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*
The Burnham Institute, La Jolla, CA 92037; and
Glycobiology Research and Training Center, Department of Medicine, University of California at San Diego, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous work on a library of bovine lung oligosaccharides led us to find a novel carboxylated glycan that was constitutively expressed on mammalian endothelial cells (2, 3). These glycans interacted with cell surface molecules on activated, but not resting, neutrophils (3). A mAb against this novel modification inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation (3). This inhibition of cell trafficking correlated with increased sequestration but reduced transmigration of leukocytes adherent to the endothelium of the mesenteric venules (3). To identify molecules involved in the process, we searched for proteins capable of binding these sugar chains. We report here that two soluble proteins of predominant leukocyte origin, which are externalized on activation and known to modulate neutrophil and monocyte extravasation interact specifically with the novel carboxylated glycans of bovine lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following materials were obtained from the sources indicated: platelet-activating factor (PAF)4 and PMA, Sigma (St. Louis, MO); ionomycin, Calbiochem (La Jolla, CA); DEAE Sephadex A25 and Sephadex G25, Pharmacia (Uppsala, Sweden); Affigel-10, Bio-Rad (Hercules, CA); Na125I, American Radiolabeled Chemicals (St. Louis, MO); Iodobeads iodination reagent, GelCode Blue, and bis-sulfosuccinimidyl suberate (BS3), Pierce (Rockford, IL); Phenyl-650C Toyopearl Resin, TosoHaas (Montgomeryville, PA); mouse anti-bovine annexin I, Biogenesis (Sandown, NH); mouse anti-human S100A8 and S100A9, Bachem Bioscience (King of Prussia, PA); purified mouse IgG1 isotype standard, BD PharMingen (La Jolla, CA); annexin I cDNA, American Type Culture Collection (ATCC; Manassas, VA; ATCC number 65115); expression vector pET23b, Novagen (Madison, WI); restriction enzymes and T4 DNA ligase, Promega (Madison, WI); nucleotide primers, Genset (La Jolla, CA); Western blotting chemiluminescence detection reagent kit, Amersham Pharmacia Biotech (Piscataway, NJ); [2-3H]mannose, American Radiolabeled Chemicals. mAb mAbGB3.1 directed against the novel carboxylated glycan was generated as described (3). Calf pulmonary artery endothelial cells (CPAE) were purchased from ATCC, and HUVECs were from Cell Applications (San Diego, CA).
Generation and fractionation of bovine lung glycopeptides
Five grams of bovine lung acetone powder was extensively digested with proteinase K at 50°C for 24 h in 0.1 M Tris-HCl, pH 7.5. The mixture was heat-inactivated by boiling at 100°C for 5 min, was centrifuged, and the supernatant was dialyzed against 3 x 4 liters of water by using a molecular mass cut-off of 3500 Da. The glycopeptides then were loaded on a 75-ml column of DEAE Sephadex A25 equilibrated in 10 mM Tris-HCl, pH 8.0, washed with 10 column volumes of starting buffer, and eluted sequentially with 0.1 M, 0.3 M, and 1 M NaCl in starting buffer. These fractions are hereafter simply called "0.1 M, 0.3 M, or 1 M" glycopeptides. The eluates were individually dialyzed extensively against water by using a molecular mass cut-off of 1000 Da and tested for their ability to inhibit binding of mAbGB3.1 to immobilized bovine lung glycans. Briefly, 96-well plates were coated with 50 ng of BSA neoglycoproteins containing total bovine lung glycopeptides coupled to BSA by using glutaraldehyde (see preparation of neoglycoproteins for immunization and screening; Ref. 3) for 4 h at 37°C. Plates were washed and blocked with 3% BSA in PBS overnight at 4°C. Wells were incubated with purified mAbGB3.1 IgG at 10 ng/ml in PBS containing 1% BSA and 0.05% Tween 20, in the absence or presence of varying concentrations of the above glycopeptides, in a total assay volume of 100 µl for 2 h at room temperature. The plates then were washed and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG, followed by development with p-nitrophenyl phosphate substrate. Absorbance was measured at 405 nm in an ELISA plate reader.
Monosaccharide compositional analysis of the 0.1 M and 0.3 M glycopeptides obtained in a similar fractionation experiment were analyzed by using a high pH anion-exchange chromatography-pulse amperometric detection system as described earlier (2). Sialic acid content was determined using resorcinol assay.
Generation of an affinity column for the isolation of cognate proteins from bovine lung
From the above fractionation, 0.1 M and 0.3 M NaCl eluates were
used individually to generate affinity columns. The glycopeptides were
first desialylated by mild acid treatment (10 mM HCl, 30 min at
100°C) to remove sialic acids, split into two equal aliquots, and
lyophilized. These are referred to in the text as "0.1 M or 0.3 M
asialo-COO- glycopeptides." The
carboxylate groups on one aliquot were modified by two cycles of
methylamidation as described earlier (2). This treatment
neutralized 
80% of the carboxylate groups (not shown). These
glycopeptides are referred to in the text as "0.1 M or 0.3 M
asialo-CONHMe glycopeptides." The glycopeptides were
coupled to BSA by using glutaraldehyde (3), and the BSA
neoglycoproteins then were coupled to Affigel-10 following
manufacturer’s recommendations. The efficacy of methylamidation of
carboxylates was determined by loss of reactivity with mAbGB3.1.
