(Received for publication, November 30, 1994; and in revised form, January 13, 1995)
From the
CD22 is a cell-surface receptor of resting mature B cells that
recognizes sialic acid (Sia) in the natural structure
Sia2-6Gal
1-4GlcNAc (Powell, L. D., Jain, R. K.,
Matta, K. L., Sabesan, S., and Varki, A.(1995) J. Biol. Chem. 270, 7523-7532). Human umbilical vein endothelial cells
(HEC) treated with inflammatory cytokines such as tumor necrosis
factor-
(TNF-
) display increases in cell-surface CD22
ligands, caused by increased expression of the enzyme
-galactoside
2,6-sialyltransferase (Hanasaki, K., Varki, A., Stamenkovic, I.,
and Bevilacqua, M. P.(1994) J. Biol. Chem. 269,
10637-10643; Hanasaki, K., Varki, A., and Powell, L. D.(1995) J. Biol Chem. 270, 7533-7542). Thus, CD22 could direct
potential interactions between mature B cells and endothelial cells
during inflammatory states. However, this would have to occur in the
presence of blood plasma, which contains many sialoglycoproteins known
to carry
2-6-linked sialic acids. We show here that human
plasma can indeed inhibit Sia-dependent binding of a recombinant
soluble chimeric form of human CD22 (CD22Rg) to TNF-
activated
HEC. Affinity adsorption of individual human plasma samples with
immobilized CD22Rg showed that, of the numerous
2-6-sialic
acid containing glycoproteins in plasma, only three polypeptides with
apparent molecular mass (under reducing conditions) of 74, 44, and 25
kDa bound, and were specifically eluted with
2-6-sialyllactose. NH
-terminal amino acid
sequencing of these high affinity CD22 ligands revealed that they are
subunits of immunoglobulin M (IgM) and haptoglobin. Purified human IgM
from pooled human plasma can be quantitatively bound by CD22Rg, and
binding is blocked by
2-6-sialyllactose, but not by
2-3-sialyllactose. Pretreatment by sialidase or by mild
periodate oxidation of sialic acid side chains abolishes these
interactions. IgM at physiological concentrations also inhibits CD22Rg
binding to TNF-
-activated HEC in a manner dependent not only upon
its sialylation but also requiring its intact multimeric structure.
These data show that CD22 is capable of highly selective recognition of
certain multimeric plasma sialoglycoproteins that carry
2-6-linked sialic acids. Notably, the two proteins that are
selectively recognized are known to be involved in immune and
inflammatory responses. Haptoglobin synthesis by the liver is markedly
increased during the ``acute phase response'' to systemic
inflammation, while IgM is the major product resulting from activation
of resting CD22-positive B cells.
CD22 is a cell-surface phosphoglycoprotein found on the majority
of resting mature B cells, and appears to be involved in
antigen-induced cell activation (1, 2) and in cell
adhesion, mediating interactions with activated blood cells, accessory
cells, and endothelial cells (3, 4, 5, 6, 7) (see also
the accompanying papers(8, 9) ). Soluble chimeric
forms of CD22 (CD22Rg)(), containing the three
amino-terminal Ig-like domains of human CD22
fused to the
COOH-terminal Fc domains of human IgG or mouse IgG (10, 11) have been previously used to identify
sialoglycoprotein ligands on activated T and B cells, which include,
among others, CD45, the leukocyte-specific receptor-linked
phosphotyrosine-phosphatase(5, 10, 12) .
These interactions involve recognition of the sialic acid (Sia)
containing structural motif
Sia
2-6Gal
1-4GlcNAc
1-(8, 11, 13, 14) .
This sequence is known to occur in varying copy numbers on the N-linked oligosaccharides of some cell-surface glycoproteins (15) . The Sia residues of these glycoprotein ligands are
essential for binding to CD22, since the interaction is blocked by
their pretreatment with sialidase or by mild periodate oxidation under
conditions which are specific for truncation of the exocyclic side
chain of sialic acid (6, 10, 11) .
Recent
studies have demonstrated some potential regulatory mechanisms that
could affect CD22-dependent adhesion events in vivo.
