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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1245-1252
Sialylation of the Sialic Acid Binding Lectin Sialoadhesin Regulates
Its Ability to Mediate Cell Adhesion
By
Yvonne C. Barnes,
Tim P. Skelton,
Ivan Stamenkovic, and
Dennis C. Sgroi
From the Department of Pathology, Harvard Medical School and the
Molecular Pathology Unit, Massachusetts General Hospital, Boston, MA.
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ABSTRACT |
The macrophage-specific cell surface receptor sialoadhesin, which is
a member of the newly recognized family of sialic acid binding lectins
called siglecs, binds glycoprotein and glycolipid ligands containing
a2-3-linked sialic acid on the surface of several leukocyte subsets.
Recently, the sialic acid binding activity of the siglec CD22 has been
demonstrated to be regulated by sialylation of the CD22 receptor
molecule. In the present work, we show that desialylation of in vivo
macrophage sialylconjugates enhances sialoadhesin-mediated lectin
activity. Herein, we show that receptor sialylation of soluble
sialoadhesin inhibits its binding to Jurkat cell ligands, and that
charge-dependent repulsion alone cannot explain this inhibition.
Furthermore, we show that the inhibitory effect of sialic acid is
partially dependent on the presence of an intact exocyclic side chain.
These results, in conjunction with previous findings, suggest that
sialylation of siglecs by specific glycosyltransferases may be a common
mechanism by which siglec-mediated adhesion is regulated.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
NUMEROUS STUDIES have demonstrated that
carbohydrates serve directly as molecular determinants responsible for
mediating molecular and cellular interactions.1-4 The
monosaccharide sialic acid, by virtue of both its location at the
terminal position on glycans associated with cell surface glycoproteins
and glycolipids, and its net negative charge at physiological pH,
serves as a potentially important regulator of these
interations.4 Polysialic acid (PSA)-mediated abrogation of
homotypic interactions between neural cell adhesion molecules (NCAM)
illustrates the potential inhibitory effect of sialic
acid.5 In contrast to its role as a regulatory inhibitor,
sialic acid can also promote interactions by providing a critical
ligand component for various sialic acid binding lectins.4 Recognition of sialic acid by lectins can be affected by a variety of
modifications of sialic acid itself, and variation in the a-ketosidic linkages to the underlying sugar chain, the structure of the chain, and
the structure of the underlying protein or lipid.
Recently, a subset of immunoglobulin superfamily receptors has been
found to behave as sialic acid binding lectins and proposed to define a
new class of adhesion receptors known as siglecs.6 Although
all members of this family, which include CD22, CD33, myelin-associated
glycoprotein (MAG), and sialoadhesin (Sn) share sialic acid binding
properties, they differ in the manner in which they recognize sialic
acid. More specifically, sialoadhesin, CD33, and MAG recognize a2-3
sialylated-, while CD22 recognizes a2-6-sialylated-glycoproteins and
glycolipids.7-9 The sialylated oligosaccharides recognized by each siglec are common to many glycoproteins and glycolipids, suggesting that siglec-mediated interactions may be highly regulated. Evidence for such regulation has been provided for interactions between
CD22 and its ligands. a2,6-sialylation of CD22 itself abrogates
CD22-mediated adhesion,10 and 9-O-acetylation of the side
chain of a2-6-linked sialic acid masks CD22 ligands in
vivo.11 These observations suggest that molecular
interactions between CD22 and its ligands may be intimately associated
with the expression and function of at least two specific transferases,
a2-6-sialyltransferase and O-acetyltransferase.
Recent evidence suggests that regulation of carbohydrate recognition by
several siglec family members may be similar to that of CD22. For
example, sialylation of cis ligands for CD33 and MAG modulates
their adhesive interaction.12,13 In addition, modification
of cell surface ligand-associated sialic acids by 9-O-acetylation has
been demonstrated to abrogate sialoadhesin-mediated binding of red
blood cells (RBC).14 Together, these findings suggest that
siglecs may use common mechanisms to regulate their adhesive
properties. In the present work, we sought to determine whether
sialylation of sialoadhesin itself regulates its binding properties. We
demonstrate that sialylation of sialoadhesin-associated glycans
abrogates sialoadhesin-receptorglobulin (Sn-Rg) binding activity and
that such negative regulation by sialoadhesin-associated sialic acid
is, in part, contingent on an intact polyhydroxyl side chain.