Briefly, 96-well plates were coated with 250 ng of the individual BSA
neoglycoproteins in PBS for 4 h at 37°C. Plates were washed and
blocked with 3% BSA in PBS overnight at 4°C. Wells were incubated
with purified mAbGB3.1 IgG at 10 ng/ml in PBS containing 1% BSA and
0.05% Tween 20, in a total assay volume of 100 µl for 2 h at
room temperature. The plates then were developed as indicated
above.
Affinity chromatography of bovine lung extracts
One hundred grams wet weight of bovine lung was homogenized with
a BioHomogenizer (Biospec Products, Barletsville, OK) in PBS
containing EDTA-free protease inhibitors, 10 mM DTT, 1 mM
CaCl2, and 1% Nonidet P-40 (NP-40). The
suspension was centrifuged at 650 x g for 15 min and
then at 10,000 x g for 30 min. The supernatants
(2.5 g of protein) first were precleared by passing over a 2-ml
BSA-Affigel column, and equal volumes then were loaded on BSA-bovine
lung glycopeptide affinity columns or the corresponding
carboxylate-neutralized columns run in PBS (10 mM phosphate, 150 mM
NaCl), pH 7.5. Unbound proteins were washed out with 10 column volumes
of starting buffer, and bound proteins were eluted with 50 mM sodium
citrate in 10 mM phosphate buffer (no change in pH or net ionic
strength over loading/wash buffer). The eluates were then dialyzed,
lyophilized, reconstituted in PBS, and analyzed on SDS-PAGE
gels.
SDS-PAGE and Western blot analysis
Affinity-purified bovine lung or neutrophil lysate proteins were separated on 12% SDS-polyacrylamide gels under reducing conditions and visualized by silver staining or by using Gelcode Blue. For immunodetection, proteins were electroblotted onto nitrocellulose membranes. The membranes were blocked overnight with 3% BSA in PBS, washed with PBS containing 0.05% Tween 20, and incubated with mouse anti-bovine annexin I, mouse anti-human S100A8, or mouse anti-human S100A9 diluted in PBS containing 1% BSA and 0.05% Tween 20 for 1–2 h at room temperature. This was followed by incubation with alkaline phosphatase or peroxidase-conjugated goat anti-mouse IgG. Bound proteins were visualized by incubating with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium, or by using chemiluminescence detection reagents.
N-terminal sequence analysis
Bovine lung proteins eluted from the glycopeptide affinity column were separated on 12% SDS polyacrylamide gels run in electrophoretic buffer containing 0.1 M thioglycolate, and transferred to polyvinylidene difluoride membranes. Transfer was conducted at 80 mV for 2 h and proteins stained with 0.1% Coomassie brilliant blue in 40% methanol. Individual bands were excised after destaining in 40% methanol and 7.5% acetic acid, and N-terminal Edman sequencing was performed on a Model 494 Procise Sequencer (Applied Biosystems, Foster City, CA).
Binding studies on human neutrophil lysates
Human neutrophils were isolated from fresh peripheral (EDTA-anticoagulated) blood from healthy volunteers by differential migration through mono-poly resolving medium (Ficoll-Hypaque d1.114) followed by hypotonic lysis of residual erythrocytes. The preparations were >98% granulocytes as confirmed by using Turk’s staining (0.01% crystal violet in 3% acetic acid). Cells were lysed by sonication in PBS containing protease inhibitors, and debris was removed by centrifugation at 10,000 x g for 30 min. Specificity of binding was determined by using three different approaches.
Binding to BSA-glycopeptides immobilized on ELISA plates. Plates (96-well) were coated with 250 ng of BSA-coupled 0.3 M asialo-COO- or asialo-CONHMe glycopeptides in PBS for 4 h at 37°C. Plates were washed and blocked with 3% BSA in PBS overnight at 4°C. The wells then were incubated with neutrophil lysate proteins in 10 mM HEPES buffer, pH 7.5, containing 150 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, and 1.3 mM CaCl2 for 2 h at room temperature. This was followed by incubation with anti-bovine annexin I Ab or anti-human S100A8 or anti-S100A9 for 2 h at room temperature. Wells then were washed and incubated with alkaline phosphatase conjugated goat anti-mouse IgG, followed by development with p-nitrophenyl phosphate substrate.
Binding to the glycopeptide affinity columns.
Lysate proteins (1 mg) were individually precleared on a BSA-Affigel
column, and equal volumes then were loaded on BSA-bovine lung
glycopeptide affinity columns or the corresponding
carboxylate-neutralized columns run in PBS (10 mM phosphate, 150 mM
NaCl), pH 7.5. Unbound proteins were washed out with 10 column volumes
of starting buffer, and bound proteins were eluted with sodium citrate
in phosphate buffer, without change in ionic strength, as described
above for bovine proteins. The unbound fractions and eluates were
concentrated on YM10 membranes and equivalent proportions of
each were analyzed after separation on 12% SDS gels by silver staining
and western blotting by using mouse anti-bovine annexin I, mouse
anti-human S100A8, or mouse anti-human S100A9 as described
above for bovine proteins.