Activation of T lymphocytes causes enhanced expression of CD22 ligands (10) , apparently via increased expression of -galactoside
2,6-sialyltransferase(16) , the enzyme which transfers
terminal
2-6-linked Sia to
Gal
1-4Glc(NAc)(17, 18) . This favors the
notion that CD22 functions as a co-receptor in T-B cell interactions (5, 12) . However, a similar enhancement in expression
of
-galactoside
2,6-sialyltransferase and of CD22 ligands
also occurs during activation of resting B
lymphocytes(10, 19, 20) . Indeed,
coexpression studies have shown that the lectin function of CD22 can be
abrogated by sialylation of CD22 itself with
-galactoside
2,6-sialyltransferase(9, 21) . Thus, it has been
suggested that CD22-ligand interactions can be positively or negatively
regulated by the expression of this
sialyltransferase(9, 21) . In addition, some CD22
ligands in cells of lymphoid tissues appear to be ``masked''
by 9-O-acetylation of
2-6-linked sialic acids, a
naturally occurring modification which markedly reduces binding to
CD22(14) . Finally, we recently found that treatment of human
umbilical vein endothelial cells (HEC) with inflammatory cytokines such
as tumor necrosis factor-
(TNF-
), causes increased expression
of
-galactoside
2,6-sialyltransferase, and enhanced
expression of CD22 ligands (detected by CD22Rg binding)(7) . As
shown in the preceding paper(9) , this is accompanied by
enhanced binding of Chinese hamster ovary cells expressing transfected
human CD22. Thus, we considered the possibility that CD22-expressing B
lymphocytes might bind to activated endothelium during inflammatory
conditions. This could theoretically mediate extravasation of B cells
into tissues, antigen transfer to B cells, and/or some other unknown
biological interactions. However, this intercellular recognition
process would have to occur in the presence of blood plasma, a rich
source of sialoglycoproteins, many of which carry
2-6-linked
sialic acids (22) . Indeed, we found that human plasma is
capable of inhibiting this interaction. In pursuing this finding, we
have identified the major high-affinity plasma ligands for CD22, which
are dependent upon
2-6-linked sialic acids for recognition.
Figure 1:
Effect of sialyllactose on CD22Rg
binding to HEC. A, effects of TNF- stimulation and
linkage-specific sLac. Confluent HEC, stimulated for 48 h with or
without 200 units/ml TNF-
, were incubated with CD22 mRg in the
absence or presence of 1 mM
2-3- or
2-6-sLac. After washing, cells were incubated with
peroxidase-conjugated goat anti-mouse IgG Ab and binding was detected
as described under ``Experimental Procedures.'' The data are
the mean ± S.E. of triplicates from a representative experiment (n = 4). B, effects of sLac concentration.
TNF-
-activated HEC were incubated with CD22 mRg in the presence of
various concentrations of sLac, and the binding detected as above. The
data are the means of duplicates from a representative experiment (n = 3).
Figure 2:
Effects of human plasma on CD22Rg binding
to TNF--activated HEC. TNF-
-activated HEC were incubated with
CD22 mRg in the presence of various concentrations of human plasma and
assayed as described in the legend to Fig. 1. Plasma samples
from four different individuals were studied. The data shown are the
mean ± S.D. of duplicates with each plasma
sample.
Figure 3:
Identification of CD22-binding proteins in
plasma by affinity adsorption. Plasma (lane 1) was applied to
a PAS column. The pass-through fraction was collected (lane
2), which was then incubated with CD22Rg-coupled to PAS (lanes
4-6), CD8Rg-PAS (lane 7), or PAS alone (lane
8). After centrifugation, the supernatants were collected as the
unbound fraction (lane 3 shows one example). The beads were
washed with TBSE and incubated with buffer alone (lane 4), 1
mM 2-3-sLac (lane 5), or 1 mM
2-6-sLac (lanes 6-8) at 37 °C. The
supernatant was collected after centrifugation. Plasma samples (lanes 1-3, originating from 0.2 µl of plasma) or
the eluted samples (lanes 4-8, originating from 20
µl of plasma) were analyzed by SDS-PAGE under reducing
conditions.
Figure 4:
Rebinding of 2-6-sLac-eluted
plasma IgM to CD22Rg and effects of sialidase or periodate. Plasma
proteins eluted by
2-6-sLac from CD22Rg-PAS (see lane 6 of Fig. 3) were treated with or without sialidase or mild
periodate as described under ``Experimental Procedures.''
Protein A-coated plates were incubated with CD22 mRg, and then
incubated with the plasma samples in the absence or presence of
2-6- or
2-3-sLac (1 mM). The plates were
incubated with biotin-labeled goat anti-human µ-chain specific Ab,
and binding was assayed using peroxidase-conjugated streptavidin. The
data are the mean ± S.E. of triplicates from a representative
experiment (n = 2).
