(Received for publication, October 20, 1995; and in revised form, December 18, 1995)
From the
Selectins are a family of lectins, that mediate tethering and
rolling of leukocytes on endothelium in vascular shear flow. Mild
periodate oxidation of the L-selectin ligand CD34, or L-selectin
ligands on leukocytes, enhanced resistance to detachment in shear and
decreased rolling velocity equivalent to an 8-fold increase in ligand
density, yet had little effect on the rate of tethering. Enhanced
interactions were also seen with mildly oxidized sialyl Lewis and sialyl Lewis
glycolipids. Enhancement was
completely reversed by borohydride reduction, yielding a strength of
interaction equivalent to that with the native ligands. No effect on
the strength of P-selectin and E-selectin interactions was seen after
mild oxidation of their ligands. Completeness of modification of sialic
acid by mild periodate was verified with monoclonal antibody to sialyl
Lewis
-related structures and resistance to neuraminidase.
The addition of cyanoborohydride to leukocytes rolling through
L-selectin on mildly oxidized but not native CD34 caused arrest of
rolling cells and formation of EDTA-resistant bonds to the substrate,
suggesting that a Schiff base was reduced. Cyanoborohydride reduction
of mildly oxidized cells rolling on P-selectin and E-selectin also
caused arrest and formation of EDTA-resistant bonds but with slower
kinetics. These data suggest that interactions with a sialic acid
aldehyde group on mildly oxidized ligands that include interconversion
to a Schiff base can occur with three selectins yet only stabilize
binding through the selectin with the fastest k
,
L-selectin.
The selectins are a family of three
Ca-dependent membrane-bound lectins that initiate
adhesion of leukocytes to platelets or endothelial cells under the
shear forces found in the venular
circulation(1, 2, 3) . L-selectin is
expressed on leukocytes (4) and binds certain sulfated
glycoproteins from lymph node high endothelial venules (HEVs), (
)initially defined by mAb MECA-79 and collectively known as
peripheral node addressin (PNAd)(5) . The components of PNAd
include the sialomucins GlyCAM-1 (6) and CD34(7) . CD34
is the major L-selectin ligand from human tonsil HEV and mediates
leukocyte tethering and rolling in shear flow(8) . L-selectin
also binds to uncharacterized ligands on neutrophils and hematopoietic
progenitor cells(9, 10) . P-selectin, expressed by
thrombin-activated platelets and endothelial cells, and E-selectin,
expressed by cytokine-activated endothelial cells, bind to carbohydrate
ligands on myeloid cells and subsets of lymphocytes. P-selectin
glycoprotein ligand (11, 12, 13) and
E-selectin ligand (14) bear selectin ligands on myeloid cells.
Selectins contain highly homologous (60-70% amino acid
identity) Ca-dependent N-terminal lectin-like
domains. The three-dimensional structure is known for
E-selectin(15) . Site-directed mutagenesis studies have
identified a number of residues that are critical to P-selectin- and
E-selectin-mediated carbohydrate
recognition(15, 16, 17) . All three selectins
bind sialylated, fucosylated lactosaminoglycans, of which the prototype
is sialyl Lewis
(sLe
;
Neu5Ac
2-3Gal
1-4(Fuc
1-3)GlcNAc)(18, 19) . O-linked glycans of GlyCAM-1 have been structurally
characterized and include a 6`-sulfosialyl Lewis
core 2
structure attached to a T-antigenic core 1 structure that also bears
sialic acid(20, 21) . E-selectin ligand is also
decorated with sLe
(14) . P-selectin glycoprotein
ligand has O-linked glycans extended with
poly-N-acetyl lactosamine, carries sLe
, and can
also bind E-selectin (12) . Sialic acid is essential to ligand
activity as shown by abolition with neuraminidase treatment of binding
by all three selectins(18, 19) .
