Generation and Application of Type-specific Anti-Heparan Sulfate
Antibodies Using Phage Display Technology
FURTHER EVIDENCE FOR HEPARAN SULFATE HETEROGENEITY IN THE
KIDNEY*
Toin H.
van Kuppevelt
§,
Michel A. B. A.
Dennissen
,
Walther J.
van Venrooij¶,
René M. A.
Hoet¶
, and
Jacques H.
Veerkamp
From the
Department of Biochemistry, 160, Faculty of
Medical Sciences and the ¶ Department of Biochemistry, Faculty
of Sciences, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
 |
ABSTRACT |
Detailed analysis of various heparan sulfate (HS)
species is seriously hampered by a lack of appropriate tools, such as
antibodies. We adopted phage display technology to generate anti-HS
antibodies. A "single pot" semisynthetic human antibody phage
display library was subjected to four rounds of selection on HS from
bovine kidney using panning methodology. Three different phage clones
expressing anti-HS single chain variable fragment antibodies (HS4C3,
HS4D10, and HS3G8) were isolated, with an amino acid sequence of the
complementarity-determining region 3 of GRRLKD (VH3
gene, DP-38), SLRMNGCGAHQ (VH3 gene,
DP-42), and YYHYKVN (VH1 gene,
DP-8), respectively. The antibodies react with HS and
heparin, but not with DNA or other glycosaminoglycans. Kd values for HS are about 0.1 µM.
The three antibodies react differently toward various HS preparations
and show different staining patterns on rat kidney sections, indicating
recognition of different HS molecules. This also holds for two
described mouse anti-HS IgMs (JM403 and 10E4; both generated by
conventional hybridoma technique) and indicates the presence of at
least 5 different HS species in the kidney. O- and
N-sulfation are important for binding of HS to HS4C3 and
HS3G8. The three single chain antibodies, but not JM403, block a basic
fibroblast growth factor binding site of HS. It is concluded that phage
display technology presents a powerful technique to generate antibodies
specific for HS epitopes. This is the first time this technique has
been successfully applied to obtain directly antibodies to
(poly)saccharides.
 |
INTRODUCTION |
Heparan sulfate (HS)1
represents a heterogeneous class of molecules within the group of
glycosaminoglycans. It has been implicated in many basic cellular
phenomena, such as cell growth, migration, and differentiation (1-4).
HS binds and modulates various proteins, including growth factors and
cytokines, enzymes, protease inhibitors, and extracellular matrix
proteins. Studies involving specific enzymatic or chemical cleavage and
subsequent analysis of the resulting oligosaccharides indicate the
existence of many HS species and the presence of domain structures
within the HS molecule (5-15). There are clues that specific
monosaccharide sequences within the molecule dictate the specific
features of a given species, e.g. a pentasaccharide for the
binding of HS/heparin to anti-thrombin III and a preferential sequence
for the binding of HS to bFGF (4, 16-19). The appreciation of the
structural diversity of HS species and its role in pathological
conditions is strongly hampered by the lack of appropriate
methodologies. Sequence strategies are not at hand, and specific
antibodies, obvious tools for studying diversity, are difficult to
raise. HS, and glycosaminoglycans in general, are almost
nonimmunogenic, and consequently, only a few specific antibodies have
been described (20, 21). To circumvent this, we adopted antibody phage
display technology because this system allows one to generate
antibodies against "self" antigens. We report here on the
generation and application of three specific antibodies against HS
species using this technique. We compared these antibodies with two
described mouse monoclonal antibodies, with regard to immunostaining on
sections of rat kidney, immunoreactivity toward various HS
preparations, and reactivity with bFGF sites on HS.
 |
EXPERIMENTAL PROCEDURES |
Materials
A "single pot" human semisynthetic phage library (22) (now
officially named synthetic scFv library 1) was generously provided by
Dr G. Winter, Cambridge University, Cambridge, United Kingdom. This
library contains 50 different VH genes with synthetic
random complementarity-determining region 3 segments, which are 4-12 amino acid residues in length. The heavy chains are combined with a
single light chain gene (DPL 16). The library contains > 108 different clones.
Two Escherichia coli strains were used: the suppressor
strain TG1 (K12, D(lac-pro), supE, thi,
hsdD5/F'traD36,
proA+B+,
laqIq, lacZDM15), and the
nonsuppressor strain HB2151 (K12, ara,
D(lac-pro), thi/F"proA+B+,
lacIqZDM15). Helper phages VCS-M13 were from
Stratagene (La Jolla, CA).