Figure 5:
Binding of CD22Rg to purified pooled IgM.
PAS-unbound IgM and CD22-bound IgM were prepared from pool IgM as
described under ``Experimental Procedures.'' All three IgM
preparations were coated in 96-well plates, incubated with CD22 mRg in
the absence or presence of 2-6- or
2-3-sLac (1
mM), and assayed as described in the legend to Fig. 1.
The data are the mean ± S.E. of triplicates from a
representative experiment (n =
2).
As shown in Fig. 6, pooled
human IgM blocked CD22Rg binding to TNF--activated HEC, with an
IC
of 170 ± 44 µg/ml (three experiments), which
corresponds to an effective sialic acid concentration of 2.9 ±
0.7 µM (the sialic acid content of purified IgM was
measured at 16.8 nmol/mg). Pretreatment of the IgM with
mild-periodate/NaBH
reduction (to selectively truncate
sialic acid side chains, leaving the rest of the molecule intact)
completely abolished CD22Rg binding activity (data not shown), as well
as its inhibitory effects on CD22Rg binding to TNF-
-activated HEC (Fig. 6). Pretreatment of IgM with proteinase K destroyed its
pentameric structure (see ``Experimental Procedures'') and
caused a marked decrease of its inhibitory effects on CD22Rg binding (Fig. 7), shifting the IC
to
1 mg/ml. Thus,
the inhibitory properties of IgM cannot be explained on the basis of
sialic acid content alone. In keeping with this, reduction and
alkylation of IgM into its component subunits gave a similar reduction
of its inhibitory potency (Fig. 8). Commercial pooled
haptoglobin also suppressed CD22Rg binding to HEC (72 ± 3%
inhibition at 1 mg/ml), whereas IgG showed no inhibition at the same
concentration (data not shown). Further studies with haptoglobin were
not pursued because of problems with the purity of the samples
(including some contamination by IgM).
Figure 6:
Effects
of mild periodate oxidation on the ability of IgM to inhibit CD22Rg
binding to TNF--activated HEC. TNF-activated HEC were incubated
with CD22 mRg in the presence of various concentrations of pooled IgM,
sham-treated IgM or mild periodate-treated IgM, and binding assayed as
described in the legend to Fig. 1. The data are the mean of
duplicates from a representative experiment (n =
2).
Figure 7:
Effect
of proteinase K digestion on the ability of IgM to inhibit CD22Rg
binding to TNF--activated HEC. TNF-
-activated HEC were
incubated with CD22 mRg in the presence of various concentrations of
pooled IgM, sham-treated IgM, or proteinase K-treated IgM, and binding
assayed as described. The data are the mean of duplicates from a
representative experiment (n = 2). Diisopropyl
fluorophosphate-treated proteinase K alone did not affect the control
binding (not shown).
Figure 8:
Effects of reduction and alkylation on the
ability of IgM to inhibit CD22Rg binding to TNF--activated HEC.
TNF-
-activated HEC were incubated with CD22 mRg in the presence of
various concentrations of pooled IgM, sham-treated IgM, or
dithiothreitol/iodoacetamide-treated IgM, and binding assayed as
described. The data are the mean of duplicate
determinations.
The preceding paper (9) shows that activated vascular
endothelium expresses increased levels of CD22-ligands bearing
2-6-linked sialic acids. Such activated endothelial cells
are potentially in a position to bind CD22-positive B lymphocytes
present in the bloodstream. However, such binding in vivo would have to occur in the presence of human blood plasma, which
has a high concentration of soluble sialoglycoproteins. Indeed, whole
human plasma is shown here to have potent inhibitory properties with
regard to CD22 lectin function. Since isolated CD22 is capable of
binding to all blood cell types under serum-free
conditions(6) , this inhibitory property of plasma might also
be important to prevent clumping of B cells with other cells in the
bloodstream. In this regard, it is noteworthy that the concentrations
of many plasma glycoproteins (particularly large molecules such as IgM)
are considerably lower in extracellular fluid than in
plasma(32) . Thus some interactions of CD22-positive
lymphocytes that are inhibited in the bloodstream might be permitted
within lymphoid tissues.