A rapid k may be important for tethering of leukocytes in
shear flow through selectins to the vessel wall, and rapid k
and k
rate constants are
thought to be responsible for the transient adhesive interactions that
allow rolling of cells on selectins in response to hydrodynamic drag
forces. Recent measurements show an unstressed k
of 1 s
for P-selectin(22) , 0.5
s
for E-selectin(23) , and a substantially
faster k
of 7 s
for
L-selectin tethers. (
)Rolling through L-selectin is faster
than through E-selectin or P-selectin, even when ligand density is
adjusted to give identical resistance of rolling adhesions to
detachment by shear. (
)These findings suggest that k
is an important determinant of rolling
velocity. When the selectin-ligand bond is stressed by the hydrodynamic
drag forces acting on the cell, k
increases only
modestly(22) .
Selectins thus have high tensile
stability, a factor that may be important for maintenance of adhesion
in the vasculature.
Mild periodate treatment of fixed cryostat sections of rat lymph nodes enhanced binding to HEV of lymphocytes and staining by L-selectin-IgG chimera(24) . Evidence was obtained for reversible Schiff bases between newly generated aldehyde groups of HEV ligands and lysine amino groups in the lectin domain of L-selectin. Mild periodate selectively oxidizes at the exocyclic C-7, C-8, and C-9 positions of sialic acid, and results in cleavage of the C-7-C-8 and C-8-C-9 bonds and a product with an aldehyde group at C-7. Other carbohydrate groups in complex glycans are unaffected by mild periodate(25, 26) . The effect of mild periodate treatment on the ligands of P-selectin and E-selectin, or on ligands for L-selectin distinct from those on HEV, remains to be determined. Furthermore, the effect of this modification on tethering and rolling of leukocytes under laminar flow conditions is not known.
In the
present study, we have investigated the effect of selective oxidization
of sialic acid in selectin ligands on tethering, rolling velocity, and
the strength of rolling adhesions under laminar flow conditions. Our
results show that interactions with several physiologic ligands through
L-selectin, but not through P-selectin and E-selectin, are enhanced
after mild periodate treatment. Leukocytes roll more slowly and possess
markedly higher resistance to shear detachment on mild
periodate-treated PNAd. However, the rate of tethering in shear flow is
unaffected. The effect on enhanced interactions of mild periodate
treatment was reversed by reduction with borohydride. Furthermore,
modification of the terminal sialic acid attached to N-acetyl
lactosamine in sLe or to its isomer lacto-N-biose
in sLe
is sufficient to enhance L-selectin interactions. We
demonstrate by reduction with cyanoborohydride that reversible Schiff
bases are formed not only between L-selectin and ligand sialic acid
aldehyde groups but also between E-selectin and P-selectin and their
mildly oxidized ligands. A labile Schiff base thus can occur in
complexes of all three selectins with mildly oxidized ligands, although
perhaps only for a brief fraction of the time that the ligand is bound
to the receptor. However, the C-7 aldehyde group of the mildly oxidized
sialic acid in the ligand only enhances the kinetics or equilibria for
the interaction with the selectin with the fastest k
, L-selectin.
Tethering in flow was measured as the
number of cells free in flow that tethered in 1 min within the field of
view and remained rollingly adherent for at least 5 s. Tethers that
were initiated after a nonadherent cell touched an adherent cell were
not counted. Cell accumulation was measured as the total number of
cells present within the field of view after 3 or 5 min as cells/unit
area. Cells were allowed to accumulate on the substrates at different
wall shear stresses, and the number of accumulated cells/unit area was
determined after 3 or 5 min. For detachment assays, cells were infused
into the chamber at 0.84 dyn/cm and allowed to adhere until
a sufficient number of cells accumulated (about 2 min). Nonadherent
cells were cleared by perfusion with H/H binding medium at 0.84
dyn/cm
for 30 s. Then the wall shear stress was increased
every 3 s for glycolipids or 10 s for other substrates to a maximum of
36 dyn/cm
to generate a detachment force. A shorter
interval was used on glycolipids because the cells rolled faster than
on other substrates, and it was difficult to distinguish detachment
from cells rolling out of the field of view. The number of cells
remaining bound was calculated as a percentage of the number of cells
rolling on the substrate at 0.84 dyn/cm
. Rolling velocities
were calculated as described previously (34) for 15-20 of
the cells observed during detachment assays.
Inhibition with L-selectin mAb or control myeloma IgG and with fucoidan and EDTA was determined as described previously(8) . Inhibition with E-selectin mAb or P-selectin mAb was carried out by incubating selectin bilayers to which tethering had already been measured with 10 µg/ml mAb for 20 min and repeating the measurements. All mAbs and inhibitors remained present during the adhesion assay.