Furthermore, our data suggest that the inhibitory effect of
sialoadhesin-associated sialic acid is not solely a result of
charge-dependent repulsion.
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MATERIALS AND METHODS |
Materials.
Cell culture media (Dulbecco's modified Eagle's medium
[DMEM]) and fetal bovine serum were purchased from
Irvine Scientific (Santa Ana, CA). L-glutamine and antibiotics were
from GIBCO (Grand Island, NY). Diethyl aminoethyl
(DEAE)-dextran, dimethyl sulfoxide (DMSO), Nonidet P-40,
aprotinin, and sodium periodate were from Sigma (St Louis, MO).
Fluorescein-labeled goat antihuman affinity-purified antibodies were
from Cappel (Malvern, PA). Oligonucleotide synthesis reagents were
acquired from Millipore (Bedford, MA). Vibrio cholerae (VC) and
arthrobacter ureafaciens (AU) sialidase were purchased from Boehringer Mannheim (Indianapolis, IN). NANase I was purchased from Glyko (Novato, CA). Sheep erythrocytes were purchased from ICN
(Costa Mesa, CA). Fluorescein isothiocyanate (FITC)-conjugated polyacrylamide substituted with a2-3 sialylactose
(3'-PAA-FITC) was obtained from GlycoTech (Rockville, MD).
COS-7 cell adhesion (rosetting) assay.
Rosetting assays were performed as described previously.15
Briefly, COS-7 cells were transfected with full-length
sialoadhesin cDNA or mock-transfected using the DEAE-dextran method as
described.15 Twelve hours after transfection, cells were
trypsinized and replated onto fresh 60-mm culture plates. Forty-eight
hours later the transfected COS-7 cells were overlaid with human or
sheep RBC, which were allowed to adhere for 20 minutes at 4°C.
Nonadherent RBCs were washed away with serum-free DMEM, and rosetting
was evaluated under an inverted microscope. Five separate transfections
performed independently on 5 different days were evaluated. To analyze
the effect of endogenous sialylation of surface expressed sialoadhesin, transfectants were treated with 50 mU/mL vibrio cholerae
sialidase (VC-sialidase) at 37°C in phosphate-buffered saline
(PBS), pH 6.9 for 1 hour before performing the rosetting assay. The
rosettes obtained under these conditions were compared with those of
untreated transfectants under identical conditions.
Splenic cryostat adhesion assay.
Splenic cryostat sections (8 µm) were placed on glass slides and air
dried for 60 minutes. Cryostat sections were preincubated with 50 mU/mL
of VC-sialidase or heat inactivated VC-sialidase for 1 hour at
37°C, washed extensively with cold PBS, and incubated with washed
sheep erythrocytes (sRBC) for 30 minutes at 4°C. The slides were
washed in PBS with gentle agitation to remove unbound sRBC from the
sections and immediately fixed in 4% formaldehyde. Binding was
examined by light microscopy. Antibody blocking studies were performed
as follows. Sialidase-treated cryostat sections were preincubated with
3 mg/mL of either the antisialoadhesin monoclonal antibody (MoAb) 3D6
(Serotec, Raleigh, NC) or the isotype-matched control anti-CD44 MoAb
KM81 for 30 minutes at room temperature (RT). The slides were washed
extensively with PBS and then subjected to the adhesion assay described above.
3'-PAA-FITC staining.
The lectin activity of cell surface sialoadhesin was examined using a
protocol similar to that previously
described.16 Briefly, Mm1 cells were washed
several times in ice-cold PBS containing 0.02% sodium azide, 1%
bovine serum albumin (BSA) (staining buffer), and the lectin activity
of cell surface sialoadhesin examined by incubating cells (untreated
and treated) for 1 hour in 100 mL staining buffer containing 1.5 mg
3'-PAA-FITC. The cells were washed in staining buffer and
analyzed with a FACscan instrument (Becton Dickinson Immunocytometry
Systems, Mountain View, CA). The treated cells were incubated with
Arthrobacter ureafaciens (AU) and VC-sialidase, 40 and 50 mU/mL, respectively, washed with staining buffer three
times, and preincubated with staining buffer alone or
staining buffer containing 5 mg/mL of the antisialoadhesin antibody,
3D6. Following these treatments, the cells were stained with
3'-PAA-FITC and analyzed as described above.