Heparan sulfate from bovine kidney, chondroitinase ABC (Proteus
vulgaris, EC 4.2.2.4), chemically modified heparan sulfate and
heparin kits, and mouse anti-heparan sulfate antibody (clone 10E4) were
from Seikagaku Kogyo Co. (Tokyo, Japan). Heparin from porcine
intestinal mucosa, chondroitin 4-sulfate from whale cartilage, chondroitin 6-sulfate from shark cartilage, dermatan sulfate from porcine skin, keratan sulfate from bovine cornea, hyaluronate from
human umbilical cord, DNA from calf thymus, dextran sulfate, bovine
serum albumin (fraction V), FITC-conjugated goat anti-mouse IgM,
FITC-conjugated goat anti-mouse IgG, heparinase I (from
Flavobacterium heparinum, EC 4.2.2.7), heparinase II (from
F. heparinum), and heparinase III (heparitinase, from
F. heparinum, EC 4.2.2.8), were from Sigma. Horseradish
peroxidase-conjugated, and alkaline phosphatase-conjugated rabbit
anti-mouse IgG were from Dakopatts (Glostrup, Denmark). Recombinant
bovine bFGF (146 amino acids) and mouse anti-c-Myc tag IgG (clone 9E10)
were from Boehringer Mannheim. PCR kit was from Promega (Madison, WI),
and the DNA sequencing kit (Sequenase version 2.0) and
[
-35S]dATP were from Amersham Pharmacia Biotech.
Restriction enzyme BstNI was from New England Biolabs (Beverly, MA).
Polystyrene Maxisorp Immunotubes were from Nunc (Roskilde, Denmark).
Various HS preparations were a generous gift from Dr. U. Lindahl,
University of Uppsala (Uppsala, Sweden).
Growth of Library
5.108 bacteria, corresponding to about
108 phage clones, were inoculated into 50 ml 2xTY medium
containing 1% (w/v) glucose and 100 µg ampicillin/ml (2XTY/glu/amp),
and grown at 37 °C while shaking until an absorbance at 600 nm of
0.6-0.8 was reached. To 10 ml of this culture, 5.1011
VCS-M13 helper phages were added, and after 30 min at 37 °C (no shaking) cells were spun down for 10 min at 3000 × g
at 37 °C. Bacteria were resuspended in 10 ml of 2xTY and added to
300 ml of preheated (30 °C) 2xTY/amp containing 25 µg of
kanamycin/ml. Incubation was for 16 h at 30 °C with
shaking.
Isolation of Phages
After spinning the culture for 10 min at 10,000 × g (37 °C), 20% (v/v) of a polyethylene glycol/NaCl
solution containing 20% (w/v) polyethylene glycol 6000 and 2.5 M NaCl was added to the supernatant. After mixing, the
solution was kept on ice for 1 h, spun (10,000 × g for 30 min at 4 °C), and resuspended in 40 ml of
ice-cold Milli Q water followed by addition of 8 ml of the polyethylene
glycol/NaCl solution. After 20 min on ice and spinning (30 min at
3000 × g at 4 °C), the supernatant was carefully
removed, and the pellet was respun to aspirate any residual
polyethylene glycol/NaCl. The pellet was resuspended in 2.5 ml of
ice-cold PBS (4 °C), spun down (10 min at 3000 × g), and filtered through a 0.45-µm filter to remove any
bacterial debris. This solution, containing the phages, was immediately
used for selection.
Selection of Heparan Sulfate Binders by Library Panning
The library was subjected to four rounds of panning. Polystyrene
tubes were coated with HS from bovine kidney using a solution of 20 µg/ml for 16 h, 4 °C. Tubes were rinsed three times with PBS,
blocked by incubation with 2% (w/v) Marvel (nonfat milk powder) in PBS
for 2 h (22 °C), and rinsed again three times with PBS. To the
tubes, 2 ml of phage suspension and 2 ml of 4% (w/v) PBS with 2%
(w/v) Marvel were added, and incubation was for 30 min under continuous
rotating, followed by standing for 90 min (22 °C). Tubes were washed
20 times with PBS containing 0.05% (w/v) Tween-20 and 20 times with
PBS. Bound phages were eluted by addition of 1 ml 100 mM
triethylamine, which was neutralized with 0.5 ml of 1 M
Tris/HCl, pH 7.4. One ml of this phage suspension was added to 9 ml of
a suspension of TG1 bacteria (A600 nm,
0.6-0.8) and incubated for 30 min at 37 °C in order to allow for
infection. Bacteria were spun down, taken into 1 ml of 2xTY, and grown
for 16 h at 37 °C on a 24 × 24-cm TYE plate (TY with
bacto-agar) containing 1% (w/v) glucose and 100 µg of ampicillin/ml.
