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INTRODUCTION |
Soluble human complement receptor type 1 (sCR1,1 TP10) has been shown
to inhibit the effects of complement activation in a variety of disease
models, including ischemia-reperfusion injury (1-3), burns and acute
lung injury (4), hyperacute rejection of transplanted organs (5),
autoimmune diseases (6, 7), and many others. sCR1[desLHR-A], a
truncated version of sCR1 lacking the C4b binding site, was shown to be
a selective inhibitor of the alternative complement pathway (8) and was
protective in several models of myocardial injury (9-11).
The selectin adhesion molecules, in particular P- and E-selectin, have
been shown to mediate cellular interactions in the required rolling
phase, which precedes the adherence and extravasation of leukocytes
from the vasculature at sites of inflammation. The selectins, and in
particular P-selectin, also mediate the adherence of platelets to
lymphocytes (12) as well as the adherence of platelets to neutrophils.
The significance of selectin-mediated processes to various immune and
inflammatory responses in vivo has been demonstrated using
selectin-deficient animals, anti-selectin antibodies, soluble bivalent
selectin-IgG chimeric proteins, and the selectin ligand sLex tetrasaccharide as well as its analogs. Many of the
same animal disease models, which have been shown to be
complement-dependent using sCR1 as described above, have
also been shown to be selectin-dependent using the
sLex tetrasaccharide. For example, myocardial
ischemia-reperfusion injury (13), cobra venom factor-induced rat lung
injury (14), and IgG immune complex-induced rat lung injury
(15) have all been ameliorated by the use of the
sLex tetrasaccharide.
Sialated, fucosylated oligosaccharides, including the sLex
tetrasaccharide, are carbohydrate ligands for the P-, E-, and
L-selectin adhesion molecules (16) and as such represent a
potential means to combine anti-selectin activity with the
anti-complement activity of a glycoprotein such as sCR1. Furthermore,
sCR1 and sCR1[desLHR-A] expressing the sLex
tetrasaccharide should have the potential not only to inhibit complement activation and selectin-mediated cellular interactions, but
also to localize these activities at sites of inflammation where
activated endothelium has up-regulated the expression of P- and
E-selectin.
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EXPERIMENTAL PROCEDURES |
Complement Proteins, Antibodies, and Cell Lines--
Purified
sCR1 and sCR1[desLHR-A] were prepared as described previously (Refs.
2 and 8, respectively). Concentrations of sCR1, sCR1sLex,
sCR1[desLHR-A], and sCR1[desLHR-A]sLex were determined
by immunoassay in a microtiter plate format using two mouse monoclonal
antibodies (mAb) specific for CR1. Microtiter plates were coated with
antibody (mAb 6B1.H12 (17)), and detection employed a horseradish
peroxidase-conjugated antibody (mAb 4D6.1 (17)). Alternatively,
concentrations of the purified proteins were determined by absorbance
at 280 nm using a molar extinction coefficient of 2.5 × 105 liter mol
1 cm
1 for sCR1 and
sCR1sLex and of 2.0 × 105 liter
mol
1 cm
1 for sCR1[desLHR-A] and
sCR1[desLHR-A]sLex, obtained from quantitative amino acid
analysis (Cornell Biotechnology Program, Ithaca, NY). The human
histiocytic lymphoma cell line U937 (18) was obtained from ATCC. The
cloned cell line PRO
LEC11.E7, referred throughout this
paper simply as the LEC11 cell line (19), was obtained from Dr. Pamela
Stanley of the Albert Einstein College of Medicine.
CHO Cell Lines Expressing Soluble P-selectin-IgG Chimera and
Cell Surface E-selectin--
The plasmid CDM7 B (20), which codes for
a chimeric protein of amino acids 1-292 of human P-selectin fused with
the H-CH2-CH3 heavy chain region of human IgG1, was a gift from Dr.
Brian Seed of the Massachusetts General Hospital. The chimeric gene was
cloned into the eukaryotic expression vector pTCSLneo, which is pTCSgpt (21) modified to carry the neomycin resistance marker, and
cotransfected with pTCS-DHFR, a plasmid containing the dihydrofolate
reductase gene, into CHO DUKX-B11 cells. Single clones that secreted
3-5 µg/ml P-selectin-IgG protein were selected for subsequent
experiments using a commercial immunoassay for human IgG1 (The Binding
Site, San Diego, CA).
The plasmid containing cDNA for human E-selectin (22) was
also provided by Dr. Brian Seed. The E-selectin cDNA fragment was
excised with XbaI. Utilization of the single, internal
XbaI restriction site at position 2717-2722 resulted in the
truncation of 1136 base pairs of the 3'-untranslated region, thus
deleting several potential mRNA-destabilizing sequence motifs such
as ATTTA (six of seven motifs). Cotransfections were performed as
described above. E-selectin expression was monitored by flow cytometry, and single clones were amplified in methotrexate.