Review of the Carbbank data base (22) indicates that many plasma glycoproteins carry multiple N-linked oligosaccharides with 2-6-linked sialic
acids, including fibrinogen (normal range 2-4.5 mg/ml),
transferrin (2-4 mg/ml),
-macroglobulin
(1.5-4.0 mg/ml), haptoglobin (1-2.5 mg/ml),
-acid glycoprotein (0.5-1.4 mg/ml), IgM
(0.6-2.5 mg/ml), hemopexin (0.5-1.2 mg/ml),
-antichymotrypsin (0.3-0.6 mg/ml), ceruloplasmin
(0.2-0.6 mg/ml), plasminogen (0.1-0.3 mg/ml), antithrombin
III (0.17-0.3 mg/ml), and the C1q component of complement
(0.1-0.2 mg/ml). Each of these sialoglycoproteins thus has the
possibility of having specific interactions with CD22. This study
demonstrates that of all of these potential ligands, IgM and
haptoglobin can selectively bind to CD22 under conditions where the
others do not.
Previously, we demonstrated that certain purified
serum glycoproteins including transferrin and fetuin (a fetal bovine
serum glycoprotein) can interact with CD22Rg in a Sia-dependent
manner(13) . However, for both of these proteins, the binding
was weak enough that they could be completely removed from CD22Rg-PAS
by repeated washing. Somewhat stronger interactions were seen with
-acid glycoprotein, with approximately one-third of
some batches of this protein surviving repeated washings after binding
by CD22Rg-PAS. However, despite its presence in normal plasma at a
concentration of 0.5-1.4 mg/ml,
-acid
glycoprotein was not detected in the present study of total plasma
proteins that bound and eluted from CD22Rg-PAS (Fig. 3). This
may be because total plasma glycoproteins contain
1 mM
2-6-Sia residues, which would act as an inhibitor of
the binding of molecules with moderate affinity. Indeed, whole plasma
blocks CD22Rg-HEC binding with an IC
of 3.7 volume %
(corresponding to
40-50 µM
2-6-Sia
residues), which is similar to the IC
for
2-6-sLac in the enzyme-linked immunosorbent assay
(30-120 µM) and the K
of
CD22Rg-
2-6-sLac binding (32 µM, see
accompanying paper(8) ). Thus, the total content of
2-6-Sia residues present on multiple plasma glycoproteins
may be sufficient to prevent (or reduce to below the level of
detection) the binding of
-acid glycoprotein,
explaining its absence here.
Given the presence of this high level
of ``low-affinity'' inhibitors in plasma, it is all the more
remarkable that IgM and haptoglobin are able to bind selectively to
CD22Rg-PAS. The binding of these two ``high-affinity''
ligands to CD22 clearly depends upon 2-6-linked Sia residues
of their carbohydrate moieties, since
2-6-sLac, but not
2-3-sLac, can suppress the interaction. In addition,
pretreatment of IgM with sialidase or mild periodate oxidation (which
selectively truncates the exocyclic side chain of sialic acids)
completely abolishes its CD22 binding activity. IgM also inhibits
CD22Rg binding to the ligands on TNF-
-activated HEC in a
sialylation-dependent manner, and is at least 18-fold more effective
than whole plasma even when considered in relation to its protein-bound
sialic acid concentration. Indeed, 50% inhibition of binding was seen
with
0.2 mg/ml pooled IgM, which is well within its physiological
concentration range.
Additional structural features of IgM seem to
be responsible for its high affinity CD22Rg binding. Extensive
destruction of the polypeptide by proteinase K, or mere dissociation of
subunits by reduction and alkylation under nondenaturing conditions
significantly decreased the ability of IgM to bind to CD22Rg (as judged
by its inhibition of CD22Rg-HEC binding). Thus, while
2-6-linked sialic acid residues and their side chains are
essential for recognition by CD22, structural features dependent upon
the pentameric structure of the (IgM)
J chain complex
are essential for its high affinity binding. Less information is
available on the structure of haptoglobin, and given its propensity for
variable sialylation and the impurity of different commercial
preparations, we did not study it as extensively as IgM. However,
haptoglobin also exists in a polymerized form with a high molecular
mass in plasma. Thus, for both pentameric IgM and haptoglobin,
high-affinity CD22Rg binding may be dependent upon the presentation of
multiple sialylated N-linked chains in a specific orientation
or conformation, with or without participation of additional
protein-protein binding. The latter would be analogous to the recently
reported differential recognition of various glycoproteins by the
mammalian lectins, mannan binding protein and conglutinin(33) .
In keeping with this possibility, some other multimeric plasma proteins
with
2-6-linked sialic acids (such as fibrinogen) were not
seen to bind. Regardless, even if IgM and haptoglobin are simply
providing a multivalent presentation of
2-6-linked sialic
acids, the fact that a subset of cell-surface CD22 exists in a
multimerized form (8) gives such a mechanism the potential to
be biologically relevant.