Periodate- or sham-treated substrates were treated with Vibrio
cholera neuraminidase (5 milliunits/ml) (Oxford Glycosystems,
Rosedale, NY) for 30 min in 50 mM sodium acetate, 4 mM CaCl, 0.1% bovine serum albumin, pH 5.5, at room
temperature by injecting the enzyme through a side port of the flow
chamber. The chamber was then washed with binding medium, and tethering
was measured to the same field.
Periodate- and sham-treated cells were treated for 30 min with or without V. cholera neuraminidase (5 milliunits/ml) in the binding medium at room temperature. Cells were washed three times and resuspended in the binding medium.
Figure 1:
Effect of periodate
treatment on selectin-mediated cell binding in shear flow. A,
lymphocytes or CHO-E cells (5 10
cells/ml) were
infused at a wall shear stress of 0.84 dyn/cm
on
polystyrene slides incubated with CD34 at 185 ng/ml to yield 150 CD34
sites/µm
, and assembled in a parallel plate flow
chamber. The number of cells that accumulated in 3 min of continuous
flow within the field of view was calculated and expressed per
mm
. Periodate, borohydride, and neuraminidase treatments of
immobilized CD34 and treatments with DREG-56 mAb to L-selectin, BB-11
mAb to E-selectin, and fucoidan were as indicated in the figure and described under ``Experimental Procedures'' and were
carried out on substrates to which binding in absence of inhibition had
previously been measured. The data represent the mean ± range of
two randomly selected 10
fields and are representative of two
independent experiments. B, lymphocytes (5
10
cells/ml) were infused at varying wall shear stresses through the
parallel wall flow chamber containing sham- or mild periodate-treated
CD34 at 150 sites/µm
. The number of tethers formed per
minute at a given shear stress was quantitated. The data points
represent the mean ± range of the number of tethered lymphocytes
in a 10
field and are representative of two independent
experiments performed in duplicate. C, human promyelocytic
HL-60 cells (5
10
/ml) were infused at 0.84
dyn/cm
through the parallel plate flow chamber containing
P-selectin (110 sites/µm
) or E-selectin (175
sites/µm
) in supported lipid bilayers, or neutrophils
were infused at the same conditions over a substrate containing
immobilized L-selectin-IgG chimera, and the number of cells that
accumulated after 3 min of continuous flow was quantitated. Cells were
treated with periodate and neuraminidase as described under
``Experimental Procedures,'' and cell accumulation on the
same microscopic field was determined. Treatments with the G1 mAb to
P-selectin and the BB11 mAb to E-selectin were as described under
``Experimental Procedures.'' The data represent the mean
± range of two randomly selected 10
fields and are
representative of two independent
experiments.
Borohydride reduces the C-7 aldehyde group in mildly oxidized sialic acid to a C-7 hydroxyl. Borohydride treatment reversed the effect of mild oxidation, reducing lymphocyte accumulation on CD34 to control levels (Fig. 1A). The aldehyde moiety at C-7 of sialic acid therefore appears to be required for enhanced L-selectin-dependent binding to mildly oxidized CD34, whereas removal of C-8 and C-9 appears to have no effect. In contrast, strong periodate treatment, which cleaves between endocyclic carbons of sugar moieties containing vicinal hydroxyls and is expected to leave more aldehyde groups per glycan than mild periodate treatment, completely abolished lymphocyte binding (Fig. 1A). These data suggest that aldehyde groups generated on sialic acid side chains of CD34 are specifically involved in the enhanced interaction.
CD34 can support E-selectin-dependent tethering and rolling of E-selectin transfected CHO-E cells(8) . However, mild periodate modification of CD34 did not affect CHO-E cell accumulation in shear flow (Fig. 1A). To extend these studies to ligands for P-selectin and E-selectin on myeloid cells, we examined mild oxidation of neutrophils, the promyelocytic cell line HL-60, and hematopoietic progenitor KG1a cells, for an effect on interaction with immobilized P-selectin and E-selectin under laminar flow conditions. Both P-selectin and E-selectin-mediated accumulation of HL-60 cells were unaffected by mild periodate treatment (Fig. 1C). Similar results were obtained with neutrophils and KG1a cells (data not shown).