LEC11 Cells Expressing sCR1sLex--
The expression
plasmid coding for sCR1 was prepared as described previously (2). The
plasmid pTCSLdhfr* coding for a mutant mouse dihydrofolate reductase
with an abnormally low affinity for methotrexate (23) was derived from
pSV2-DHFR* (BamHI-HindIII, ~1.5-kilobase
fragment) cloned into pTCSLneo by direct substitution of the neomycin
gene. LEC11 cells were cotransfected with pTCSLdhfr* and the plasmid
coding for sCR1. Clones producing high concentrations of
sCR1sLex were selected in growth medium containing methotrexate.
LEC11 Cells Expressing sCR1[desLHR-A]sLex--
The
expression plasmid pT-CR1c6A coding for sCR1[desLHR-A] was prepared
as described previously (8). Using methods described previously (24),
LEC11 cells were cotransfected with pT-CR1c6A and pTCSLdhfr*.
High-expressing clones were selected in growth medium containing methotrexate.
Cell Culture Production and Purification of
sCR1sLex and sCR1[desLHR-A]sLex--
LEC11
cells secreting sCR1sLex or
sCR1[desLHR-A]sLex were cultured in roller bottles, as
described previously (8), and typically yielded 5-10 µg/ml
glycoprotein in the medium. sCR1sLex and
sCR1[desLHR-A]sLex were purified from cell culture
supernatants by cation exchange chromatography (SP-Sepharose, Amersham
Pharmacia Biotech or HS50, Perseptive Biosystems), ammonium sulfate
precipitation, hydrophobic interaction chromatography (butyl-Toyopearl
650M, Tosohaas), buffer exchange chromatography (Sephadex
G-25, Amersham Pharmacia Biotech), anion exchange chromatography (DEAE,
Tosohaas), size exclusion (HW65S, Tosohaas), or weak cation exchange
(CM, Tosohaas) chromatography, and concentration by ultrafiltration.
Overall purification yields were 40-70% for both
sCR1[desLHR-A]sLex and sCR1sLex, resulting in
typical lot sizes of up to 0.5-1.0 g of purified glycoprotein. Protein
purity by Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis
was >95%, and in most cases >99%, for both proteins.
Western Blot Analysis--
Each of the purified glycoproteins
(0.5 µg) was subjected to nonreducing SDS-polyacrylamide gel
electrophoresis followed by transfer to polyvinylidene fluoride
membranes (Millipore Corp., Bedford MA) using a semi-dry blotting
system. The membranes were blocked with 5% dry milk in Dulbecco's PBS
(phosphate-buffered saline without Ca2+ and
Mg2+, Life Technologies, Inc.) and incubated with a mAb
specific for CR1 (mAb 3C6.D11 (17)) or specific for sLex
(mAb 2F3, PharMingen or mAb KM93, Kamiya Biomedical Co., Thousand Oaks,
CA) at 5 µg/ml in 3% bovine serum albumin followed by washing and
detection using horseradish peroxidase-conjugated goat anti-mouse IgM
antibody (Southern Biotechnology Associates) or goat anti-mouse Ig
(Cappel) as appropriate.
Monosaccharide Analysis by HPLC--
The neutral and amino sugar
composition of the glycoproteins was determined by trifluoroacetic acid
hydrolysis, reductive amination with anthranilic acid, separation by
C18 reverse phase HPLC, and fluorescence detection (25). Sialic acid
content was determined by sodium bisulfate hydrolysis, reaction with
o-phenylenediamine, separation by C18 reverse phase HPLC,
and fluorescence detection (26).
Oligosaccharide Characterization by Mass
Spectrometry--
Glycoprotein samples were prepared and analyzed by
electrospray ionization mass spectrometry as described previously
(27-30) and as summarized below.
A 25-µg aliquot of glycoprotein was reduced, denatured, and digested
with N-glycosidase-F (Boehringer Mannheim) for 16 h at 37 °C, in the presence of 0.15% SDS (Bio-Rad) and 0.3% Nonidet P-40 (Sigma). The SDS and protein were removed after digestion by
elution of the released glycans from a C18 Sep-Pak. The Sep-Pak cartridge (Waters number 20515) was attached to a 5-ml syringe (Hamilton number 1005) and rinsed with 7-8 ml of acetonitrile followed
by 7-8 ml of 90% H2O, 10% AcCN by use of gentle air
pressure. The digest was loaded onto the cartridge, and the glycans
were eluted with 4 ml of 90% H2O, 10% AcCN. The volume
was reduced on a Speed Vac Concentrator (Savant), and the sample
transferred to a 1.5-ml glass Reacti-Vial, dried, and vacuum-desiccated
prior to permethylation.