Depletion of annexin I and S100 proteins from human neutrophil lysates by incubation with bovine lung glycopeptides. Plates (96-well) were coated with BSA-coupled 0.3 M asialo-COO- lung glycopeptides and blocked as above. Human neutrophil lysates were sequentially incubated in each of four wells for 30 min/well at 37°C in the above-mentioned buffer, in presence or absence of 200 µM 0.3 M asialo-COO- glycopeptides or asialo-CONHMe glycopeptides. After incubation, the supernatants from the incubation wells were collected and protein depletion analyzed by immunoblotting by using the respective Abs. For detection of S100A8/A9 complexes, the proteins in the supernatants were stabilized by chemical cross-linking by using BS3 according to Staros (4). The cross-linker was added from a fresh 2-mM stock solution to neutrophil lysates from the above depletion experiments to a final concentration of 200 µM, and the mixture was incubated at room temperature for 30 min. Reactions were quenched by the addition of 1 M Tris to a final concentration of 10 mM and samples stored at -80°C before assay. Cross-linked samples were incubated with 2-ME before separation on SDS-PAGE.
Neutrophil adhesion assays
Microtiter plates (96-well) were coated with 250 ng/well of BSA-conjugated 0.3 M asialo-COO- glycopeptides for 4 h at 37°C. Control wells had BSA alone. Plates were washed and blocked with 3% BSA in PBS overnight. Neutrophils were isolated as described above. Cells were resuspended at 5 x 106 cells/ml in HBSS buffer containing 0.2% BSA and used within 30 min after isolation. A total of 0.5 x 106 neutrophils in the above buffer containing 1 µM PAF were added to each well and incubated for 30 min at 37°C in the presence and absence of varying amounts of modifiers, in a total volume of 200 µl. Unbound cells were removed by washing with HBSS. Bound cells were quantitated by assaying myeloperoxidase as described (3). Assays were performed in duplicate.
Flow cytometric assays
Neutrophils were isolated as above. Cells were resuspended at 3 x 106/ml in HBSS, incubated in the presence or absence of 1 µM PAF or 100 ng/ml PMA/1 µM ionomycin for 15 min at 37°C, and pelleted by centrifugation at 300 x g for 10 min. Activated and unactivated neutrophils were incubated with mouse monoclonal anti-annexin I or anti-S100A8 (both IgG1 mouse Abs) or isotype IgG1 mouse control Ab in HBSS containing 1% BSA (each Ab at 1 µg/million cells), followed by FITC-conjugated anti-mouse IgG. They were analyzed by flow cytometry with a FACScan (Becton Dickinson, Mountain View, CA) equipped with CellQuest software, and gated by the side scatter and forward scatter filters.
Purification of human S100A8/A9 complex from peripheral human neutrophils
All steps were conducted at 4°C unless otherwise indicated. Ammonium sulfate precipitation was conducted as described (5). Briefly, 100 ml of peripheral blood was obtained from healthy volunteers and neutrophils was obtained by Ficoll-Hipaque centrifugation as indicated above. The cells were lysed in PBS containing 1 mM DTT, 1% NP-40, and protease inhibitors and subjected to ultracentrifugation at 160,000 x g for 30 min. The protein in the supernatant was adjusted to 2 mg/ml with the above buffer, and the solution stirred on ice. Solid ammonium sulfate was slowly added to a final concentration of 75% (w/v) and stirred for 1 h. The solution the was centrifuged at 10,000 x g for 30 min and the supernatant dialysed against 3x 4 liters of the above buffer for 24 h. The preparation was concentrated on YM10 membranes and applied to a 10-ml column of Phenyl-650C resin equilibrated in 0.1% trifluoroacetic acid (6). The unbound proteins were washed off with 3 column volumes of starting buffer and the bound proteins eluted with a gradient of 0–50% acetonitrile in the starting buffer. S100A8/A9 proteins eluted at 35–50% acetonitrile and were >90% pure as determined by SDS gels. The pooled fractions were lyophilized and reconstituted in PBS containing protease inhibitors and stored at -20°C.
Expression and purification of recombinant human annexin I
Nucleotide sequences encoding human annexin I were amplified by
PCR by using a cDNA clone containing the complete coding sequence
as template and the oligonucleotides Ann1_up:
5'-GTCGATAACATATGGCAATGGTATCAGAATTCC-3' and Ann1_down:
5'-AACGTACTTCATATGTTAGTTTCCTCCACAAAGAGCC-3' as primers. This
resulted in a 1067-bp fragment containing the entire annexin I cDNA
flanked by NdeI restriction sites. The fragment was digested
with NdeI and cloned into the expression vector pET23b by
using standard procedures (7), resulting in the plasmid
pWE151. Proper insertion and DNA sequences were confirmed by
established DNA sequencing methods by using primers oVW144: 5'-GAT GCT
GAT GAA CTT-3', oVW145: 5'-AAG TTC ATC AGC ATC-3', oVW146: 5'-CCA CAA
CTT CGC-3', and oVW147: 5'-GCG AAG TTG TGG-3'. This revealed a conflict
from the published sequence (GenBank accession number NM_0007000) at
position 288 (T288G). The sequences of two other
clones showed the same alteration. However, the T to a G does not alter
the encoded amino acid and thus could be a polymorphism. The expression
construct then was used to transform Escherichia coli strain
BL21(DE3). Three hours after induction with 1 mM
isopropyl-1-thio-
-D-galactopyranoside, the
cells, grown in Luria-Bertani medium supplemented with 100
µg/ml ampicillin, were harvested by centrifugation and resuspended in
25 mM Tris-HCl buffer, pH 8.0. Soluble fractions were obtained by
freeze-thaw lysis in three to five cycles in the above buffer. The
lysates were loaded on a DEAE Sephadex A25 column equilibrated with the
above buffer, and the flow-through fractions were collected. The
molecular mass of purified annexin I was determined by SDS gels and
matrix-assisted laser desorption ionization (MALDI) time-of-flight mass
spectrometry (Perseptive Biosystems, Cambridge, MA).