Inhibition studies were
performed to confirm that the interactions with mild periodate-treated
substrates were specific. Dreg-56 mAb to human L-selectin(29) ,
fucoidan, and chelation of Ca with EDTA abrogated the
binding of lymphocytes to both sham- and periodate-treated CD34 (Fig. 1A), whereas a class-matched control antibody had
no effect (not shown). Similarly, both sham- and periodate-treated
neutrophil and KG1a cell interactions with immobilized L-selectin were
abrogated with Dreg-56 mAb (not shown). E-selectin and
P-selectin-mediated interactions with their ligands were abolished by
EDTA and by pretreatment with mAb BB11 to E-selectin and mAb G1 to
P-selectin, respectively (Fig. 1C).
To further confirm
oxidation of sialic acid by periodate treatment, several mAbs directed
against sialyl Lewis-related structures were tested on
sham- and periodate-treated neutrophils, HL-60, and KG1a cells.
Neuraminidase treatment of sham-treated cells completely abrogated
binding by all mAbs (Fig. 2). Furthermore, the epitopes of
CSLEX-1 and HECA-452 mAb, but not of the FH6 mAb, were sensitive to
mild periodate oxidation. Moreover, mild periodate treatment rendered
the FH6 epitope completely resistant to neuraminidase, confirming that
sialic acid side chains were indeed quantitatively modified.
Figure 2:
Effect of mild periodate and neuraminidase
on carbohydrate antigens related to sialyl Lewis. Periodate
and neuraminidase treatments of HL-60 cells were carried out as
described under ``Experimental Procedures.'' Cells were
stained with mAbs that bind to sialyl Lewis
or related
structures, CSLEX-1, HECA-452, and FH6, and with mAb to CD63 and the
myeloma X63 as binding and nonbinding controls, respectively. Cells
were stained with fluorescein isothiocyanate goat anti-mouse Ig and
subjected to immunofluorescent flow
cytometry.
Figure 3:
Effect of mild periodate treatment on
selectin-mediated accumulation of cells on glycolipids. A,
L-selectin-dependent binding of SKW3 cells. B,
E-selectin-dependent binding of CHO-E cells. Cells (5
10
/ml) were infused at 0.84 dyn/cm
on
polystyrene slides containing immobilized glycolipids assembled in a
parallel plate flow chamber. The number of cells that accumulated after
3 min of continuous flow was calculated and expressed per
mm
. Periodate treatment of immobilized glycolipids was
carried out as described under ``Experimental Procedures.''
DREG-56 mAb to L-selectin, BB-11 mAb to E-selectin, and 5 mM EDTA were used as described under ``Experimental
Procedures.'' The data represent the mean ±
S.D.
Figure 4:
Reduction of the Schiff base formed
between selectins and mild periodate-treated ligands. A,
rollingly adherent lymphocytes on mild periodate-treated or
sham-treated CD34 at 150 sites/µm were treated by
perfusion with 5 mM sodium cyanoborohydride at 0.84
dyn/cm
, and rolling velocities of 20-25 randomly
selected cells were determined as described under ``Experimental
Procedures.''
, sham + cyanoborohydride;
,
periodate;
, periodate + cyanoborohydride. B,
rolling adherent sham- or mild periodate-treated neutrophils on
E-selectin at 0.84 dyn/cm
were subjected to perfusion with
5 mM cyanoborohydride at t = 0, and rolling
velocities were determined for 10-15 cells. C, as in B, except on P-selectin. Data points in A, B, and C are mean rolling velocity (stationary cells
are included with velocity = 0) and are representative of two to
three independent experiments. D, rollingly adherent
lymphocytes on sham- or mild periodate-treated CD34 or sham- or mild
periodate-treated neutrophils rollingly adherent on P-selectin and
E-selectin were treated by perfusion with 5 mM cyanoborohydride at 0.84 dyn/cm
for 10 min and then
with EDTA. Bound cells were enumerated before and after perfusion with
medium containing 5 mM EDTA.