Bacteria were scraped from the plate using a glass spreader and 5 ml of
ice-cold 2xTY/15% (v/v) glycerol. Of this suspension, 50 µl was
inoculated into 50 ml of 2xTY/glu/amp and grown at 37 °C until an
absorbance at 600 nm of 0.6-0.8 was reached. Phages were rescued after
addition of helper phages as described and used for further rounds of
selections. A total of four selections were carried out. In one case,
an additional negative selection step was introduced. In this case,
after the second round of selection, 1 ml of phages was added to a tube containing 3 ml of PBS containing 30 µg of dermatan sulfate and 30 µg of chondroitin 4-sulfate and incubated for 1 h at 22 °C under continuous rotation. Two ml of this suspension was then subjected
to two additional rounds of panning on HS.
Screening for Phages Expressing Heparan Sulfate Binding
Antibodies
Bacteria picked from single colonies after the last round of
selection were grown in 200-µl 2xTY/0.1% (w/v) glu/amp in 96-well polystyrene round bottom plates for about 3 h at 37 °C until
bacterial growth was visible. Antibody production was induced by adding 25 µl of 2xTY containing 9 mM
isopropyl-
-D-thiogalactopyranoside. Plates were
centrifuged, and the supernatant containing soluble antibodies was
applied to wells of polystyrene microtiter plates previously coated
with HS and blocked with 2% (w/v) PBS with 2% (w/v) Marvel. Bound
antibodies were detected using a mouse monoclonal antibody (9E10)
directed against the c-Myc tag, followed by incubation with horseradish
peroxidase-conjugated rabbit anti-mouse IgG. Peroxidase activity was
detected using tetramethylbenzidine as a substrate. The enzymatic
reaction was stopped after 5 min with 2 M
H2SO4, and absorbance was measured at 450 nm.
Screening Phage Clones for Unique Antibody Inserts Using PCR,
Fingerprinting, and Sequencing
Phages displaying anti-HS antibodies were checked for the
presence of full-length inserts by 25 cycles of PCR using phagemid DNA
as template. Taq polymerase was used; LBM3
(5'-CAGGAAACAGCTATGAC) was used as the backward primer, and fd-SEQ1
(5'-GAATTTTCTGTATGAGG) was used as the forward primer (23). The primers
span a region containing the VH, linker, and VL
elements (about 1 kilobase pair). Fingerprinting was performed using
BstNI as the restriction enzyme. PCR and fingerprinting
analysis were performed using 1% (w/v) and 4% (w/v) agarose gels,
respectively. To establish the complementarity-determining region 3 and
the germ line VH gene DNA segments, unique clones were
sequenced using the dideoxy method of Sanger et al. (24) using FOR LINK SEQ RIC (5'-GCCACCTCCGCCTGAACC) as the primer (located in the linker region between the VH and the VL
genes). For this purpose, single-stranded DNA was isolated using
standard procedures.
Source of Antibodies
To obtain optimal amounts of soluble anti-HS antibodies, phages
were allowed to infect the nonsuppressor E. coli strain
HB2151. Unless stated otherwise, culture medium was used as source of antibodies in ELISAs, whereas the periplasmatic fraction was applied in
immunohistochemical experiments. The latter, in which antibodies are
more concentrated, was prepared as follows. Bacteria were grown for
22 h at 37 °C in 5 ml 2xTY/glu/amp, subsequently added to 500 ml of the same medium containing 0.1% (w/v) instead of 1% (w/v)
glucose, and incubated until an absorbance of 0.5-0.8 was reached.
Induction was obtained by addition of
isopropyl-
-D-thiogalactopyranoside (final concentration,
1 mM). After incubation at 30 °C for 3 h, the
culture was left on ice for 20 min, centrifuged (3000 × g for 10 min at 4 °C), and the pellet was resuspended in
5 ml of ice-cold 200 mM sodium-borate buffer (pH 8.0)
containing 160 mM NaCl and 1 mM EDTA. After
centrifugation (3000 × g for 10 min at 4 °C) and
recentrifugation (48,000 × g for 30 min at 4 °C), the supernatant was filtered through a 0.45 µm filter and dialysed versus PBS. The preparation thus obtained is the
periplasmatic fraction.