For permethylation, desiccated samples were dissolved in 100 µl of a NaOH/Me2SO suspension, prepared by vortexing
Me2SO and powdered sodium hydroxide. After 1 h at room
temperature, 30 µl of methyl iodide was added, and the solution was
held for 1 h at room temperature with occasional vortexing.
Glycans were partitioned by adding 100 µl of chloroform, and the
suspension was back-extracted six times with approximately 0.5 ml of
H2O and the chloroform layer taken to dryness.
Permethylation was repeated. All samples were stored at
20 °C.
Periodate oxidation was performed using a 9 mM solution of
NaIO4 buffered with 0.1 M sodium acetate at pH
5.5 in a dark cold room (4 °C) for 3 days. The reaction was quenched
with 3 µl of ethylene glycol and allowed to stand overnight under the
same conditions. The sample was neutralized with 0.1 M NaOH
and reduced by the direct addition of 5 mg of solid
NaB1H4 or NaB2H4 (which
remained at room temperature for an additional 16 h). Excess
reducing agent was destroyed by the addition of 5 µl of acetic acid
and the solution dried in a vacuum centrifuge. Borate was removed as
its ester by repeated addition and drying with methanol. The sample was
vacuum-desiccated overnight prior to methylation.
Electrospray ionization mass spectrometry (ES-MS) was performed on a
Finnigan-MAT TSQ-700 (Finnigan-MAT Corp., San Jose, CA) instrument
equipped with an electrospray ion source. Methylated glycans were
dissolved in water/methanol solutions (40:60 v/v) containing 0.25 mM NaOH and analyzed by syringe pump flow injected at a
rate of 0.85 µl/min directly into the electrospray chamber through a
stainless steel hypodermic needle. The voltage difference between the
needle tip and the source electrode was
3.7 kV. A unique feature of
ES-MS is the generation of multiply charged ions from a single
molecular species. Unlike fast atom bombardment mass spectroscopy,
which has difficulty in obtaining spectra of high molecular weight
glycans, multiple charging brings the instrumentally measured mass
(m/z) of large glycopeptides and glycans to an
easily determined lower value. ES-MS data were analyzed directly or
deconvoluted by an algorithm designed to extract molecular weight
information from the peak spacing exhibited by parent ion multiply
charging. This computational approach was accomplished by pattern
recognition using a relative, or level-2, entropy algorithm (28).
Treatment of the data in this manner suppresses the artifacts
associated with alternative deconvolution algorithms.
Carbohydrate Analysis by Fluorophore-assisted Carbohydrate
Electrophoresis (FACE®, Glyko Inc. Novato, CA)--
The
N-linked oligosaccharides were released intact from the
glycoprotein by digestion with the recombinant enzyme peptide N-glycosidase F (Glyko). The glycoprotein was denatured by boiling for
5 min in 0.1% SDS, 50 mM
-mercaptoethanol. Nonionic
detergent Nonidet P-40 was added to 0.75% followed by incubation with
0.5 milliunit of peptide N-glycosidase F overnight at
37 °C. The protein was removed by ethanol precipitation, and the
supernatant containing the oligosaccharides was dried prior to
labeling. The released oligosaccharides were fluorescently labeled by
the addition of 0.15 M disodium
8-aminonapthalene-1,3-disulfonate in 15% acetic acid followed by an
equal volume of 1.0 M NaBH3CN. The
8-aminonapthalene-1,3-disulfonate-labeled oligosaccharides were then
separated by electrophoresis in a 21% polyacrylamide gel and imaged
and analyzed using the FACE® Imager and software.
Assays of Complement Regulatory Activity--
The inhibition of
complement-mediated lysis of antibody-sensitized sheep erythrocytes
(classical pathway) or of guinea pig erythrocytes in
EGTA-Mg2+ buffer (alternative pathway) was assessed as
described previously (8).
Radioligand Binding of sCR1sLex to CHO Cells
Expressing E-selectin--
CHO DUKX-B11 cells expressing E-selectin
(CHO/E-selectin) were harvested using PBS with 1 mM EDTA
and washed with PBS containing 1% bovine serum albumin (BSA), 1 mM CaCl2. E-selectin expression was monitored
with a fluorescein-labeled anti-ELAM1 mAb (Southern Biotechnology,
Birmingham, AL).
sCR1sLex and sCR1 were radiolabeled with 125I
using the Bolton-Hunter reagent (NEN Life Science Products) to yield a
specific activity of 1-2 × 105 cpm/µg of labeled
protein. Mixtures of labeled and unlabeled protein (1:5) over a
concentration range of 0.2-3 µM were incubated for
3 h at room temperature with 5 × 106 CHO cells
expressing E-selectin in a sample volume of 0.25 ml. Bound and free
ligand were separated by centrifuging the samples through a 0.2-ml
layer of dibutyl/dioctyl phthalate (7/4 volume ratio) and quantitated
using a
-counter. Samples were run in triplicate. Nonspecific
binding was determined using sCR1, and the apparent dissociation
constant (Kd(app)) for specific binding of
sCR1sLex was estimated as the concentration yielding
half-maximal binding.