Figure 5:
Effect of mild periodate treatment on
selectin-mediated resistance to cell detachment by shear. A,
lymphocytes were allowed to tether at 0.84 dyn/cm at the
following site densities of sham- or periodate-treated CD34:
,
150 sites/µm
(sham);
, 150 sites/µm
(+periodate);
, 60 sites/µm
(sham);
, 60 sites/µm
(+periodate);
, 35
sites/µm
(sham);
, 35 sites/µm
(+periodate). *, 290 sites/µm
(no
treatment). Wall shear stress was then increased every 10 s to a
maximum of 36 dyn/cm
, and the percentage of cells remaining
bound at each shear was determined. B, periodate, borohydride,
and neuraminidase treatments of immobilized CD34 at 150
sites/µm
were carried out in the order described under
``Experimental Procedures'' prior to the detachment assay.
The data points represent the mean ± range of the number of
lymphocytes that remained bound in two randomly selected 10
fields and are representative of two to three different independent
experiments.
, sham;
, +periodate;
,
+periodate + borohydride;
, +periodate +
neuraminidase;
, sham + neuraminidase. C,
L-selectin-IgG chimera and P-selectin-IgG chimera were immobilized on
plastic substrates at 1.5 and 2 µg/ml, respectively. The P-selectin
density was about 150 sites/µm
. Periodate or
sham-treated neutrophils were allowed to tether at 0.84
dyn/cm
, and detachment was measured by increasing the wall
shear stress every 10 s to a maximum of 36 dyn/cm
. The data
represent mean ± S.D. of two to three independent experiments.
, sham-treated, P-selectin;
, periodate-treated,
P-selectin;
, sham-treated, L-selectin;
, periodate-treated,
L-selectin. D, human hematopoietic progenitor KG1a cells after
sham and periodate treatments were infused at 0.84 dyn/cm
on substrates containing L-selectin-IgG or P-selectin-IgG
chimeras immobilized at 2.5 µg/ml, and after sufficient
accumulation of cells, the wall shear stress was increased every 20 s
to a maximum of 42.7 dyn/cm
and the percentage of cells
remaining bound at each shear was determined. The density of P-selectin
was 180 sites/µm
. The data represent the mean ±
S.D.
, sham-treated, P-selectin;
, periodate-treated,
P-selectin;
, sham-treated, L-selectin;
, periodate-treated,
L-selectin. E and F, human promyelocytic HL-60 cells
after sham, periodate, and neuraminidase treatments were allowed to
accumulate on supported lipid bilayers containing P-selectin (110
sites/µm
) (E) or E-selectin (175
sites/µm
) (F) at 0.84 dyn/cm
, and
the detachment assay was performed by increasing wall shear stress
every 10 s to a maximum of 36 dyn/cm
. Data represent mean
± S.D. of three independent experiments.
, sham;
,
+periodate;
, +periodate + neuraminidase;
,
+neuraminidase. G, SKW3 T cells were allowed to tether at
0.84 dyn/cm
on sham- or periodate-treated glycolipid
substrates. Wall shear stress was then increased every 3 s to a maximum
of 36 dyn/cm
, and the percentage of cells remaining bound
at each shear was determined. Data represent mean ± S.D.
, sLe
(sham);
, sLe
,
(+periodate);
, sLe
(sham);
, sLe
(+periodate).
Neuraminidase treatment of sham- but not periodate-treated CD34 abrogated lymphocyte resistance to detachment (Fig. 5B). Similar results were obtained with P-selectin and E-selectin ligands (Fig. 5, E and F). Borohydride reduction after modification reversed the increased strength of lymphocyte rolling interactions on CD34, further confirming the specific involvement of aldehyde groups (Fig. 5B).