Evaluation of Specificity by ELISA
Reactivity of the anti-HS antibodies with various molecules was
evaluated in ELISA in two ways: (a) by application of
antibodies to wells of polystyrene plates coated with the test
molecules (1 µg/ml coating solution), and (b) by an
inhibition assay in which the antibodies were incubated with the test
molecule (40 µg/ml, unless stated otherwise) for 16 h at
22 °C in PBS, followed by transfer to wells previously coated with
HS (bovine kidney). Test molecules included HS from bovine kidney,
aorta, lung, intestine, human aorta, pig intestine, and whale lung;
heparin; dermatan sulfate; chondroitin 4- and 6-sulfate; keratan
sulfate; dextran sulfate; hyaluronate; BSA; Marvel (the nonfat milk
preparation using as a blocking reagent during panning); and DNA. In
addition, HS (bovine kidney) digested with heparinase I, II, or III or
with HNO2 at pH 1.5 was evaluated. Furthermore, chemically
modified heparan sulfates (bovine kidney) and heparins (porcine
intestine) were analyzed, viz. preparations that were
completely desulfated and N-acetylated, preparations that
were completely O-desulfated and N-sulfated, and
preparations that were N-desulfated and
N-acetylated. Bound antibodies were detected using
anti-c-Myc tag mouse monoclonal antibody 9E10 and alkaline
phosphatase-conjugated rabbit anti-mouse IgG antibody. Enzyme activity
was detected using p-nitrophenyl phosphate as the substrate,
and absorbance was read at 405 nm.
Determination of Affinity
Kd values of the antibodies were determined
using an indirect competition ELISA, essentially according to Friquet et al. (25). Briefly, bacterial culture supernatants
containing 0.1 µg of antibodies/ml were incubated for 16 h at
22 °C in wells of microtiter plates with various amounts of HS from
bovine kidney. Next, 100 µl was transferred to a corresponding well
previously coated with HS and incubated for 1.5 h at 22 °C.
Bound antibodies were detected as described for ELISA. By plotting the
reciprocal of the fraction of antibodies bound versus the
reciprocal of the concentration HS used, a Klotz plot can be generated
in which the slope represents the Kd. For HS, a
molecular mass of 20 kDa was taken.
Immunohistochemistry
Immunofluorescence--
Specimens from rat kidney (Wistar, male,
3 months old) were snap-frozen in liquid isopentane cooled with liquid
nitrogen and stored at
70 °C. Cryosections (6 µm) were
rehydrated for 10 min in PBS, blocked with PBS containing 1% (w/v) BSA
(PBS/BSA), and incubated with anti-HS antibodies for 90 min at
22 °C. As a control, antibodies (single chain variable fragments)
against filaggrin were used. Filaggrin is a nonrelated protein not
present in kidney (26). Phage display antibodies were detected by
incubation with mouse anti-c-Myc tag antibodies (9E10, culture
supernatant), followed by FITC-labeled goat anti-mouse IgG (1:100 in
PBS/BSA), both for 90 min at 22 °C. After each incubation, sections
were washed in PBS (3 × 10 min). Additional controls were the
omission of primary or conjugated antibody. In addition to the phage
display-derived antibodies, two mouse anti-HS IgMs, JM403 (21) and 10E4
(20), were used, both obtained by conventional hybridoma techniques. They were detected using FITC-conjugated goat anti-mouse IgM antibodies (1:100 in PBS/BSA).
Enzyme Immunohistochemistry--
Samples from human kidney and
uterine myometrium (rich in heparin-containing mast cells) were fixed
in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin. Sections
were deparaffinized and treated for 30 min with 1% (v/v) hydrogen
peroxide in methanol to remove intrinsic peroxidase activity. After
rehydration, sections were incubated with anti-HS antibodies, which
were detected by incubation with mouse anti-c-Myc tag antibodies (9E10,
culture supernatant), followed by peroxidase-conjugated rabbit
anti-mouse IgG (1:100 in PBS containing 1% (w/v) BSA) for 90 min.
Sites of peroxidase activity were revealed using diaminobenzidine as a substrate. Sections were counterstained with Harris's hematoxylin. The
same controls were used as described under immunofluorescence.
Evaluation of Specificity by Immunofluorescence
Cryosections were pretreated with heparinase III (digests HS;
0.02 units/ml; 50 mM NaAc/50 mM
Ca(Ac)2, pH 7.0), and with chondroitinase ABC (digests
dermatan and chondroitin sulfate; 1 unit/ml; 25 mM Tris/HCl, pH 8.0) for 30 min at 37 °C. As control, sections were incubated in the reaction buffer without enzyme. After rinsing three
times with PBS and blocking with PBS/1% BSA, sections were incubated
with antibodies and processed for immunofluorescence as described. The
efficacy of chondroitinase ABC treatment was evaluated by incubation of
sections with antibodies against chondroitin sulfate "stubs,"
generated by chondroitinase ABC (Ab 2B6 from Seikagaku Kogyo Co.,
Tokyo, Japan)
Specificity of the anti-HS antibodies was further tested by
preincubation of the antibodies with various molecules, viz.