Development of receptorglobulin fusion proteins.
Soluble receptor-immunoglobulin fusion proteins (termed Rg for
"receptorglobulin") were prepared according to the methods of
Aruffo et al.17,18 Development of murine SnRg19
CD44Rg,18 and CD8Rg18 have been described
previously; the SnRg contains the first four Ig-like domains of Sn. COS
cells were transfected with either SnRg, CD44Rg, or CD8Rg by the
DEAE-dextran method,17 and 12 hours after transfection,
culture media replaced with serum-free DMEM, which was maintained for
an additional 72 hours. The supernatant was harvested, and the fusion
proteins purified by protein-A sepharose (PAS) chromatography, eluted
with sodium citrate buffer pH 3.0, dialyzed against PBS, concentrated
with ultrafree-CL filters (Millipore, Bedford, MA), and quantitated
with a Bio-Rad (Richmond, CA) protein assay
kit.8 In addition, SnRg and CD8Rg were also purified from
two separate Chinese hamster ovary (CHO) cell lines stably transfected with the SnRg-coding and CD8Rg-coding plasmids.
Cell lines and cell culture.
The Jurkat T-cell line was obtained from American Type Culture
Collection (ATCC, Rockville, MD) and grown in RPMI (Irvine Scientific) supplemented with 10% fetal bovine serum. Human
erythrocytes were obtained from healthy donors, washed in PBS, and used
directly in COS-7 cell rosetting assays. The Mm1 cell line was a
generous gift from Paul Crocker (University of Dundee, Dundee,
Scotland) and grown in RPMI (Irvine Scientific) supplemented with 10%
fetal bovine serum.
Determination of the effect of sialidase treatment on sialoRg
function.
COS-7 cells were transfected with cDNA coding for SnRg by the
DEAE-dextran method, and supernatants were harvested 4 days posttransfection as described above. SnRg was bound batchwise to PAS
beads at 4°C overnight, washed with PBS, and the PAS-bound SnRg was
subjected to digestion by different types of sialidases. PAS-bound SnRg
was resuspended in 0.5 mL of 0.1 mol/L sodium acetate pH 5.5, 0.1 mol/L
NaCl, 1 mmol/L CaCl2 (AU sialidase buffer), and digested
with 100 mU of Arthrobacter ureafaciens sialidase (AU
sialidase) or VC-sialidase for 6 hours at 37°C. An equal amount of
PAS-bound SnRg was resupended in 0.5 mL of 50 mmol/L sodium phosphate
pH 6.0 and digested with 250 mU of NANaseI (Glyko) for 10 hours at
37°C. After the sialidase digest, the PAS beads were extensively
washed with PBS, SnRg eluted, dialyzed, concentrated, and quantitated
as described above. Untreated PAS-bound SnRg was resuspended in
AU-sialidase buffer and incubated in the absence of sialidase for 10 hours at 37°C and purified as described above. Jurkat cells were
incubated with 50 mg/mL sialidase-treated SnRg, untreated SnRg, or
control (CD44Rg and CD8Rg) receptorglobulins for 1 hour on ice. Cells
were washed in cold PBS, resuspended in PBS, 0.02% sodium azide and 5 mg/mL fluorescein-conjugated, affinity-purified goat antihuman antibody
for 30 minutes on ice, washed, fixed with PBS containing 4%
formaldehyde, and analyzed by a FACscan instrument (Becton Dickinson
Immunocytometry Systems).
Coupling of glutamic acid to VC-sialidase-treated SnRg with
dithiobis(sulfosuccinimidylproprionate) (DTSSP).