Figure 6:
Effect of mild periodate treatment on
rolling velocity. A, lymphocytes were allowed to tether at
0.84 dyn/cm for 2-3 min at the following site
densities of sham- and periodate-treated CD34:
, 150
sites/µm
(sham);
, 150 sites/µm
(+periodate); ┌, 150 sites/µm
(+periodate + borohydride);
, 60
sites/µm
(sham);
, 60 sites/µm
(+periodate);
, 35 sites/µm
(sham);
, 35 sites/µm
(+periodate). *, 290
sites/µm
(no treatment). Wall shear stress was then
increased every 10 s to a maximum of 36 dyn/cm
, and rolling
velocities were measured for 15-20 cells. The data points
represent the mean rolling velocity ± S.E. and are
representative of two to three different independent experiments. B, SKW3 T cells were tethered to sham and periodate-treated
glycolipid substrates and rolling velocities of 25-30 cells were
determined during detachment assays as described in the legend of Fig. 5. Data represent mean ± S.D. of rolling velocities
of cells measured in two independent experiments.
, sLe
(sham);
, sLe
, (+periodate);
,
sLe
(sham);
, sLe
(+periodate). C and D, sham-, periodate-, and neuraminidase-treated
promyelocytic HL-60 cells were allowed to accumulate on supported lipid
bilayers containing P-selectin (110 sites/µm
) (C) or E-selectin (175 sites/µm
) (D)
at 0.84 dyn/cm
and subjected to increased shear in
detachment assays as described in Fig. 5. Rolling velocities of
15-20 cells were determined in each experiment, and the data
represent mean ± S.D. of rolling velocities of cells in two to
three independent experiments.
, sham;
, +periodate;
, +periodate + neuraminidase;
,
+neuraminidase.
In this study, we have determined the effect of mild
periodate oxidation of the carbohydrate ligands of L-selectin,
E-selectin, and P-selectin on adhesive interactions in hydrodynamic
shear flow. A previous study showed enhanced lymphocyte binding under
static conditions to mild periodate-treated fixed cryostat sections of
lymph node HEVs and enhanced binding of L-selectin IgG to mildly
oxidized PNAd(24) . We have quantitated the effect of mild
periodate oxidation on several measures of L-selectin-dependent
interactions in shear flow. We have extended observations to L-selectin
ligands on leukocytes and to the simple L-selectin ligands sLe and sLe
. Furthermore, we demonstrate that
interactions through L-selectin but not P-selectin or E-selectin are
enhanced by mild periodate treatment and that Schiff bases are formed
with all three selectins.
To determine the effect of mild periodate
treatment on dynamic selectin-mediated interactions, we monitored four
different parameters, stable tethers, cell accumulation, rolling
velocity, and resistance to shear detachment. Stable tethers are
initial interactions between a cell in flow and the substrate that
result in rolling interactions that last for at least 3 s. We
hypothesize that the rate of the initial formation of the tether is
related to the on rate of the reaction between the selectin and ligand;
the requirement for subsequent rolling would also be influenced by the
off rate. The finding that mild oxidation had little effect on
formation of stable tethers on CD34 suggests that k either was not increased or was not rate-limiting in the assay
system. Cell accumulation may reflect both the kinetic constants and
the equilibrium constant; its enhancement for L-selectin by mild
periodate oxidation of CD34 suggests an increase in K
= k
/k
.
Rolling velocity is hypothesized to reflect the average number of
receptor-ligand bonds between the cell and the substrate, governed by k
/k
, and the rate at which
bonds break (k
). Receptor-ligand dissociation
during rolling is thought to allow the cell to move forward in response
to hydrodynamic drag until it is held by other bonds. Additionally, how k
responds to tensile force on the bond will
influence rolling velocity(22) . Resistance to detachment by
hydrodynamic shear force is hypothesized to be influenced by the same
parameters as rolling velocity. The hydrodynamic drag force experienced
by a cell near a wall in shear flow is proportional to and can be
calculated from the wall shear stress (41) . It is interesting
that rolling velocity and resistance to detachment by shear were the
parameters most dramatically influenced by mild periodate oxidation;
rolling velocity was decreased 3-4-fold, and resistance to
detachment was increased approximately 10-fold higher in wall shear
stress. Changes in both parameters were equivalent in effect to an
8-fold increase in CD34 density on the substrate. Based on these
results, we suspect that periodate oxidation either diminished the
L-selectin:ligand k
with no effect on k
or diminished k
much
more than k
. It will be important to
experimentally test this prediction.