HS (bovine kidney), heparin, dermatan sulfate, chondroitin 4- and 6-sulfate, keratan sulfate, dextran sulfate, hyaluronate, and DNA.
Incubation was for 16 h at 22 °C at concentrations of 0.5-1.0 mg/ml periplasmatic fraction. Processing for immunofluorescence on
sections was as above.
Inhibition of Antibody Binding by bFGF
To study whether the HS epitope recognized by the antibodies is
involved in the binding of bFGF, 100 µl of a solution containing bFGF
(1.7 µg/ml TBS containing 0.1% (w/v) Tween-20 and 1% (w/v) BSA) was
applied for 8 h at 22 °C to wells of polystyrene microtiter plates previously coated with HS from bovine kidney. After washing, anti-HS antibodies (0.04 µg of protein/ml) were applied for 16 h
at 22 °C, and bound antibodies were detected as described for ELISA.
Alternatively, cryosections of rat kidney were incubated for 16 h
with bFGF (20 µg/ml PBS/1% (w/v) BSA) and, after washing, incubated
for 90 min with anti-HS antibodies. Detection of bound antibodies was
as described for immunofluorescence.
 |
RESULTS |
Selection of Anti-heparan Sulfate Antibodies
After four rounds of panning, 19 clones expressing anti-HS
antibodies and containing full-length DNA inserts were isolated. The 19 clones included 3 unique clones expressing antibody HS4C3, HS4D10, and
HS3G8. The clone expressing antibody HS3G8 was obtained after
additional negative selection on chondroitin sulfate and dermatan
sulfate. Sequencing analysis revealed an amino acid sequence of the
VH complementarity-determining region 3 of GRRLKD for
antibody HS4C3, of SLRMNGCGAHQ for HS4D10, and of YYHYKVN for HS3G8.
The VH family and germ line segments are VH3
and DP-38 for HS4C3, VH3 and DP-42
for HS4D10, and VH1 and DP-8 for HS3G8
(nomenclature according to Ref. 27).
Characterization of Anti-heparan Sulfate Antibodies
Evaluation of Specificity and Affinity Using ELISA--
Antibodies
from all three clones reacted with HS from bovine kidney (the
"antigen") (Table I). No reactivity
was observed with chondroitin 4-sulfate, chondroitin 6-sulfate,
dermatan sulfate, keratan sulfate, dextran sulfate, hyaluronate, DNA,
BSA, Marvel, or polystyrene (data not shown). Major cross-reactivity
with heparin was observed for HS4C3 and HS3G8. All three antibodies
reacted differently with HS preparations isolated from different
organs, each displaying its own pattern (Table I). Antibody HS4C3
preferentially reacted with HS isolated from bovine intestine and with
fraction B of HS from bovine kidney, which eluted from an
anion-exchange column between 1.1 and 1.25 M NaCl. HS4D10
preferentially reacted with HS isolated from whale lung and with
fraction B of HS from bovine kidney. HS3G8 has a reactivity toward
different HS preparations that is comparable, but not identical, to
that of HS4C3. There appears to be no correlation between antibody
reactivity and the ratio of sulfate groups/disaccharide (Table I).
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Table I
Reactivity of anti-HS antibodies with various HS preparations and
heparin in ELISA
Culture media from bacteria expressing anti-HS antibody HS4C3, HS4D10,
and HS3G8 were incubated with various molecules immobilized on the
wells of microtiter plates. Bound antibodies were visualized using
mouse anti-c-myc IgG, followed by phosphatase-conjugated rabbit
anti-mouse IgG. In addition, the mouse anti-HS IgM antibodies JM403 and
10E4 were evaluated and visualized using alkaline phophatase-conjugated
goat anti-mouse IgM. The ratio of sulfate groups to disaccharide is
indicated in parentheses. Values (in % of reactivity with HS (bovine
kidney) represent mean ± S.D. (n = 4).
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To elucidate some chemical aspects of the epitope, reactivity of the
antibodies was evaluated with chemically/enzymatically treated HS and
heparin preparations using an inhibition ELISA. Reactivity with
antibodies HS4C3 and HS3G8 was abolished when HS or heparin was
completely desulfated and N-acetylated, completely desulfated and N-sulfated, or N-desulfated and
N-acetylated (Fig. 1).
