) . Thus, the
ST6N enzyme can regulate CD22-mediated adhesion either negatively (if
expressed in cells with CD22) or positively (if expressed on potential
target cells). Similar abrogation of lectin function in cis by
endogenous ligands has been reported for CD33 and MAG (43) but
not for sialoadhesin(37) . With the latter, it is suggested that
the length of the molecule allows its functional domains to protrude
above the cellular glycocalyx containing the potential
ligands(14, 40) . Since the extracellular domains of
CD33 and CD22 contain many N-linked oligosaccharides, they
could potentially express their own ligands. Indeed, CD22Rg secreted
from transfected cells co-expressing the ST6N sialyltransferase is
functionally inactive until reactivated by sialidase
digestion(66) . It remains to be determined whether, in the
native situation, an intermolecular inactivation is more important,
involving recognition of other sialoglycoproteins. Regardless of the
mechanism, the biological purpose of this
``autoinactivation'' of lectin function remains obscure. With
NCAM, interactions of the fourth Ig-like domain with high mannose
oligosaccharides on other cell surface molecules have been suggested to
modulate the binding properties of other adhesion molecules such as
L1(57) .
2-6 sialyllactose gave a
stoichiometry of
2:1, indicating that each native CD22 molecule
has a single sialic acid-binding site (46) and predicting a
single binding site for each of the other sialic acid-binding I-type
lectins. The apparent affinity of the CD22-
2-6 sialyllactose
interaction is
30 µM.
2-6-linked sialic acids interact better with CD22
in a column binding assay(30, 46) . However, even though
many cell surface and plasma glycoproteins carry such oligosaccharides,
relatively few of these appear to be high affinity
ligands(28, 29) . Indeed, from among the large number of
sialoglycoproteins in human plasma bearing
2-6-linked sialic
acids, only two (IgM and haptoglobin) appear to bind with high affinity
to bivalent recombinant CD22, and binding to IgM is diminished upon
disruption of its pentameric structure(68) . While the
significance of these observations is uncertain, it shows that certain
glycoproteins are able to create superior ligands for CD22 when
properly sialylated. How does this high affinity recognition occur?
Likely explanations are multivalency involving specific arrangements of
2-6-linked sialic acids or additional protein-protein
interactions. Given the plethora of ligands to which CD22 has been
reported to bind, the latter appears less likely. The former is given
considerable support by the finding that at least a portion of the CD22
on cell surfaces is presented in a multimeric form(46) . These
non-covalent homomultimers are found in non-lymphoid cells expressing
transfected CD22, indicating that their formation does not require any
other B cell-specific proteins(46) .
2-6-linked sialic acids, and some could potentially act as
inhibitors or false ligands in cis. Likewise, CD33 is
expressed on myeloid cells known to express
2-3-linked
sialic acids (43). Furthermore, these lectins must function in a milieu
of natural biological fluids (plasma, extracellular fluid, or lymph)
that contain high concentrations of sialylated glycoproteins. Indeed,
human plasma contains
1 mM concentration of protein-bound
2-6-linked sialic acids (capable of strongly inhibiting
CD22-based interactions) and
0.5 mM concentration of
2-3-linked sialic acids(68) . Interestingly, the
major Sia-dependent plasma ligands for CD22 are haptoglobin (an acute
phase reactant produced by the liver in inflammatory conditions) and
IgM, the major downstream product of activation of CD22-positive
B-lymphocytes(68) . However, a large number of other plasma
sialoglycoproteins can also interact with CD22 but with a lower
affinity(30) . Thus, although CD22 shows exquisite specificity
in oligosaccharide recognition, it must function under markedly
different conditions than other vertebrate lectins such as the
asialoglycoprotein receptor(1) , the mannose 6-phosphate
receptors(2) , and the hepatic receptor for sulfated
oligosaccharide(6) . In the latter cases, the cognate ligands
are relatively rare structures that the receptors specifically
recognize among a large excess of other non-competing glycoproteins.
However, with CD22, CD33, and Sn (and possibly MAG), the primary
oligosaccharide motif recognized is a common sequence found on the
majority of glycoproteins encountered in the surrounding milieu.
Nonetheless, the evolutionary conservation of the lectin properties
indicates that they mediate specific biological functions (presumably
mediated by high affinity ligands) despite this large excess of low
affinity ligands. It is also notable that some large plasma
sialoglycoproteins are present at much lower concentrations in
extracellular fluid. Thus, functioning of these lectins might only be
triggered when the level of soluble inhibitors falls below a threshold
in certain privileged tissue compartments(68) . Regardless of
the precise significance of the low affinity inhibitors, in vitro assays done in sialoglycoprotein-poor fluids could show binding
phenomena that do not necessarily predict biological relevance in
vivo. The same caveat applies to the plethora of potential
biological ligands for these lectins that can be detected in
vitro. In vivo assays are required to determine which of
these are biologically important.
Table: Animal lectins and families
Table: Established and
putative members of the I-type lectin family
-galactoside
-2,6-sialyltransferase; NCAM, neural cell-adhesion molecule.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.