The amine selective reagent, DTSSP (Pierce, Rockford, IL), was used
according to the manufacturer's recommended protocol to covalently
cross-link glutamic acid to VC-sialidase-treated SnRg. Briefly,
VC-treated SnRg (400 mg), glutamic acid, and DTSSP were added to PBS at
1:1:50 molar ratio and incubated at 37°C for 30 minutes. The
reaction was terminated with the addition of Tris-HCl to a final
concentration of 50 mmol/L. The reaction products were separated and
isolated by FPLC gel filtration.
Gel filtration chromatography.
Protein A-sepharose affinity-purified NAN-SnRg and glutamate
cross-linked-NAN-SnRg (Glut-SnRg) were individually injected onto a
Superose-6 HR 10/30 gel filtration column (Pharmacia LKB, Uppsala,
Sweden) and subjected to a constant flow rate of 0.5 mL PBS/minute
using an FPLC System (Pharmacia). Elution fractions were collected and
protein detected by spectral absorbance at 226 nm. Elution profiles
were generated by plotting protein concentration (spectral absorbance,
A226nm) versus volume (mL).
Capillary electrophoresis.
Capillary electrophoresis was performed on a model 3850 capillary
electropherograph (Isco, Lincoln, NE) using an uncoated silica
capillary (75 µm inner diameter, 62.5 cm length, 39 cm inlet to
detector window, ISCO), a 5-second vacuum injection (10 nL), 15kV (95 mA), and 210 nm ultraviolet (UV) absorbance detection in 50 mmol/L
NaPO4, pH 7.4. Samples (10 mL) contained 2 mg SnRg and 480 mg/mL mesityl oxide (MO). Mobility (m) of a given molecule is defined
as its anodal migration relative to MO, the neutral marker, and is
determined from migration times by: m = ld(lc/V)(1/teo  1/t); where
ld is the inlet to detector window length, lc
is the capillary length, V is the applied voltage, teo is
the migration time for MO, and t is the migration time for SnRg.
Mild periodate oxidation of Jurkat cells and PAS bound SnRg.
A fresh stock of 100 mmol/L sodium metaperiodate in PBS, pH7.2, was
prepared for each experiment. Jurkat cells were washed and resuspended
in ice-cold PBS with 2 mmol/L sodium metaperiodate at 1 × 106 cells/mL and incubated on ice in the dark for 15 minutes as previously described.8,11 The cells were washed
with ice-cold PBS, stained with SnRg or CD44Rg, and analyzed by FACscan.
SnRg (400 mg) was batch adsorbed onto PAS beads overnight at 4°C,
washed with ice-cold PBS, resuspended in 20 mL of ice-cold PBS with 2 mmol/L sodium metaperiodate and incubated on ice in the dark for 20 minutes. The beads were then extensively washed with PBS, the SnRg
purified as described above, and the periodate-treated SnRg (50 mg/mL)
used to stain cells.
Immunoprecipitation and silver stain.
Untreated and sialidase-treated SnRg (10 mg) were batch adsorbed onto
PAS beads overnight at 4°C in PBS. The beads were washed four times
in PBS/0.05% Nonidet P-40 and precipitates eluted by boiling in sample
buffer in the presence of 2% 2-mercaptoethanol. Samples were subjected
to sodium dodecyl sulfate (SDS)/8% polyacrylamide (PAGE), the gel
fixed, and proteins detected by silver nitrate staining.20
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RESULTS AND DISCUSSION |
Sialoadhesin-mediated adhesion in COS-7 cells.
Expression of sialoadhesin (Sn) in COS-7 cells promoted minimal binding
of human RBCs as demonstrated by both the small size (<20
RBCs/rosette) and number (<10% of sialoadhesin-bearing COS-7 cells)
of rosettes (Fig 1A). Because sialylation
of the siglec CD22 has been demonstrated to inhibit its sialic acid
binding lectin activity,10 we sought to determine if
sialylation of sialoadhesin by COS-7 cells modulates
sialoadhesin-mediated binding of RBCs. To address this possibility,
Sn-expressing COS-7 cells were pretreated with VC sialidase before the
binding assay. Sialidase pretreatment resulted in high levels of RBC
binding as demonstrated by numerous large rosettes (Fig 1B); greater
than 85% of the sialoadhesin-expressing cells supported RBC rosettes
each consisting, on average, of more than 30 cells. The specificity of
the adhesion is underscored by the observation that VC
sialidase-treated mock-transfected COS-7 cells did not support RBC
binding (Fig 1C).