Radioiodination of proteins
Purified proteins were radioiodinated by using Na125I and Iodo-Beads Iodinating Reagent as per the manufacturer’s protocols. Unbound label was removed on a Sephadex G25 column, followed by concentration of the void volume fractions by using Centricon10 concentrators.
ELISAs with purified proteins
Immunoassays with purified proteins in the absence or presence of varying concentrations of 0.3 M asialo-COO- glycopeptides or 0.3 M asialo-CONHMe glycopeptides were done as described above for neutrophil lysates.
Precipitation of mannose-labeled glycoproteins
CPAE cells were labeled with
([2-3H]mannose at 20 µCi/ml for 24 h in
-MEM containing 10% FBS. Cells were harvested by trypsinization,
washed three times in PBS, and lysed in PBS containing protease
inhibitors and 0.5% NP-40. Protein-rich lysates were prepared by
ultrafiltration on YM10 membranes. Purified S100A8/A9 or annexin I
proteins were immobilized on Affigel-10 and mixed with the labeled
glycoproteins from CPAE cells in 10 mM HEPES buffer (described above
for neutrophil lysate ELISAs) after preclearing the lysates with
BSA-Affigel 10. For immunoprecipitations with mAbGB3.1, lysates were
precleared with normal mouse IgG-protein G beads and then incubated
with protein G coupled to mAbGB3.1 IgG in PBS. After overnight
incubation at 4°C, the individual pellets were washed free of all
unbound label until no more counts appeared in the washes, and the
radioactivity associated with the pellets was measured. For
cross-binding, label associated with each pellet first was eluted by
using high salt (5 M MgCl2, which eluted >70%
bound label). The eluates were repeatedly desalted on YM10 membranes
and resuspended in the respective buffers. Label eluted from mAbGB3.1
gels was mixed with immobilized S100A8/A9 or annexin I and vice versa.
Again, after overnight incubation at 4°C, the individual pellets were
washed free of all unbound label and counted.
Surface binding of iodinated S100A8/A9 to endothelial cells
HUVECs were grown in endothelial cell growth medium (Cell Applications) at 37°C in 5% CO2. Cell staining and Western blotting were performed to confirm expression of mAbGB3.1 Ags. HUVECS at passages 3–4 were used in the binding assays. Cells were grown to confluence in 12-well plates and activated with 1 µM PAF for 4 h. Activated and unactivated cells were washed with cold HBSS and then incubated with 4–80 nM 125I-S100A8/A9 in 1 ml of HBSS or 20 nM labeled complex in the absence and presence of modulators after activation at 4°C for 2 h. The reaction was stopped by rapid removal of unbound label, and the cells were washed three times with cold HBSS. They were solubilized in 1 M NaOH followed by addition of an equal volume of 1 M HCl, and the radioactivity associated with the lysates was measured with a gamma counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation and characterization of bovine lung glycopeptides for affinity chromatography
We had earlier shown that mAbGB3.1 specifically recognizes
carboxylate groups (3). We made an affinity column
enriched for the novel carboxylate on bovine lung glycopeptides.
Proteinase K-digested glycopeptides from bovine lung acetone powder
were fractionated on a DEAE Sephadex column into 0.1 M, 0.3 M, and 1 M
NaCl fractions as indicated in Materials and Methods. Each
anionic fraction in the above preparation inhibited binding of the Ab
to bovine lung proteins, but the neutral glycopeptides did not (Fig. 1
). Based on earlier work, the 1 M
fractions probably include a complex array of glycosaminoglycan chains
(2). For this reason, and because the most consistent
results were obtained with 0.1 M and 0.3 M fractions, the latter were
further processed. Also, for most inhibition assays described in the
text, desialylated 0.3 M glycopeptides were used at 100–200 µM final
concentration.
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), but with no evidence of any uronic
acids typical of glycosaminoglycan chains. In separate experiments, to
determine the proportion of the unknown carboxylate groups in the two
fractions, oligosaccharides were released from the 0.1 M or 0.3 M
glycopeptides by hydrazinolysis, desialylated by mild acid treatment,
and end-labeled with [3H]sodium borohydride.
Labeled oligosaccharides were subjected to methanolysis to remove
sulfate esters and convert the carboxylates to methyl esters. The
neutralized oligosaccharides then were subjected to base treatment to
regenerate the carboxylate groups. Charge fractionation before and
after the treatments showed that 29% and 33% of the glycans in 0.1
M and 0.3 M fractions contained the novel carboxylate species
respectively. This was further confirmed by QAE analysis of
[3H]acetic anhydride-labeled desialylated 0.1 M
or 0.3 M glycopeptides before and after carboxylate neutralization by
methylamidation by using
1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide/N-hydroxysuccinimide
(EDC/NHS) and methylamine (both results not shown).
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Based on the above findings, and to eliminate any sialic
acid-dependent binding, we chose to use desialylated
carboxylate-enriched glycopeptides to make the affinity column.
Henceforth, these are referred to in the text as "0.1 M or 0.3 M
asialo-COO- glycopeptides." To
serve as negative controls, carboxylate groups on aliquots of the
glycopeptides were neutralized by methylamidation by EDC/NHS and
methylamine (2). Henceforth, these are referred to in the
text as "0.1 M or 0.3 M asialo-CONHMe glycopeptides."