Binding of sCR1sLex to CHO Cells Expressing
E-selectin by Flow Cytometry--
CHO cells were removed from flasks
using trypsin and washed. The buffer used for flow cytometry was 0.2%
BSA, 0.02% NaN3 in PBS, which included 0.9 mM
Ca2+ and 0.5 mM Mg2+. Fluorescein
isothiocyanate (FITC)-labeled glycoproteins were incubated at 500 µg/ml for 60 min at 4 °C with CHO/E-selectin cells (3 × 106 cells/ml), washed, and resuspended for analysis on a
Becton Dickinson FACScan flow cytometer. Negative controls included
untransfected CHO cells and sCR1-FITC. Expression of E-selectin was
confirmed using a mAb-specific for human E-selectin (Serotec) and an
isotype-matched irrelevant control mAb, which were incubated with the
transfected CHO cells at 2 µg/ml for 30 min at 4 °C followed by a
FITC-labeled goat anti-mouse Ig antibody (Becton Dickinson) for an
additional 30 min.
Inhibition of U937 Cell Adhesion to Activated Endothelial
Cells--
Human aortic endothelial cells (Clonetics), confluent in
96-well microtiter plates, were stimulated with tumor necrosis
factor-
(100 units/ml) and phorbol 12-myristate 13-acetate (20 ng/ml) for 4 h at 37 °C to up-regulate E-selectin. The cells
were then washed twice with Dulbecco's modified Eagle's medium
containing 1% fetal bovine serum. Serial dilutions of
sCR1[desLHR-A]sLex or sCR1[desLHR-A] were added to
achieve final concentrations up to 2.7 µM. To each well,
5 × 105 U937 cells in Dulbecco's modified Eagle's
medium were added and incubated for 20 min at 37 °C. The wells were
filled with media, sealed, and centrifuged inverted at low speed for 5 min. The seal was removed, the plates blotted, and the number of bound
cells in three microscope fields was determined and reported as a
function of glycoprotein concentration with S.E. values.
Inhibition of U937 Cell Adhesion to Immobilized P-selectin-IgG
Chimera--
Flat bottom, black 96-well microtiter plates were coated
with 2 µg/ml goat anti-human IgG
, incubated with
P-selectin-IgG-containing supernatants, and blocked using 2.5% BSA in
PBS. U937 cells at 2 × 106 cells/ml in PBS were
incubated 15 min at 37 °C with 2 µg/ml calcein-acetoxymethyl ester
and washed three times with Dulbecco's PBS with 0.9 mM
Ca2+ and 0.5 mM Mg2+. Varying
concentrations of sCR1 or sCR1sLex were added to the
P-selectin-IgG-coated microtiter plates followed by the fluorescently
labeled U937 cells at 2 × 105 cells/100 µl in each
well. The plates were then centrifuged very slowly (<600 rpm) for 5 min followed by a 15-30 min incubation at room temperature. The wells
were completely filled with PBS with Ca2+ and
Mg2+, sealed, inverted, and centrifuged at low speed as
before. The plate sealer was removed to empty the wells. Adherent cells
remaining behind in the microtiter wells were lysed with 100 µl of
TRAx® lysis buffer (Avant) and the fluorescence was measured
(
ex = 485 nm,
em = 530 nm; Cytofluor II,
Perseptive Biosystems) and reported as means and S.E. The control mAb
CSLEX1 which is specific for sLex was obtained as a
hybridoma line (ATCC) and used as ascites fluid.
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RESULTS |
Western Blot Analysis--
The presence of sLex on the
purified glycoproteins derived from LEC11 cell expression is clearly
evident by staining with two different anti-sLex mAb (Fig.
1, lanes 2, 4, 10, 12). The
glycoproteins derived from both the LEC11 and DUKX B11 cells show
comparable reactivity using anti-sCR1 mAb (lanes 5-8).

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Fig. 1.
Western blots of sCR1 (lanes 1, 5, 9), sCR1sLex (lanes 2, 6, 10),
sCR1[desLHR-A] (lanes 3, 7, 11), and
sCR1[desLHR-A]sLex (lanes 4, 8, 12) with
anti-sLex mAb KM93 or 2F3 and anti-sCR1 mAb 3C6.D11 as
indicated. To the left of the blot are the apparent
molecular masses from markers run and blotted in the
left-most unlabeled lanes of each blot.