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| Fig 1.
Sialidase pretreatment of sialoadhesin-transfected COS
cells unmasks their ability to mediate red blood cell
binding. (A) Untreated sialoadhesin-transfected COS cells. (B)
Vibrio Cholerae sialidase-treated sialoadhesin-transfected
COS-7 cells. (C) Vibrio Cholerae sialidase-treated
mock-transfected COS cells.
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In vivo sialoadhesin-mediated adhesion is enhanced by sialidase.
Although sialoadhesin-mediated adhesion in COS cells is augmented by
sialidase pretreatment, we sought to demonstrate this phenomenon in
macrophages in vivo. Therefore, we performed a sheep erythrocyte
binding assay using sialidase treated- and untreated-cryostat sections
of mouse spleen. We demonstrate that untreated cryostat sections of
mouse spleen bind sRBC minimally under our experimental conditions
(Fig 2A). Interestingly,
pretreatment of cryostat splenic sections with sialidase resulted in
marked erythrocyte binding to the splenic marginal zone, an area that
coincides with the expression of sialoadhesin (Fig 2B).21
Mouse splenic cryostat sections pretreated with heat-inactivated
sialidase did not enhance sRBC binding (data not shown). Preincubation
of the sialidase-treated cryostat section with the antisialoadhesin
MoAb 3D6 resulted in the loss of sRBC adhesion (Fig 2C), demonstrating
the sialoadhesin-dependent nature of this binding; an isotype-matched
antibody did not inhibit sRBC binding under the same experimental
conditions (data not shown).
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| Fig 2.
Sialidase pretreatment of splenic cryostat sections
enhances sialoadhesin-mediated adhesion of sheep erythrocytes. (A)
Murine splenic cryostat section (untreated). (B) Murine splenic
cryostat section pretreated with VC-sialidase; arrow denotes adherent
sheep erythrocytes in the splenic marginal zones. (C) VC-sialidase
treated splenic cryostat section pretreated with the antisialoadhesin
blocking MoAb, 3D6.
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Recently, a probe consisting of polyacrylamide substituted with
multiple copies of Siaa2-6Galb1-4Glcb1 has been successfully used to
directly measure the lectin activity of cell surface
CD22.16 With this in mind, we sought to probe the lectin
activity of cell surface sialoadhesin with commercially available
synthetic conjugate of fluorescein-labeled polyacrylamide substituted
with multiple copies of Siaa2-3Galb1-4Glc (3'-PAA-FITC);
sialoadhesin has been previously demonstrated to specifically recognize
the sugar moieties Siaa2-3Galb1-4GlcNAc or Siaa2-3
Galb1-3GalNAc.9 Untreated Mm1 cells, a macrophage cell
line, which constitutively expresses sialoadhesin,22 did
not stain with 3'-PAA-FITC, however pretreatment of such cells
with sialidase unmasks the sialoadhesin-lectin activity (Fig 3). The specificity of
3'-PAA-FITC for sialoadhesin is demonstated by the observation
that preincubation of Mm1 cells with the adhesion-blocking antisialoadhesin MoAb 3D623 abrogates 3'-PAA-FITC
binding (Fig 3); the isotype-matched rat MoAb to CD44 fails to inhibit
3'-PAA-FITC binding. These results indicate that desialylation of
in vivo sialylconjugates enhances sialoadhesin-mediated lectin activity and that 3'-PAA can be used as a specific ligand for detecting unoccupied sialoadhesin lectin molecules.
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| Fig 3.
Detection of cell surface sialoadhesin lectin activity on
Mm1 cells using a multivalent 3'-sialylactosylated probe
(3'-PAA-FITC). Cultured Mm1 cells, a murine macrophage cell line,
were washed, untreated, Arthrobacter Ureafaciens
(AU)/VC-sialidase treated, or AU/VC-sialidase treated followed by
preincubation with the 3D6 adhesion-blocking MoAb, and stained with
FITC-conjugated 3'-PAA. Staining was detected using single color
flow cytometry (fluorescence-activated cell sorting [FACS]
analysis).