These glycopeptides then were individually conjugated to BSA by using
glutaraldehyde. The reactivity of 0.1 M and 0.3 M
BSA-asialo-COO- glycopeptides against
mAbGB3.1 was substantially reduced (75% and 95%, respectively)
after carboxylate neutralization (not shown). These
carboxylate-enriched or neutralized glycopeptide-BSA neoglycoproteins
then were individually coupled to Affigel-10 to make the affinity
columns.
A very small fraction of the proteins from whole bovine lung bind to the columns in a carboxylate-specific manner
A high concentration of immobilized negatively charged sugars
could function simply as an ion exchanger rather than as an affinity
matrix. To minimize such nonspecific ionic interactions, we conducted
initial binding under physiological ionic strength and pH by using
phosphate buffer in the presence of 150 mM NaCl. Because citrate is an
inhibitor of mAbGB3.1 binding to bovine lung proteins (3),
we then eluted the column with 50 mM citrate in phosphate buffer,
(i.e., keeping the pH and ionic strengths unchanged). Under these
conditions, <0.0001% of the loaded proteins bound to and eluted from
the column. Of the five or more protein bands that were detected on
silver stained gels, three proteins of masses 35, 37, and 30 kDa did
not bind to columns on which carboxylate groups were neutralized (Fig. 2A). As seen in Fig. 2B, the three proteins completely rebound to the unmodified
column after dialysis to remove citrate, and could be eluted with as
little as 5 mM citrate. Bands of >48 kDa also were present in the
unbound fractions, and their binding was not carboxylate-dependent
(Fig. 2A).
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Fig. 3 shows the N-terminal
sequences of the bound proteins. BLAST searches for sequence similarity
of the proteins in the GenBank data bases showed that the sequence of
the 37-kDa band was >80% similar to a sequence beginning with the
13th residue of a leukocyte calcium binding protein, annexin I. Native
annexin I is known to be blocked at the N terminus (8).
The identity of annexin I was further confirmed in immunoblots by using
a commercially available mouse mAb against bovine annexin I. A doublet
was recognized on the blots, the native (37 kDa), and a
"clipped"(35.5 kDa) form, which is devoid of the first 12 amino
acids, as deduced from the protein sequencing data (not shown).
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59% sequence homology
with that of the mouse (9, 10).
S100A8 has a molecular mass of 10.8 kDa. However, the protein we
isolated with the N-terminal sequence of S100A8 had a molecular mass of
35 kDa. It is known that the protein often exists and physiologically
functions as a Ca2+-dependent complex with S100A9
(or MRP14), another protein of the S100 family (11). The
complexes have molecular masses of 24.5 kDa [S100A8 · S100A9
heterodimer], 35 kDa [(S100A8)2·S100A9
heterotrimer], and 48.5 kDa
[(S100A8)2·(S100A9)2
heterotetramer]. Though disulfide-linked complexes can be generated in
vitro from purified S100A8 and S100A9 proteins, heteromeric complexes
formed in vivo are known to be noncovalently linked and break down on
SDS gels under both reducing and nonreducing conditions
(11). They have been identified by electrospray ionization
mass spectrometry (12) and the two-hybrid system
(13). It remains to be seen how we were able to isolate
the 35-kDa [(S100A8)2·S100A9] trimer as a
nondissociable complex. We did not obtain a sequence for S100A9 on the
35-kDa band. This could be attributable to an N-terminal block, as is
known with native human S100A9 (14). Table II compares the N-terminal sequences of a
few mammalian S100A8 and annexin I proteins.
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-chain of hemoglobin and also variable overlapping sequences that
could not be matched with any known protein(s). Human homologues of the S100 proteins and annexin I also bind to the novel carboxylated bovine lung glycans
The two affinity-isolated bovine proteins showed specific binding to bovine lung glycans immobilized on microtiter plates, as detected by using radiolabeled bovine S100A8/A9, or an Ab against annexin I (data not shown).
Next, we sought to determine whether human homologues of S100A8/A9
complexes and annexin I showed similar binding by using three different
approaches. First, we checked and confirmed specific binding of the
proteins from human neutrophil lysates to carboxylated but not to
neutralized glycans, by direct binding to immobilized glycans on ELISA
(Fig. 4, A and B).
Next, we analyzed binding of human neutrophil lysate proteins to the
carboxylated and carboxylate-neutralized glycopeptide columns under the
same conditions that were used for bovine proteins. Both S100A8/A9 and
annexin I bound specifically to the carboxylated, but not the
neutralized species (Fig. 4, insets). Bound proteins also
consisted of few other minor bands that were detected on silver-stained
gels (not shown). The identity of these were not determined and is
beyond the scope of the present study. Approximately 95% of the total
loaded proteins did not bind to the columns, and because S100A8,
S100A9, and annexin I together account for 35% of the total
neutrophil cytosolic protein, the load probably exceeded the column
capacity. Analysis was done on reducing gels to see whether any of the
unusually nondissociable S100 complexes (as seen with the bovine
S100A8/A9 heterotrimer) were present in the bound fractions. In fact, a
small fraction of the bound human S100 proteins consisted of a stable
heterodimer, the identity of which was confirmed by a separate
immunoblot with S100A9 (not shown). However, a majority of the proteins
broke down to monomeric forms. Also, the identity of a set of 42- to
45-kDa anti-S100A8 reactive bands that bound to the column could
not be determined because they did not react with anti-S100A9, and
their masses did not correspond to any known S100A8/A9 complexes.