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Monosaccharide Analysis--
Table I
summarizes the monosaccharide content for typical lots of each
glycoprotein. As might be expected, the most striking difference
between the LEC11- and DUKX B11-derived proteins was found in the
fucose content, which was more than doubled for the LEC11-derived
proteins, most likely due to
(1,3)-fucosyltransferase activity,
which is a primary distinction between these cell lines. In general,
the LEC11 versions of the proteins also had higher sialic acid content.
This higher sialic acid content may reflect the fact that, in addition
to sCR1 expression, the LEC11 clones were selected for high
sLex expression and hence high sialic acid. Both versions
of sCR1 tended to have higher glucosamine, galactose, and mannose than did the versions of sCR1[desLHR-A], reflecting a greater number of
complex oligosaccharide chains on the larger protein.
Oligosaccharide Analysis by Mass Spectrometry--
Table
II summarizes the N-linked
oligosaccharide content of typical lots of each glycoprotein as a mole
percent of each glycan species. Only complex type N-linked
oligosaccharides were observed. Significant quantities of high mannose
oligosaccharides were not observed. No evidence of unusual
oligosaccharide types or sulfated forms was observed. While measurable
differences were obtained between separate lots of the same protein
(data not shown) or between each of the four different proteins (Table
II), certain general trends were clearly evident. For example, the
predominant species of complex N-linked oligosaccharide was
typically biantennary (70-80%), followed by triantennary (12-25%)
and tetraantennary (1-10%). Approximately 97% of oligosaccharides
had core
(1-6)-fucose.
If it is assumed that the sialic acid and
(1-3)-fucose distribute
randomly between branches, the mole percent of sLex species
can be estimated from these data. To test the assumption of a random
sialic acid and fucose distribution, the oligosaccharides from
sCR1[desLHR-A]sLex were treated with a fucosidase that
removes
(1-3)-fucose only from branches that lack terminal sialic
acid. ES-MS analysis of the biantennary glycans with a single sialic
acid (BiNA1, BiNA1F1, and
BiNA1F2) after such fucosidase treatment
yielded quantities for each form that were within 10% of the expected
values, based on the quantities prior to treatment and an assumption of
random distribution (data not shown). Using the assumption of random sialic acid and
(1-3)-fucose distribution for the sample lots reported here, both sCR1sLex and
sCR1[desLHR-A]sLex yielded an sLex mole
percent of 60% (Table II).
Carbohydrate Analysis by Fluorescence-assisted Carbohydrate
Electrophoresis--
Typical profiles of the intact oligosaccharide
chains for sCR1[desLHR-A] (Fig. 2,
lane 1) and sCR1 (lane 3) were dominated by three
major bands corresponding to biantennary structures, which have zero
(BiNA0), one (BiNA1), or two
(BiNA2) terminal sialic acid moieties. This was consistent
with ES-MS results described above in which biantennary structures were
the predominant species (Table II). The
sCR1[desLHR-A]sLex (Fig. 2, lane 2) and
sCR1sLex (lane 4) FACE® profiles included these
same bands at lower relative intensities, but had additional bands from
variously fucosylated forms of these structures. The lower intensities
of the nonfucosylated bands for the LEC11 glycans occur because the
predominant biantennary species have been apportioned among various
fucosylated versions having zero, one, or two branched
(1-3)-fucoses. Thus the FACE® oligosaccharide profiles clearly
demonstrated the striking differences in oligosaccharide structures
obtained from glycoproteins expressed by DUKX-B11 cells (lanes
1 and 3) as compared with those derived from LEC11
cells (lanes 2 and 4). These differences can be
attributed to
(1,3)-fucosyltransferase activity.

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Fig. 2.
Oligosaccharide profile using FACE®.
From the left, the four lanes profile the carbohydrate from
sCR1[desLHR-A] in lane 1, sCR1[desLHR-A]sLex
in lane 2, sCR1 in lane 3, and
sCR1sLex in lane 4. The predominant structures
associated with each band in lanes 1 and 3 are
indicated to the left of the image; those associated with
bands in lanes 2 and 4 are to the
right of the image. The structural abbreviations are defined
in the footnote to Table II. Sequencing of the individual bands
indicated that some bands include contributions from minor components.
For example, the band indicated to be predominantly
BiNA1F2 includes the minor component
TriNA2F1. Similarly, the
BiNA1F1 band includes the minor component
TriNA3F1. The band labeled BiNA1,
BiNA2F2 includes contributions from roughly
equivalent amounts of the components BiNA1 and
BiNA2F2.
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In addition, the individual bands from the oligosaccharide profile were
cut out and subjected to further digestion and separation in order to
sequence and quantitate the various species. By such methods, the mole
percent of each oligosaccharide species, as well as the mole percent of
sLex per oligosaccharide chain, were determined and shown
to be in good agreement with data obtained by ES-MS for these
glycoproteins (31).