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Sialylation of the sialoadhesin receptorglobulin (SnRg) modulates its
binding activity.
There are at least two possible interpretations of the above
experimental results. First, an a2,3-sialylated COS glycoprotein or
glycolipid on the same cell surface (in cis) may bind
sialoadhesin, thereby preventing its interaction with ligands on
adjacent cells (RBCs). Second, sialylation of sialoadhesin itself may
interfere with its ability to recognize its ligands. To address the
latter possibility, cDNA encoding the soluble recombinant
sialoadhesin-immunoglobulin fusion protein (SnRg) was transiently and
stably expressed in COS and CHO cells, respectively. The SnRg was
purified from the supernatants, subjected to sialidase or mock
digestion, analyzed by SDS-PAGE and assessed for binding to Jurkat
cells by flow cytometry. In comparison to mock-digested SnRg, VC
sialidase-treated SnRg (VC-SnRg) displayed a decrease in molecular
mass, consistent with the notion that COS-7-derived SnRg is sialylated
(Fig 4, lane 4) and a significant increase
in Jurkat cell binding activity (Fig 5A);
soluble CD44Rg (Fig 5), as well as soluble CD8Rg
(Fig 6) served as negative controls.
Previous work has demonstrated that the a2,6-sialic acid binding
activity of CD22 is specifically inhibited by CD22-associated
a2,6-linked sialic acid.8 To determine if the observed
inhibition of SnRg-mediated binding may be sialic acid linkage
specific, PAS-bound SnRg was treated with different sialidases, eluted,
analyzed by SDS-PAGE, and tested for binding to Jurkat cell-surface
ligands. SnRg treated with AU sialidase (AU-SnRg), which hydrolyzes
a2-3, 2-6-and 2-8-linked sialic acid, displayed a significant decrease
in molecular mass (Fig 4, lane 5) and an approximately 10-fold increase
in binding activity (Fig 5B). Interestingly, SnRg treated with NANaseI
(NAN-SnRg) enhanced its binding activity (Fig 5B), despite undergoing a
slight decrease in molecular mass (Fig 4, lane 1); NANaseI specifically
and selectively hydrolyzes a2,3-linked sialic acid. The differential
sialidase hydrolysis suggests that SnRg expressed in COS-7 cells is at
least a2,3- and a2-6-sialylated, but that a2,3-sialylation, rather than a2-6-sialylation, may play a dominating role in regulating ligand binding activity. The difference in binding activity of native (or
untreated) SnRg in Figs 5 and 6 may be explained by the fact that the
SnRg in Fig 5 was derived from transient COS cell transfectant supernatants, whereas the SnRg used for Fig 6 was derived from the
supernatants of CHO cells stably tranfected with SnRg. The COS-derived
SnRg was harvested 3 days after transfection, whereas CHO-derived SnRg
was harvested 8 days after plating in serum-free medium. Thus, the
binding differences between the COS- and CHO-derived SnRg may be
explained by variable release of soluble neuraminidase from cells
cultured under suboptimal nutrient conditions.24,25 Neuraminidase released into the culture medium after CHO cell lysis can
desialylate recombinant soluble glycoproteins.24
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| Fig 4.
SnRg produced in COS-7 cells is sialylated. SnRg was
precipitated with protein A-sepharose (PAS) beads and subjected to mock
(lane 2), NANaseI (lane 1), Vibrio Cholerae sialidase (lane 4),
Arthrobacter Ureafaciens sialidase (lane 5) digestions, or mild
periodate oxidation (lane 3). Treated and nontreated SnRg (10 mg/lane)
were subjected to 7% SDS-PAGE and protein detected by silver nitrate
staining. Molecular mass markers (in kD) are shown.
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| Fig 5.
Sialidase treatment of SnRg enhances reactivity to the
Jurkat T-cell line. Jurkat (1 × 106) cells were incubated
in (A) with 50 mg/mL of CD44Rg, untreated sialoRg (UT-SnRg) or
Vibrio Cholerae sialidase-treated SnRg (VC-SnRg) and in (B)
with 50 mg/mL UT-sialoRg, Arthrobacter Ureafaciens
sialidase-treated SnRg (AU-SnRg) or NANaseI-treated SnRg.