Selective enhancement of
L-selectin interactions by mild periodate oxidation of PNAd was
generalized to other L-selectin ligands. L-selectin ligands on HEV, i.e. PNAd and the CD34 fraction of PNAd studied here, bear the
sulfation-dependent MECA-79 carbohydrate
epitope(42, 43) ; however, ligands for L-selectin are
present on human neutrophils and human hematopoietic progenitor KG1a
cells (8, 9, 10) that lack this epitope.
Therefore, we also studied interactions of sham- and mild
periodate-treated neutrophils and KG1a cells with L-selectin adsorbed
to a substrate, which mediates rolling of these cells. Consistent with results with HEV-specific ligands,
L-selectin-mediated interactions of both neutrophils and KG1a cells
were enhanced after mild periodate treatment. Both cell types
accumulated better, rolled slower, and were more resistant to shear
detachment after mild periodate treatment. Our studies were further
extended to sLe
and sLe
. O-linked
glycans of an HEV-specific L-selectin counter-receptor have been shown
to have two terminal sialic acids, one each on the T-antigen and N-acetyl lactosamine carbohydrate structures(21) .
Therefore, we tested the effect of mild periodate treatment on
sLe
and sLe
-containing glycolipids, which have
sialic acid only on the counterpart of the N-acetyl
lactosamine moiety of the HEV ligands. Mild periodate modification of
both of these glycolipids enhanced L-selectin-mediated binding of SKW3
T cells, as reflected in slower rolling velocity and higher resistance
to shear detachment. These results support the participation of the N-acetyl lactosamine sialic acid in the enhanced interactions
with HEV ligands of L-selectin, although we cannot rule out an
additional interaction with the terminal sialic acid on the T-antigen
structure. Furthermore, neuraminidase treatment of immobilized CD34,
KG1a cells, and neutrophils completely abolished ligand activity. These
results show that enhanced interaction after mild periodate treatment
is a general phenomenon for L-selectin ligands and may reflect
modification of the terminal sialic acid on the N-acetyl
lactosamine structure.
Control studies with EDTA, mAb specific for E-selectin, P-selectin, and L-selectin and with fucoidan, which blocks L-selectin but not E-selectin interactions, showed that interactions with mildly oxidized substrates were specific. CD34 treated with neuraminidase prior to periodate modification was inactive, confirming the selective involvement of modified sialic acids in enhanced interactions with L-selectin. On the other hand, periodate modification rendered sialic acid insensitive to neuraminidase and protected ligand activity for E-selectin, P-selectin, and L-selectin. Results with the mAbs CSLEX and HECA-452 show that their epitopes include C-8 and C-9 of sialic acid, unlike all three selectins and the epitope of the FH6 mAb. These results and results on inhibition by mild periodate oxidation of digestion by neuraminidase showed that essentially all sialic acid side chains required for E-selectin, P-selectin, and L-selectin ligand activity, and for the CSLEX-1, HECA-452, and FH6 epitopes, were modified by mild periodate.
Mild periodate oxidation of carbohydrate
ligands did not enhance interactions with E-selectin or P-selectin. The
sialomucin CD34 is also a ligand for E-selectin and mediates tethering
and rolling interactions of CHO-E cells that express E-selectin.
However, E-selectin-mediated interactions were not affected by mild
periodate treatment of CD34 as shown by lack of effect on rolling
velocities and detachment profiles. Interactions of CHO-E cells with
sLe and sLe
glycolipids were also unaffected by
mild periodate oxidation. Moreover, mild periodate oxidation of HL-60
and KG1a cells had no effect on rolling interactions with P-selectin
and E-selectin, in contrast to enhancement of interactions with
L-selectin. Subsequent reduction with borohydride also had no effect on
interaction with E-selectin or P-selectin, in agreement with findings
that truncation of the sialic acid exocyclic side chain does not
destroy recognition by E-selectin (44) .
Our results suggest
that an aldehyde group on mildly oxidized ligands is responsible for
the enhanced interaction with L-selectin. Sialic acid is a nine-carbon
sugar containing an exocyclic chain; mild periodate oxidation under the
conditions used here quantitatively generates the seven-carbon aldehyde
form(26) . Borohydride reduction subsequent to the mild
periodate oxidation reduces the 7-aldehyde to a primary alcohol.