The sialic acid linkage was determined by digestion with the
neuraminidases NANase I (
2-3-specific) and NANase III
(
2-3,6,8-specific). The banding patterns of both digestions were
identical (31), and the changes in mobility were consistent with the
loss of NeuNAc, indicating that the NeuNAc linkage is
2-3.
Fucose linkage was also determined using a combination of fucosidase
and galactosidase digestions. After neuraminidase treatment, fucose was
removed using FUCase III, indicating the linkage to be either
1-3
or
1-4 to GlcNAc. After removing Fuc, galactosidases were used to
determine the position of the remaining Gal and, by inference, the
linkage of Fuc (31). Digestion with GALase III (Gal
1-4-specific)
changed the band migration pattern, indicating that Gal is linked
1-4, consistent with the presence of Fuc
1-3. Digestion with
GALase (Gal
1-3-specific) did not change the banding pattern,
indicating that Gal is not
1-3-linked, also consistent with the
presence of Fuc
1-3 (31).
Inhibition of Complement Activation in Vitro--
Having
established the presence of the sLex tetrasaccharide on the
LEC11 glycoproteins, it was important to examine the effects of such
glycosylation on complement inhibitory function. In general, the
concentrations required to inhibit human complement-mediated lysis of
erythrocytes were similar for glycoproteins expressed by LEC11 cells
(sLex versions, open squares and
circles, Fig. 3) compared with
those expressed by DUKX-B11 cells (non-sLex version,
closed squares and circles). A representative
titration of each of the four glycoproteins is depicted in Fig. 3 in
both an assay of classical pathway mediated lysis (Fig. 3, A
and C) as well as in an assay of alternative pathway
mediated lysis (Fig. 3, B and D). As expected,
both versions of sCR1 (squares) and sCR1[desLHR-A]
(circles) were similar in their capacity to inhibit alternative pathway mediated lysis, but the sCR1 versions were much
more effective than the sCR1[desLHR-A] versions in the inhibition of
classical pathway lysis.

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Fig. 3.
Representative assays of complement
regulatory activity. Inhibition of cell lysis by the classical
(A and C) or alternative (B and
D) pathway as a function of the concentration of sCR1
(filled squares, A and B), and
sCR1sLex (open squares, A and
B) or of sCR1 [desLHR-A] (filled circles,
C and D) and sCR1[desLHR-A]sLex
(open circles, C and D).
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A single such titration can be characterized by the concentration of
glycoprotein required for 50% inhibition of maximal lysis (IC50) so that a lower IC50 value would be
indicative of a more effective inhibitor in these assays. From an
examination of the means and S.D. values of the IC50 values
from numerous such titrations, the mean concentration of
sCR1sLex required to yield half-maximal lysis
(IC50 = 0.27 ± 0.082 nM, n = 31) of sensitized sheep erythrocytes was somewhat
higher than required for sCR1 (IC50 = 0.21 ± 0.060 nM, n = 65). The difference in mean
IC50 values between sCR1sLex and sCR1 is
statistically significant (p = 10
5 by
analysis of variance), indicating a possible effect of differing glycosylation in these assays. The actual difference, however, is
smaller than the differences observed between lots of either glycoprotein or even between multiple assays of a single lot. The most
important aspect of these results is that both sCR1sLex and
sCR1 are potent inhibitors of classical complement activation as
indicated by the subnanomolar IC50 values in these in
vitro assays.
Similarly, in the assay of alternative pathway lysis of guinea pig
erythrocytes, sCR1sLex (IC50 = 38 ± 16 nM, n = 37) appeared somewhat less
effective than sCR1 (IC50 = 19 ± 6.6 nM,
n = 10), which again was statistically significant
(p = 0.0012 by analysis of variance). The relatively higher IC50 values obtained in the alternative
versus the classical pathway assays reflect the higher
concentrations of serum complement used in the alternative pathway
assay. Nevertheless, both sCR1sLex and sCR1 were shown to
be potent inhibitors of alternative pathway activation as reflected in
IC50 values of approximately 10
8
M.
Analogous results were obtained for the two versions of
sCR1[desLHR-A]. In the classical pathway assay, sCR1
[desLHR-A]sLex (IC50 = 140 ± 32 nM, n = 3) was somewhat less effective than sCR1[desLHR-A] (IC50 = 58 ± 38 nM,
n = 4, p = 0.033) but, as expected, both were much less effective than either version of sCR1 which yielded subnanomolar IC50 values. In the alternative
pathway assay, sCR1[desLHR-A]sLex (IC50 = 46 ± 9.1 nM, n = 4) was again
somewhat less effective than sCR1[desLHR-A] (IC50 = 37 ± 6.2 nM, n = 4, p = 0.16) but comparable with either version of sCR1.