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| Fig 6.
The addition of a negatively charged moiety to
sialidase-treated SnRg does not inhibit its binding activity. Jurkat (1 × 106) cells were incubated with 50 mg/ml of CD8Rg, SnRg,
VC-SnRg, or Glut-SnRg and FACS analyzed for binding reactivity.
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Because sialidase treatment results in the removal of negative charge,
we addressed the possibility that the negative charge inherent to the
carboxylate at the 1-carbon position of sialic acid (at physiological
pH) may modulate binding through electrostatic repulsion, as has been
demonstrated for neural cell adhesion molecule (NCAM).5
Sialic acid was removed from SnRg with VC-sialidase, which hydrolyzes
sialic acid irrespective of its linkage, and the net negative charge
reconstituted by covalently coupling glutamic acid to the VC-sialidase
treated SnRg (VC-SnRg) using the amine reactive homobifunctional
N-hydoxysuccimide ester (NHS-ester), DTSSP. The cross-linking reaction
was performed with an equal molar ratio of glutamic acid to VC-SnRg,
and a 50 molar excess of DTSSP to optimize the coupling of glutamic
acid to SnRg (Glut-sialoRg) while minimizing the production of VC-SnRg
dimers. Primary amines of amino acid side chains are the principle
targets for DTSSP, and while five amino acids have nitrogen in their
side chains, only the e-amine of lysine reacts significantly with
DTSSP.26 Theoretically, under our reaction conditions, one
end of DTSSP reacts with the amine group of one molecule of glutamic
acid, while the other end of the cross-linker reacts with the e-amine of a lysine residue in one molecule of VC-SnRg. Thus, as a result of
this reaction, each VC-SnRg molecule will acquire a net negative charge
of 3 for each molecule of glutamic acid added: one molecule of glutamic
acid contains two free carboxyl groups, and the positively charged
lysine of VC-SnRg is neutralized by its reaction with DTSSP.
Furthermore, because DTSSP is in 50 molar excess, it will react with
and neutralize the positive charge associated with additional lysine
residues of VC-SnRg and result in an even greater net negative charge.
The uncoupled glutamic acid, VC-SnRg dimers and Glut-SnRg products were
separated and isolated using FPLC gel filtration. The Glut-SnRg eluted
in the same separation fractions and displayed SDS-PAGE mobility
similiar to that of VC-SnRg (data not shown). Furthermore, the isolated
Glut-SnRg displayed a significant increase in net negative charge
relative to native SnRg and VC-SnRg as assessed by capillary
electrophoresis (Table 1), in agreement with the expected result. Interestingly, despite the greater overall negative charge, Glut-SnRg retained its ligand binding activity (Fig
6), implying that charge-dependent repulsion is not likely to be solely
responsible for regulating the lectin activity of SnRg. Thus, the
inhibitory mechanism by which sialylation affects binding of
sialoadhesin differs from that which operates on NCAM.5
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Table 1.
The Relative Net Negative Charge Due to Sialic Acid or
Cross-Linked Glutamate on CHO-Derived SnRg Molecules Determined by
Capillary Electrophoresis
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Mild periodate oxidation of SnRg partially unmasks its binding
activity.
Modifications of the exocyclic side chain of sialic acid have been
shown to affect a wide spectrum of biological phenomena.27 We have previously demonstrated that the sialic acid side chains of B-
and T-cell CD22 ligands are required for recognition by CD22,8,28 and others have demonstrated that 9-O-acetylation of CD22 ligands inhibits binding to CD22.11 Similarly,
modifications of sialoadhesin ligand-associated sialic acid have been
demonstrated to alter sialoadhesin-mediated recognition.22
In light of these observations regarding ligand sialylation, we
addressed the possibility that the sialic acid residues on the
receptor, SnRg, may require an intact exocyclic side chain to provide
their inhibitory activity. PAS-bound SnRg was subjected to mild sodium
metaperiodate oxidation, and the resulting receptorglobulin binding
activity assayed. Under these mild conditions, sodium metaperiodate
selectively oxidizes the exocyclic side chain of sialic acid to produce
the eight and seven carbon products,
5-acetamido-3-4dideoxy-D-galactosyloctuosonic and
galactosylheptulosonic acids, respectively, while leaving the ring
structure of sialic acid and the underlying oligosaccharides intact.29 The binding activity of periodate-treated SnRg
was fourfold higher than that of untreated SnRg and threefold lower than AU-sialidase-treated SnRg (Fig 7),
suggesting that the integrity of endogenous sialic acid side chains is
required for optimal inhibition. Capillary electrophoretic mobility of
the periodate-treated SnRg was similar to that of untreated SnRg (Table
1) confirming that the periodate treatment did not change the charge of
the molecule. This latter observation, in conjunction with the
increased binding activity of periodate-treated SnRg, further supports
the notion that charge-dependent repulsion alone cannot explain the inhibitory effect of sialic acid. Taken together, our results suggest
that although the general mechanism of sialic acid inhibition is common
among two siglec members, additional regulatory requirements based on
recognition of specific sialic acid structural components (ie, side
chain modifications) may exist within this family.