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, S100A8 homodimer as well as S100A8/A9
heterodimer and heterotrimer were depleted from the incubation mixture
after binding to the glycopeptides (lane 2). The
binding of the heterodimer and homodimer, but not the heterotrimer, was
blocked if the incubation was done in the presence of bovine lung 0.3 M
asialo-COO- glycopeptides but not by
asialo-CONHMe glycopeptides (lanes 3 and
4, respectively). A separate immunoblotting with
anti-human S100A9 confirmed the identity of the heteromeric
complexes (lane 5). Therefore, the differences in the
binding and stability of the human and bovine heteromeric complexes
could be a species difference. S100A8 monomer, arising from breakdown
of any residual noncross linked complexes, also showed similar binding
specificity. The monomer probably does not represent the component
involved in the actual binding, because complex formation is considered
an essential prerequisite for the biological functions of these S100
proteins (14).
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, annexin I from the neurophil lysates
appeared as the native (37 kDa) and the clipped (35.5 kDa) doublet,
which is devoid of the first 12 amino acids, based on our sequencing
data. Whereas the intact protein was >90% depleted from the
neutrophil preparations after binding to the carboxylate-enriched
glycopeptides, the truncated protein was not (lane
2), suggesting that the N-terminal 1–12 amino acids may be
important for optimal binding under these conditions. Reactivity of the
intact human protein was blocked in the presence of 0.3 M
asialo-COO- glycopeptides
(lane 3), suggesting specificity for the novel
carboxylate.
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We previously showed that when the novel carboxylated glycans are
immobilized on plastic, they supported the binding of freshly activated
human neutrophils (3). This binding was completely blocked
by mAbGB3.1 and by soluble
asialo-COO- glycopeptides, but not by
the asialo-CONHMe glycopeptides. To determine whether this
interaction was in fact mediated by annexin I or the S100 proteins,
here we tested the effects of anti-annexin I, anti-S100A8, and
anti-S100A9 on the binding of neutrophils to BSA-conjugated 0.3 M
asialo-COO- glycopeptides. As shown
in Fig. 7, although anti-annexin I
and anti-S100A8 had little or no inhibitory effects,
anti-S100A9 significantly reduced binding, as did mAbGB3.1,
indicating that neutrophil binding to the novel glycans may be
principally mediated by S100A9.
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S100A8 and S100A9 are cytosolic proteins, but membrane-associated
forms have been identified in monocytes on differentiation or
activation (15, 16). It is conceivable that neutrophil
binding to immobilized glycans is attributable to translocation of
S100A8/A9 complexes to the surface on activation. To test this
possibility, we determined surface expression of annexin I and
S100A8/A9 before and after activation of neutrophils by flow cytometric
analysis. S100A8 and annexin I are not expressed on resting neutrophils
but are detected on the cell surface within 15 min of activation with
PMA/ionomycin (Fig. 8) or PAF (data not
shown). This also explains our earlier findings that neutrophils
acquire surface binding sites for the glycans on short-term activation
(3).
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Purified human S100A8/A9 complex and annexin I show specific binding to the novel glycans
S100A8/A9 was purified from fresh human neutrophil cytosolic
fractions because they contain abundant amounts of the proteins
(30–45% of total cytosolic proteins; see Ref. 17). We
exploited the high solubility of S100 proteins in ammonium sulfate
(5) to obtain a preliminary enrichment of 80% before
further purification of the proteins (to 95% purity) on a
hydrophobic column (6). The proteins stayed as heteromeric
complexes through purification, were even retained on YM10 and YM30
ultrafiltration membranes, and could not be separated into individual
proteins without denaturation. The molecular masses of the purified
proteins as determined by SDS gels were 11 kDa and 14 kDa for S100A8
and S100A9, respectively (not shown). Purified S100A8/A9 complex showed
the same specificity of binding as the proteins from the initial human
neutrophil cytosol (Fig. 9, top). Binding to BSA-coupled
asialo-COO- bovine lung glycopeptides
immobilized on microtiter plates was inhibited in the presence of 0.3 M
asialo-COO- glycopeptides but much
less effectively by the asialo-CONHMe glycopeptides.
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, bottom). Again, the specificity of this binding was
demonstrated by a dose-dependent inhibition in presence of
asialo-COO- glycopeptides, but not by
asialo-CONHMe glycopeptides. Purified S100A8/A9 complex and annexin I bind to a subset of endothelial glycoproteins recognized by mAbGB3.1
We previously showed that a major fraction of the
mAbGB3.1-reactive carboxylated epitopes are carried on
N-linked glycans and that mAbGB3.1 recognized several
proteins on endothelial cells (3). In keeping with this,
mAbGB3.1 consistently immunoprecipitated 13–15% of
[2-3H]mannose-labeled glycoproteins from CPAE
cells (Table III). To determine whether
the same glycoproteins were also recognized by the S100A8/A9 complex or
annexin I, cross-binding assays were conducted as described in
Materials and Methods. Essentially, purified proteins were
immobilized on Affigel-10 and mixed with
[2-3H]mannose-labeled glycoproteins from CPAE
cells (precleared with BSA-Affigel-10). A separate immunoprecipitation
of labeled glycoproteins was simultaneously conducted by using mAbGB3.1
after preclearing the lysates with normal mouse IgG-protein G
Sepharose. As shown in Table III, S100A8/A9 complex precipitated 9% of
mannose-labeled glycoproteins from CPAE cells as compared with 14% of
label immunoprecipitated by the Ab. When the precipitated proteins were
eluted either from the S100A8/A9 complex or mAbGB3.1 and cross-bound,
S100A8/A9 complex precipitated about one-third of the label eluted from
mAbGB3.1, whereas mAbGB3.1 immunoprecipitated almost two-thirds of the
label eluted from the S100A8/A9 complex. These experiments showed that
the S100A8/A9 complex bound to a subset of endothelial glycoproteins
recognized by mAbGB3.1. In comparison, immobilized annexin I
precipitated only 2–3% of mannose-labeled glycoproteins from
endothelial cells (not shown). This may indicate differences in binding
affinities or may suggest that more of the annexin I-reactive epitopes
could be present on O-glycans.