While we have not directly measured the K values of the binding of either of these
glycoproteins to CD22Rg, CD22Rg-HEC binding is blocked by pentameric
IgM with an IC
corresponding to
3 µM
2-6-linked Sia residues, while
2-6-sLac
blocks the same interaction with an IC
of
80
µM. The IC
values measured in solid phase
binding assays do not reflect the true solution phase binding
constants(8) . Since the directly measured K
of CD22Rg for
2-6-sLac is
15-30
µM(8), the actual K
of pentameric
IgM-CD22Rg binding may be considerably better than 3 µM.
IgMs are large multimeric glycoproteins with about 10% carbohydrate
that carry more sialic acid than IgG, IgA, and IgE(34) . Each
µ-heavy chain has five N-glycosylation sites to which two
types of N-linked oligosaccharides (high mannose and complex)
can be attached, whereas the light chains usually lack
glycosylation(35, 36) . Structural analyses of
oligosaccharides have shown that each pentameric molecule of IgM
possesses more than 15 residues of sialic acids, all of which are in
the 2-6 linkage on biantennary
chains(35, 36, 37) . Notably, even the N-linked oligosaccharides on the J chain of IgM carry such
2-6-sialylated oligosaccharides(38) . This may be
explained by the finding that B cell activation (which occurs prior to
the onset of IgM secretion) (39) is accompanied by
up-regulation of the
-galactoside
2,6-sialyltransferase(19, 20) , which is known to
have a B cell-specific promoter(20, 40) .
It is
particularly interesting that of all the plasma sialoglycoproteins it
is IgM, the major downstream product of B cell activation(39) , that is capable of binding
selectively to a cell-surface receptor of resting B cells.
Thus, although this interaction was discovered serendipitously while
exploring the inhibitory effects of plasma on CD22 interactions with
endothelial cells, it might well be that its functional significance is
in a different arena. Indeed, it is tempting to speculate that the
soluble IgM pentamer might be part of a feedback loop, multivalently
cross-linking CD22 molecules to regulate antigeninduced responses
and/or B cell aggregation in lymphoid tissues. In this regard, it is
noteworthy that the IgM concentration in lymph (and presumably in
lymphoid tissues) is estimated to be about 20-45% of the serum
concentrations(32) , which may be still within the effective
range found here. Moreover, local and regional concentrations could be
higher or lower in subcompartments of the immune system. Finally,
recent studies have indicated an association of CD22 with
membrane-bound IgM within the plasma membrane of resting B
cells(41, 42) , and the consequent tyrosine
phosphorylation of CD22 is thought to be involved in antigen-induced
cell activation(42, 43) . The possibility that this
association is also mediated by 2-6-linked sialic acid on
membrane IgM must be considered.
In this study, we have focused
mainly upon the interactions of IgM with CD22. The interaction with
haptoglobin also deserves further exploration. The latter is the major
hemoglobin-binding protein of plasma, and is primarily produced by the
liver(25, 26) . The carbohydrates found on the
-chain constitute about 20% of total molecular mass and the ratio
of
2-6- and
2-3-linked sialic acid is about
4:1(26) . Haptoglobin is well-known as a classic ``acute
phase reactant''(44) , whose concentration is
substantially elevated in certain inflammatory states(24) .
Notably, hepatic expression of the sialyltransferase
-galactoside
2,6-sialyltransferase is also elevated under these
circumstances(45) . Further studies of the potential role of
the CD22-haptoglobin interaction in regulating B cell biology during
inflammation are required. It also remains to be seen if the lower
affinity sialoglycoproteins ligands in the plasma are of biological
relevance.
Finally, while CD22 shows exquisite specificity for the primary oligosaccharide sequence that it recognizes, it functions under markedly different conditions than do other vertebrate lectins such as the asialoglycoprotein receptor(46) , the mannose 6-phosphate receptors (47) , and the hepatic receptor for the sulfated oligosaccharides of pituitary hormones(48) . In all these instances, the cognate ligands are relatively rare components among a large excess of other non-competing glycoproteins. In the case of CD22, the primary structural motif recognized is a very common sequence found on the majority of glycoproteins that it encounters. A challenge for the future is to understand how the lectin property of CD22 can 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 seems important to focus attention upon the ligands with the highest apparent affinity, such as the two reported here.