A general observation was that in all of these assays, the
sLex versions were somewhat less effective inhibitors of
complement mediated cell lysis than were the non-sLex
versions, but that these differences in mean IC50 values
were in all cases modest (less than 60%). The greatest difference was observed in the assays of classical pathway mediated lysis for the
sLex and non-sLex versions of sCR1[desLHR-A],
a selective inhibitor of alternative complement activation. These
assays have large inherent variability, especially when comparing
different lots of serum complement, different lots of target
erythrocytes, or different lots of complement inhibitory glycoproteins.
Most important, despite measurable differences in IC50
values, both sCR1sLex and sCR1 are potent inhibitors of
classical and alternative complement activation, and both
sCR1[desLHR-A]sLex and sCR1[desLHR-A] are potent
inhibitors of alternative complement activation.
Radioligand Binding of sCR1sLex to CHO Cells Expressing
E-selectin--
Having established the presence of sLex in
the N-linked complex oligosaccharide of
sCR1sLex, it was important to demonstrate its capacity to
function as a selectin ligand. The binding of
125I-sCR1sLex and 125I-sCR1 to
CHO/E-selectin cells as a function of glycoprotein concentration is
shown in Fig. 4. Specific
sCR1sLex binding was obtained by subtracting sCR1 binding,
which was assumed to be nonspecific, from the observed
sCR1sLex binding. A Kd(app) for the
binding of sCR1sLex was estimated from the concentration
yielding half-maximal specific binding and found to be 1.4 µM.

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Fig. 4.
Radioligand binding of sCR1sLex
to CHO cells expressing cell surface E-selectin. Binding of
125I-sCR1sLex (circles) and
125I-sCR1 (squares) is shown as a function of
free ligand concentration.
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Binding of sCR1sLex to CHO Cells Expressing E-selectin
by Flow Cytometry--
Because the low affinities of
sCR1sLex were near the limits of typical radioligand
binding methods, a second method was used to confirm the binding of
sCR1sLex to cell surface E-selectin on transfected CHO
cells. Nearly identical staining by sCR1sLex-FITC and
sCR1-FITC indicated no binding of either labeled protein to the control
CHO cells (Fig. 5A).
Similarly, anti-E-selectin mAb-FITC and an irrelevant control mAb-FITC
showed no evidence of binding to the control CHO cells (not shown). As
expected, the anti-E-selectin mAb-FITC stained the CHO/E-selectin cells compared with the control mAb-FITC (not shown), confirming the presence
of cell surface E-selectin. Significantly greater staining of
CHO/E-selectin cells was observed for sCR1sLex-FITC as
compared with sCR1-FITC (Fig. 5B), demonstrating specific binding of sCR1sLex-FITC to cell surface E-selectin. The
addition of 1.0 mM EDTA essentially eliminated the
selective staining of CHO/E-selectin by sCR1sLex-FITC (data
not shown), which is consistent with
Ca2+-dependent binding as expected for the
selectins. In similar experiments, sCR1[desLHR-A]sLex-FITC, but not sCR1
[desLHR-A]-FITC, was also shown to bind CHO/E-selectin.

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Fig. 5.
Binding of sCR1sLex to CHO cells
expressing cell surface E-selectin by flow cytometry. Cell counts
(frequencies) are shown as a function of fluorescence intensity for
sCR1 (broken line) or sCR1sLex (solid
line) for control CHO cells (A) or CHO cells expressing
E-selectin (B).
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In related flow cytometry experiments, the mean fluorescence from
CHO/E-selectin cells incubated with 0.1 µM
sCR1sLex-FITC was reduced by approximately 50% by
competition with 0.5 µM sCR1[desLHR-A]sLex,
with 1.5 µM BSAsLex, or with 0.7 µM anti-E-selectin mAb, but was unaffected by 0.4 µM sCR1 (not shown).
Inhibition of U937 Cell Adhesion to Activated Endothelial
Cells--
In addition to binding cell surface E-selectin, the
blocking of E-selectin-mediated cell-cell interactions was examined in static cell binding assays. The capacity of
sCR1[desLHR-A]sLex to inhibit the binding of the
monocyte-like human lymphoma line U937 to activated human aortic
endothelial cells was examined in static binding assays. Activation of
endothelial cells leads to the up-regulation of E-selectin after 4 h, which was confirmed by flow cytometry using anti-E-selectin
mAb-FITC. As shown in Fig. 6,
sCR1[desLHR-A]sLex, but not sCR1[desLHR-A], inhibited
the binding of U937 cells in a concentration-dependent manner
(IC50 = 0.7 µM).

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Fig. 6.
Inhibition of U937 binding to activated human
aortic endothelial cells. The number of cells bound by microscopic
inspection is shown as a function of concentration of sCR1[desLHR-A]
(squares) and sCR1[desLHR-A]sLex
(circles).