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We found dose-dependent and saturable binding of
125I-S100A8/A9 on PAF-activated HUVECs. Binding
to unactivated cells was 2-fold less (Fig. 10, top). Binding to
activated cells was specific because it was competed by increasing
amounts of unlabeled ligand, with half-maximal competition of specific
binding at 500 nM (Fig. 10, middle), and also by
mAbGB3.1, the blocking being as effective as competition with unlabeled
ligand, whereas a control Ab had minimal effect (Fig. 10, bottom). Binding was also inhibited by anti-S100A8 and
anti-S100A9 (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Annexin I present in the bovine lung extract could have originated from
the lung tissue, the blood cells, or both. The lung also contains
annexins II and V (20), but repeated isolation of annexin
I alone on the columns showed that its binding was specific. The
specificity of binding may be imparted by the biologically active N
terminus (40 aa in length), which is unique for each member of the
annexin family (21). The N-terminal alanine of intact
annexin I is acetylated and resistant to Edman degradation
(8); the presence of the 
1–12 annexin I enabled
identification of the sequence, though the intact form was also
isolated (as found in immunoblots, not shown). In contrast, the
S100A8/A9 complex present is exclusively in leukocytes (17, 22) and probably originated from the trapped blood or
sequestered neutrophils in bovine lung. Subsequently, we found that
human homologues of annexin I and S100A8 from neutrophil lysates also
bound to the glycopeptide affinity columns and immobilized glycans in a
carboxylate-dependent manner (Fig. 4).
The two migration inhibitory factor-related proteins S100A8 and S100A9
belong to the large family of S100 proteins, the members of which have
the EF hand calcium-binding structures (for a recent review, see Ref.
14). These heteromeric complexes are found in sera of
patients with cystic fibrosis, chronic bronchitis, and rheumatoid
arthritis, suggesting extracellular roles (23, 24, 25).
Well-defined functions of S100A8 and S100A9 either as individual
proteins or as complexes has been elusive. Existing evidence suggests
that they may be involved in macrophage differentiation and
inflammation. When monocytes extravasate through the endothelium and
become resident macrophages, these Ags are lost (26).
However, at sites of chronic inflammation in patients with rheumatoid
arthritis, sarcoidosis, and tuberculosis, tissue macrophages continue
to express S100A8/A9 (26, 27). These proteins have also
been found coating vascular endothelium adjacent to marginating
neutrophils and monocytes, suggesting that secretion of S100A8/A9 on to
vessel walls is involved in the migration of leukocytes into tissues
(28). Kerkhoff et al. (29) report that only
monocytes expressing the S100A8/A9 complex preferentially migrate
through the microvascular endothelium. How the S100 proteins mediate
these interactions with the endothelium is not clearly understood.
Recent studies show that S100A8/A9 binds to unsaturated fatty acids
with high affinity and may be involved in arachidonic acid metabolism
(30, 31, 32). Cell surface S100A8/A9 binding sites have also
been detected on human leukemia cell lines (6). S100A9
also is shown to modulate adhesion of neutrophils to fibrinogen via
2 integrin Mac1, by recognizing a distinct but
uncharacterized pertussis toxin-sensitive G protein-coupled receptor on
neutrophils (33).
In this study, we provide several lines of evidence that S100A8/A9
could function by directly interacting with novel carboxylated glycans
on endothelial cells. First, S100A8/A9 heteromeric complexes of
S100A8/A9 are depleted from neutrophil lysates after binding to the
glycans, and this binding is carboxylate-dependent (Figs. 4B
and 5). Adhesion of neutrophils to the immobilized glycans is
effectively inhibited by both anti-S100A9 and mAbGB3.1 (Fig. 7).
Purified S100A8/A9 complex I binds to the novel glycans in a
carboxylate-dependent manner (Fig. 9). S100A8/A9 complex cross-reacts
with mannose-labeled glycoproteins immunoprecipitated by mAbGB3.1
(Table III). Finally, specific binding of S100A8/A9 to endothelial cell
surface is blocked by mAbGB3.1 (Fig. 10). Recombinant S100A8 alone also
does not show specific binding to the glycans (data not shown),
suggesting that heteromeric assembly of the S100 proteins was necessary
for this interaction. The binding also appears exclusive to S100A8/A9
because binding of two other S100 proteins S100A1 and S100B remained
constant between carboxylated and neutralized glycans under these
conditions (data not shown).
Annexin I, also called lipocortin I, is a member of a family of 13 distinct calcium-binding proteins characterized by a common C-terminal core structure consisting of four or eight highly conserved repeating motifs, which bind calcium and phospholipids, and a variable N terminus (21). Annexin I is distributed in different tissues, but is abundant in cells of the hemopoietic lineage, particularly granulocytes and monocytes, where it makes up as much as 2–4% of total cytosolic protein (34).