Treatment of HS from bovine kidney with heparinase I, II, or III or
with nitrous acid at pH 1.5 also destroys reactivity with these
antibodies (data not shown). Antibody HS4D10 could not be analyzed in
the inhibition ELISA, probably due to the low amount of HS species
containing the epitope in the kidney HS preparation used.

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Fig. 1.
Inhibition immunoassay to identify certain
chemical modifications influencing the epitope recognized by antibody
HS4C3. Antibodies were incubated with the test molecule and then
transferred to wells previously coated with HS (bovine kidney). Bound
antibodies were detected using anti-c-Myc tag mouse monoclonal antibody
9E10 and alkaline phosphatase-conjugated rabbit anti-mouse IgG
antibody. Enzyme activity was detected using p-nitrophenyl
phosphate as the substrate, and absorbance was read at 405 nm. Test
molecules were heparin ( ), desulfated/N-acetylated
heparin ( ), O-desulfated/N-sulfated heparin
( ), N-desulfated/N-acetylated heparin ( ), and heparan
sulfate (from bovine kidney) (+). Similarly modified HS preparations
reacted similarly to the modified heparins (data not shown). Antibody
HS3G8 reacted similarly to HS4C3. Bars indicate S.D.
(n = 3).
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Mouse monoclonal IgM JM403 reacted primarily with HS from bovine and
human aorta and from bovine lung. Chemical modification of HS or
heparin destroyed immunoreactivity, as did heparinase II and III.
Heparinase I, however, did not eliminate the reactivity of HS with
JM403. These data are in accordance with published data (28). Mouse
monoclonal 10E4 reacted strongly with HS from human aorta and bovine
intestine.
The dissociation constants (Kd) of antibody HS4C3
and HS3G8 for bovine kidney HS, as deduced from Klotz plots, are 0.12 and 0.15 µM, respectively. No value could be obtained for HS4D10.
Evaluation of Specificity Using Immunohistochemistry--
Anti-HS
antibodies stained basement membranes in rat kidney with different
specificity (Fig. 2, Table
II). Antibody HS4C3 predominantly stained
basement membranes of the glomerulus and of peritubular capillaries,
whereas HS4D10 reacted mainly with HS present in basement membranes of
tubules and of smooth muscle cells. HS3G8 had a rather promiscuous
staining behavior: it reacted with most basement membranes in kidney.
Mouse monoclonal antibody JM403 preferentially stained basement
membranes of the glomerulus and of smooth muscle cells, whereas mouse
monoclonal antibody 10E4 reacted mainly with Bowman's capsule and
basement membranes of smooth muscle cells. Staining with HS3G8 (Fig.
3), HS4C3, and HS4D10 (not shown) was
abolished by treatment of sections with heparinase III but not with
chondroitinase ABC. Staining could be precluded by preincubation of
antibodies with HS and heparin but not with hyaluronate, dermatan
sulfate, chondroitin 4- and 6-sulfate, keratan sulfate, dextran
sulfate, and DNA. Antibodies could also be used on paraffin sections
(Fig. 4). Antibody HS4C3 reacted strongly
with heparin-containing granules in mast cells (Fig.
4b).

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Fig. 2.
Immunostaining of rat kidney with anti-HS
antibodies. Cryosections were incubated with periplasmatic
fractions of bacteria expressing anti-HS antibody HS4C3 (a),
HS4D10 (b), HS3G8 (c), and anti-filaggrin
(control) (d). Bound antibodies were visualized using mouse
anti-c-Myc IgG followed by FITC-conjugated goat anti-mouse IgG. In
addition, the mouse anti-HS IgM JM403 (e) and 10E4
(f) were evaluated and visualized using FITC-conjugated goat
anti-mouse IgM. Bar, 25 µm. G, glomerulus.
Arrow in a, peritubular capillary;
arrow in f, smooth muscle cells.
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Table II
Immunofluorescence pattern of rat kidney by anti-HS antibodies
Periplasmatic fractions of bacteria expressing anti-HS antibodies
HS4C3, HS4D10, and HS3G8 were applied to cryosections of rat kidney.
Bound antibodies were visualized by incubation with mouse anti-c-myc
IgG followed by FITC-conjugated goat anti-mouse IgG. Staining: ++,
strong; +, moderate; +/ , weak; , absent.
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Fig. 3.
Specificity of anti-HS antibody HS3G8.
Prior to incubation with HS3G8 (periplasmatic fraction), rat kidney
cryosections were treated with heparitinase (a),
heparitinase incubation buffer (b), chondroitinase ABC
(c), and chondroitinase ABC incubation buffer
(d). Bound antibodies were visualized using mouse anti-c-Myc
IgG followed by FITC-conjugated goat anti-mouse IgG. Bar, 50 µm.