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Inhibition of U937 Cell Adhesion to Immobilized P-selectin-IgG
Chimera--
The capacity of sCR1sLex to inhibit
P-selectin-mediated cell adhesion was examined in static binding
assays. As shown in Fig. 7, the increased
fluorescence obtained upon lysing the adherent U937 cells, which had
been incubated with PBS buffer or with 12 µM sCR1,
reflects binding to the immobilized P-selectin-IgG. As expected, this
U937 binding was inhibited by removing Ca2+ using 20 mM EDTA or by blocking with the sLex-specific
mAb CSLEX1. Similar inhibition of U937 binding to immobilized P-selectin-IgG was obtained using 12 µM
sCR1sLex (Fig. 7). Inhibition of U937 binding was dependent
on sCR1sLex concentration with an IC50 = 3.1 µM (not shown).

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Fig. 7.
U937 binding to immobilized
P-selectin-IgG. Fluorescently labeled U937 cells were incubated
with immobilized P-selectin-IgG in the presence of PBS, 12 µM sCR1, 12 µM sCR1sLex, 20 mM EDTA, or anti-sLex mAb CSLEX1 as
indicated.
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DISCUSSION |
We have expressed the recombinant human protein sCR1, and its
deletion mutant sCR1[desLHR-A], in the LEC11 cell line in order to
incorporate sLex glycosylation in the resulting mature
glycoproteins. Cotransfection with a plasmid coding for a mutant mouse
dihydrofolate reductase enabled gene amplification to increase protein
expression using methotrexate. This approach is a practical method
enabling the large scale production of recombinant glycoproteins
bearing a desired, specific carbohydrate moiety, namely, the
sLex tetrasaccharide. Alternative approaches, such as
chemical (32) or enzymatic (33) glycosylation of purified proteins, may
not be as suitable for the preparation of biological drugs. The general applicability of the approach has been exemplified using two different, although related, glycoproteins.
sCR1 appears especially well suited for the incorporation of multiple
sLex moieties because of its elongated, flexible structure
bearing 25 potential N-linked glycosylation sites, which
should facilitate multivalent binding to cell surface selectins. This
elongated flexible structure is characteristic of proteins comprised of short consensus repeat domains such as CR1, CR2, membrane cofactor protein (MCP, CD46), decay accelerating factor (DAF, CD55), C4-binding protein, and even the selectins themselves.
Because only N-linked, complex-type oligosaccharides were
observed in the mass spectroscopy analysis, an estimate of the number of oligosaccharide chains per glycoprotein can be made from the mannose
content, assuming a trimannosyl core structure for each chain. Such a
calculation yields 12.1 and 13.7 oligosaccharide chains on
sCR1[desLHR-A] and sCR1[desLHR-A]sLex, respectively,
and 20.3 and 16.2 chains on sCR1 and sCR1sLex. Combined
with the mole percent sLex (Table II), the average
incorporation is 8 and 10 moles of sLex per
sCR1[desLHR-A]sLex and sCR1sLex, respectively.
sCR1[desLHR-A]sLex inhibited the
E-selectin-dependent binding of U937 cells to tumor
necrosis factor-
-activated human aortic endothelial cells in a
concentration-dependent manner with an IC50 of
1.3 µM. These results are similar to the IC50
values of 1 µM reported for the inhibition of HL60 cell
binding to immobilized E-selectin by SLeX16BSA and by
SLeX11BSA, bovine serum albumin conjugates with 16 and 11 mol of sLex tetrasaccharide/mol of protein, respectively
(32). It should be noted, however, that the sLex on these
serum albumin conjugates was not part of a natural oligosaccharide. The
IC50 values obtained for blocking E-selectin mediated cell adhesion reflect dissociation constants for multivalent
sLex binding to E-selectin, which was directly confirmed by
radioligand binding studies of sCR1sLex
(Kd(app) = 1.4 µM).
sCR1sLex also inhibited the binding of U937 cells to
surface-adsorbed P-selectin-IgG chimera in a
concentration-dependent manner (IC50 = 3.1 µM).
In summary, sCR1sLex and sCR1[desLHR-A]sLex
have been expressed in LEC11 cells and purified from culture
supernatants. Carbohydrate analysis presented here and elsewhere (31)
confirmed the presence of the desired sLex tetrasaccharide.
These sLex-containing glycoproteins retain potent
complement inhibitory activity that is comparable with that of the
non-sLex versions expressed by DUKX-B11 cells. The
sLex-containing glycoproteins functioned as selectin
ligands in vitro by binding to cells expressing surface
E-selectin and by blocking selectin-mediated cell adhesion in a static
system. The in vivo targeting and anti-inflammatory
activities of these glycoproteins are currently being examined in
animal models of lung injury, stroke, and myocardial
ischemia-reperfusion injury.