A growing body of evidence from studies on inflammation in animal
models and humans suggests that annexin I potently inhibits neutrophil
extravasation (reviewed in Ref. 35). Annexin I also is
strongly induced by glucocorticoids and is considered to mediate some
of the well-known antiinflammatory effects of these hormones
(36). The N terminus of annexin I has been shown to play
an important modulatory role in the biological effects of the protein.
For example, it has been shown by several studies that the N-terminal
peptide Ac 2–26 mimics the potent antiinflammatory effects of intact
annexin I (35). The amino-terminal domain regulates
interactions with membranes (37), and is directly involved
in binding to S100 proteins (38, 39). Most notably, the
N-terminal peptide Ac2–12, which in our study appears essential for
binding to the novel glycans (Fig. 6), has been shown to be as
effective in detaching adherent neutrophils from murine postcapillary
venules as the intact protein (40). An endogenous protease
catalyzes cleavage at tryptophan 12 of intact annexin I in a
Ca2+-dependent manner (41), and
proteolysis is believed to regulate the action of externalized annexin
I (36). The mechanisms by which annexin I and its peptide
mimetics affect neutrophil extravasation have remained elusive so far.
Annexin I binding sites on neutrophils and monocytes have been
identified, and the protein has recently been shown to be a ligand for
the formyl peptide receptor (42, 43). Annexin I also has
been reported to regulate the mitogen-activated protein
kinase/extracellular signal-regulated kinase signaling pathway
(44).
Here we provide evidence that annexin I could function by interacting
with novel carboxylated glycans. Annexin I from human neutrophil
lysates shows carboxylate-dependent binding to the glycans (Fig. 4A). Depletion experiments suggest that the N-terminal 1–12
amino acids of annexin I may be important for optimal binding (Fig. 6).
Recombinant full-length human annexin I also binds to the novel glycans
in a carboxylate-dependent manner (Fig. 9). However, the precise
presentation or the structure of the glycans recognized by annexin I
and S100A8/A9, and the physiological processes mediated in vivo by the
two proteins could differ. For example, annexin I precipitates only
2–3% of mannose-labeled glycoproteins from endothelial cells
compared with 9% by S100A8/A9 (not shown), suggesting that some
recognition epitopes preferred by annexin I could be on
O-glycans. Also, unlike S100 proteins, annexin I does not
modulate neutrophil adhesion to endothelial monolayers but still
impedes neutrophil emigration (Ref. 45 ; G.S. and H.H.F.,
unpublished results). It recently has been shown that annexin I
inhibits monocyte adhesion to microvascular endothelial cells, this
again involving its N-terminal domain (46). This may
explain why we find increased adhesion of leukocytes to endothelial
cells in the presence of mAbGB3.1 after induction of peritonitis in
mice (3).
We previously had shown neutrophils acquired carboxylate-dependent
binding sites for the novel glycans after short-term activation
(3). The novel glycans also bind to secretions from
activated neutrophils (not shown). Annexin I and S100 proteins are
cytosolic and lack classical leader peptide sequences and transmembrane
domains. We observe that S100A8/A9 and annexin I are expressed on the
surface of neutrophils immediately after activation (Fig. 8). Earlier
studies have found that S100A8 and S100A9 translocate from the cytosol
to the membrane and intermediate filaments in a calcium-dependent
manner (14, 15, 16). Neutrophil activation with opsonized
zymosan also correlates with translocation of S100A8 to plasma membrane
(47). Secretion of the S100 proteins is believed to be
tubulin-, energy-, and protein kinase C-dependent (48).
Cell surface expression of annexin I after neutrophil adhesion to
endothelial monolayers also has been demonstrated (34, 44, 45). These findings conform with the characteristics of annexin
I and S100A8 and S100A9 as a class of proteins that includes growth
factors and the S-type lectins or galectins, which are secreted by a
Golgi-independent pathway (49, 50).
Our findings that the novel carboxylated endothelial glycans bind to soluble leukocyte proteins that are secreted on activation and that are known to mediate inflammatory responses suggest the existence of a novel pathway in acute inflammation. Efforts are now underway in our laboratories to isolate and characterize endothelial glycoproteins that carry the novel carboxylated glycans, to identify binding partners involved in mediating the functions of S100A8/A9 and annexin I, and to decipher the signaling mechanisms by which these proteins bring about their inflammatory responses in vivo.
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Acknowledgments |
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Footnotes |
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2 Current address: Neose Technologies, Inc., La Jolla, CA 92037.
3 Address correspondence and reprint requests to Dr. Hudson H. Freeze, The Burnham Institute, 10901, North Torrey Pines Road, La Jolla, CA 92037.
4 Abbreviations used in this paper: PAF, platelet-activating factor; 0.1 M or 0.3 M asialo-COO- or asialo-CONHMe glycopeptides, bovine lung glycopeptides charge fractionated on a DEAE column and desialylated, or desialylated and carboxylate-neutralized by methylamidation; NP-40, Nonidet P-40; BS3, bis-sulfosuccinimidyl suberate; MALDI, matrix-assisted laser desorption ionization; CPAE, calf pulmonary artery endothelial cells.
Received for publication August 22, 2000. Accepted for publication January 19, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and superoxide production. J. Leukocyte Biol. 57:63.[Abstract]