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Fig. 4.
Application of anti-HS antibodies on paraffin
sections. a, section of human kidney incubated with antibody
HS4D10 (periplasmatic fraction) (bar, 100 µm).
b, section of human uterine myometrium incubated with
antibody HS4C3 (periplasmatic fraction) (bar, 50 µm). Note
the strong immunoreactivity of mast cells due to the cross reactivity
of the antibody with heparin. Bound antibodies were visualized using
mouse anti-c-Myc IgG, followed by peroxidase-conjugated rabbit
anti-mouse IgG.
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Inhibition of Antibody Binding by bFGF
Preincubation of HS-coated wells with bFGF partially inhibits the
binding of all anti-HS antibodies obtained by phage display. Binding of
antibody HS4C3 was blocked by 59 ± 2% (mean and range) (n = 2), of antibody HS3G8 by 64 ± 1%, and of
HS4D10 by 92 ± 2%. bFGF had no influence on the binding of mouse
JM403 antibody to HS (0 ± 2%). Preincubation of rat kidney
sections with bFGF largely precluded binding to HS by the three
antibodies obtained by phage display (Fig.
5). No effect was observed for mouse
monoclonal antibody JM403.

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Fig. 5.
Inhibition of HS staining of kidney sections
by preincubation with bFGF. Rat kidney cryosections were
preincubated with 1% BSA in PBS without (a-c) or with
(a'-c') bFGF, followed by incubation with periplasmatic
fractions of bacteria expressing anti-HS antibody HS4C3 (a
and a'), HS4D10 (b and b'), or with
mouse anti-HS IgM JM403 (c and c'). Phage
display-derived antibodies were visualized using mouse anti-c-Myc IgG
followed by FITC-conjugated goat anti-mouse IgG. JM403 was detected
using FITC-conjugated goat anti-mouse IgM antibodies. Bar,
50 µm.
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DISCUSSION |
We describe the generation of three anti-HS antibodies from a
semisynthetic phage display library. To our knowledge, this is the
first time this technique has been successfully used for the direct
generation of antibodies against (poly)saccharides. All three
antibodies recognize different types of HS molecules, as indicated by
their different staining pattern and different reactivity toward
various HS preparations. HS biosynthesis starts with the formation of a
precursor polysaccharide heparosan, consisting of a glucuronic
acid-N-acetylglucosamine polymer, which is then subjected to
a number of modifications (4). All three antibodies generated by phage
display technology did not react with the bacterial K5 polysaccharide,
which has the same structure as heparosan. This indicates that the
epitopes involved represent additional modifications. Modification of
the HS precursor polysaccharide starts with N-deacetylation
and N-sulfation of N-acetylglucosamine residues,
followed by epimerization of glucuronic acid to L-iduronic acid (IdoA) residues and O-sulfation at various positions,
including C-2 of the IdoA residues and C6 of GlcN residues (4). The
modification reactions are incomplete, so not all disaccharides are
similarly modified. In addition, less frequent modifications have been
reported, including sulfation at C2 and C3 of glucuronic acid residues, sulfation of C3 of GlcN residues, and the presence of an unsubstituted amino group on GlcN. At least 19 different hexuronic acid-GlcN and 12 GlcN-hexuronic acid disaccharides have been identified in HS/heparin
(29). Because an HS chain may be composed of over a 100 disaccharides,
a large potential of different HS species can be generated. This may
form the basis of the specific reactivity of HS toward a vast array of
proteins, including cytokines and growth factors.
Sulfation as such is not sufficient to create the epitope recognized by
the phage display antibodies, because dextran, chondroitin, and
dermatan sulfate are not immunoreactive. Clearly, some specific modification(s) give rise to epitope formation. Antibody HS4C3 reacted
well with HS from bovine intestine and with heparin but poorly with HS
from bovine aorta. The former molecules are characterized by a
relatively high amount of N-sulfation and a high number of contiguous but a low number of spaced N-sulfated
disaccharides (14). HS from bovine aorta has the opposite
characteristics. This indicates that in the HS4C3 epitope heparin-like
sequences, characterized by disaccharides formed by
6-O-sulfated GlcNSO3 and 2-O-sulfated
IdoA, are of importance. The importance of sulfation is also indicated
by the loss of epitope caused by chemical modifications of HS and
heparin preparations. Presence of O-sulfation, as well as
N-sulfation, seems to be necessary for antibody-HS/heparin interaction. However, because heparinase III, which cleaves HS at sites
of glucuronic acid rather than IdoA residues, abolishes immunoreactivity, other modifications are involved. It should be
stressed here that the HS preparations used herein were not pure, but
were mixtures of various HS species.