Borohydride reduction reversed the effect of mild oxidation of CD34,
showing that the enhanced interaction with L-selectin is not due to
side-chain truncation but required the 7-aldehyde group. Furthermore,
equivalent binding to the native and truncated, reduced structures
suggests that the three selectins do not interact with the C-8, C-9
diol moiety of sialic acid. These results extend a previous study that
found an unexplained disruption by borohydride of complexes of
L-selectin chimera with both native and mildly oxidized
ligand(24) . Our data and those of Norgard et al.(24) suggest that the C-7 aldehyde on sialic acid
specifically interacts with L-selectin. This interaction is very likely
with the -amino group of a specific lysine residue. The
interaction may consist of several different types of bonds that
rapidly interconvert. It may include a hydrogen bond of a lysine
-amino hydrogen with the C-7 aldehyde oxygen, which would be
predicted to be stronger than a hydrogen bond with the C-7 hydroxyl
oxygen. Interconversion could occur to a partially covalent bond, a
single bond, and a double bond or Schiff base between the lysine
-N and the C-7 carbon. Predominance as a Schiff base is unlikely,
because this requires stabilization by resonance with other double
bonds or with aromatic groups. We could demonstrate a Schiff base (24) by reduction with cyanoborohydride, as shown by formation
of an EDTA-resistant bond between leukocytes rolling on mildly oxidized
CD34 but not on sham-treated CD34. Furthermore, the cells stopped
rolling on the mildly oxidized substrate after cyanoborohydride
reduction. These findings suggest that covalent bond(s) were formed
between L-selectin and CD34.
Oxidation of E-selectin and P-selectin
ligands had no effect on measures of interactions in shear flow that
appear to reflect both the kinetics and equilibria of selectin binding;
nonetheless, Schiff base formation occurred, as shown by reduction with
cyanoborohydride. Cyanoborohydride caused cells rolling on E-selectin
and P-selectin to arrest and to form an EDTA-resistant bond to the
substrate. The kinetics of reduction of the Schiff base were somewhat
slower for E-selectin and P-selectin than for L-selectin, as determined
by the kinetics of the arrest of the rolling cells. The data show that
interconversion to a Schiff base structure can occur in all three
selectin-ligand complexes, although interconversion might be less
frequent for E-selectin and P-selectin based on the kinetics of
reduction. There may be no effect of mild oxidation on E-selectin and
P-selectin rolling behavior because gain of interaction with the C-7
aldehyde moiety is compensated for by loss of another interaction,
whereas with L-selectin there is a gain with no compensating loss.
Another way of looking at this is that the stabilizing interaction that
includes the Schiff base may only be noted kinetically with L-selectin
because of its considerably faster k with native
ligands.
The highly homologous lectin domains of selectins may use a
common recognition site for sugars and may bind in the same manner the
sLe motif that is common to all three ligands, with other
contacts that provide specificity for distinctive elements in the
ligand structures. The N-terminal lectin domains of the three selectins
have 10-14 lysine residues. Those at positions 32, 55, 67, 96,
111, and 113 are conserved in all three selectins. Although no
carbohydrate ligand has yet been cocrystallized with a selectin,
studies on a cocrystal of the homologous mannose binding protein (45) and docking of sLe
to E-selectin suggest that
lysines at positions 111 and 113 are closest to the sialic acid of
sLe
(15) . Mutation at Lys
completely
abolishes E-selectin and P-selectin ligand-binding function, and
mutation at Lys
severely decreases but does not abolish
function(15, 16, 17) . One possible model is
that Lys
forms an ionic hydrogen bond to the sialic acid
carboxylate in sLe
in all three selectins and Lys
forms a hydrogen bond to this carboxylate or to the nearby
anomeric oxygen or C-7 hydroxyl oxygen, which perhaps is more favorable
in E-selectin and P-selectin than L-selectin. In this model,
Lys
is available in all three selectins for formation of
a hydrogen bond to the C-7 aldehyde oxygen of the mildly oxidized
sialic acid, and for interchangeable formation of a Schiff base.
Specific assignments of the lysine(s) that form(s) Schiff base(s) in
E-selectin, P-selectin, and L-selectin would extend knowledge of how
these molecules bind their carbohydrate ligands.