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| Fig 7.
Mild periodate treatment of SnRg unmasks its binding
reactivity to Jurkat cells. Jurkat cells were incubated with 50 mg/mL
of untreated SnRg, AU-sialidase-treated SnRg, and sodium
periodate-treated SnRg and tested for reactivity as assessed by
indirect immunofluorescence and FACS analysis.
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|
The in vivo regulation of siglec binding to its ligands is undoubtedly
complex. In vitro studies have demonstrated that sialylation of
cis ligands regulates binding of CD22, CD33, and
MAG.10,12,13 Similarly, the observation that macrophages
express sialoadhesin ligands has led to speculation that the lectin
binding site of sialoadhesin may be occupied in a cis
configuration by sialo-oligosaccharides within the macrophage
glycocalyx.30 It has been hypothesized that the 17 Ig-like
domains of sialoadhesin project its functional domain above the
cellular glycocalyx thereby reducing the inhibitory potential of these
cis ligands and allowing adhesive interactions to occur between
macrophages and cells bearing appropriate
counterreceptors.30 Attractive as this hypothesis may be,
the relevance of cis interactions as an in vivo regulatory
determinant of sialoadhesin binding remains to be resolved.
Furthermore, based on the observation that sialylation of recombinant
CD22 abrogates its binding activity,10 it has been
hypothesized that sialylation of siglecs may be an additional mechanism
by which to regulate their binding activity. Herein, we provide in
vitro evidence to support this latter theory, and we now present
additional evidence to suggest that sialylation of siglecs may be a
common mechanism governing siglec binding.
We and others have hypothesized that the reciprocal nature of
receptor-ligand sialylation appears to play an important role in
defining the regulatory process among siglecs, and this reciprocity may
be intimately related to expression and function of specific glycosyltransferases.7,13,31 Thus, the degree to which
specific glycosyltransferase activity modulates siglec-mediated
adhesion may be related to the differentiation and activation state of the cell. This concept is supported by the observation that expression of a2,6-sialyltransferase (ST) in B cells is cell cycle-dependent, and
under the control of a B-cell specific a2,6ST promoter, which is
induced on B-cell activation.32 Speculatively, regulation of a2,6ST expression and activity in B cells and a2,3ST expression and
activity in macrophages may, in turn, regulate the ligand binding
ability of CD22 and sialoadhesin as a function of the B cell and
macrophage activation states, respectively. In vivo confirmation of
this mechanism remains to be determined, and thus a major challenge in
the future will be to understand the temporal changes in
glycosyltransferase activity as they relate to siglec-ligand interactions during the growth, differentiation, and activation states
of hematopoietic cells.
| |
FOOTNOTES |
Submitted November 4, 1997; accepted October 13, 1998.
Supported by National Institutes of Health Clinical Scientist
Development Award AI/01252 (to D.S.) and U.S. Public Health Services
Grant GM/AI 48614 (to I.S.). I.S. is a scholar of the Leukemia Society
of America.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Dennis C. Sgroi, MD, Molecular
Pathology Unit, Massachusetts General Hospital, 149 13th St, 7th Floor,
Charlestown Navy Yard, Boston, MA 02129.
| |
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Top
Abstract
Introduction