Antibody HS3G8 has a reactivity toward HS preparations, including
chemically/enzymatically modified HS/heparin, that resembles that of
HS4C3. It shows, however, a more promiscuous staining behavior. For
instance, HS3G8 reacted with HS from the renal peritubular basement
membranes, which is unreactive toward HS4C3. This indicates that the
epitopes for both antibodies are different, but share similarity. Thus,
the antibodies may discriminate between minor structural differences,
not easily detected by other means. Antibody HS4D10 is quite different
from HS4C3 both in immunostaining and in reactivity toward HS
preparations. It reacted well with HS from bovine kidney and with HS
from whale lung. It reacted poorly with HS from bovine intestine and
with heparin, both of which were highly reactive with HS4C3. HS4D10
could not be analyzed in an inhibition ELISA, precluding identification
of chemical modifications in HS important in epitope formation. In
general, epitope mapping of glycosaminoglycans poses a major
challenge.
The antibodies were different with respect to their DP number (27) and
the sequence of the VH complementarity-determining region
3. The sequence GRRLKD of antibody HS4C3 fits into the glycosaminoglycan binding site XBBXBX
(B, basic amino acid residue; X, any amino acid residue),
which has been found in a subset of heparin-binding proteins (30). The
sequence SLRMNGCGAHQ for HS4D10 resembles another motif observed in a
number of heparin-binding proteins, in which two basic amino acid
residues are at the extreme end of the motif (31). However, the
sequence YYHYKVN for HS3G8 does not fit into any of the proposed
sequences for heparin binding, and clearly, a linear motif is not a
prerequisite for heparin binding.
All three antibodies reacted differently from the described monoclonal
IgM antibodies JM403 and 10E4. All five antibodies have their own
reactivity toward various HS preparations and display an unique
staining pattern in the kidney. This indicates the presence of at least
five different HS species in kidney.
Binding and modulation of bFGF is a well defined characteristic of HS,
including kidney HS (4, 32-34). On the part of HS, 2-O-sulfated IdoA residues promote binding, whereas
6-O-sulfation of the GlcN residue seems inhibitory. This
would be in line with the almost complete blockage by bFGF of the
binding between HS and antibody HS4D10, which reacts poorly with
heparin in which 6-O-sulfated GlcN residues are prominent.
The other two phage display antibodies are much more reactive toward
heparin and are less efficiently blocked by bFGF. The epitope
recognized by mouse monoclonal antibody JM403, obtained by conventional
hybridoma techniques, does not bind bFGF. All epitopes recognized by
the phage display antibodies, however, do bind bFGF. Because binding of
bFGF to HS seems to be a general characteristic of HS, this indicates
that the antibody phage display technique may generate antibodies
directed to more common epitopes on the HS molecule, whereas those
obtained by hybridoma techniques may be directed to "rare"
epitopes. The more restricted staining of kidney basement membranes by
the mouse monoclonal antibodies JM403 and 10E4 support this idea.
In conclusion, phage display technology presents a powerful technique
to generate antibodies specific for HS epitopes. The availability of
specific antibodies and their coding DNAs may be instrumental to
further explore the realm of HS and of glycosaminoglycans in general,
as is indicated for kidney in this study.
 |
ACKNOWLEDGEMENTS |
We express our gratitude to Dr G. Winter,
Cambridge University (Cambridge, United Kingdom), for providing the
phage display library; Dr. U. Lindahl, University of Uppsala (Uppsala,
Sweden), for providing HS preparations and for stimulating discussions; Drs. J. van den Born and J. Berden, Dept. of Nephrology, University of Nijmegen, The Netherlands, for providing the JM403 antibody and
sections of human kidney; and Dr. A. Benders for helpful suggestions for affinity measurements.
 |
FOOTNOTES |
*
This study was supported in part by Grant C 96.1587 from
the Dutch Kidney Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 31-24-3616759;
Fax.: 31-24-3540525; E-mail: A.vankuppevelt{at}bioch.kun.nl.
Supported by The Netherlands Foundation for Chemical Research
(Scheikundis Ohderzoek Nederland) with financial aid from The Netherlands Technology Foundation.
1
The abbreviations used are: HS, heparan sulfate;
IdoA, iduronic acid; Kd, dissociation constant; PBS,
phosphate-buffered saline; 2xTY/glu/amp, 2xTY medium containing 1%
(w/v) glucose and 100 µg ampicillin/ml; bFGF, basic fibroblast growth
factor; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum
albumin; FITC, fluorescein isothiocyanate.
 |
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