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Antibodies binding the ADAM10 substrate recognitiondomain
inhibit Eph function
Lakmali Atapattu1, Nayanendu Saha2, Carmen Llerena1, Mary E.
Vail1, Andrew M. Scott3, Dimitar B. Nikolov2,Martin Lackmann1,*,`
and Peter W. Janes1,*1Department of Biochemistry and Molecular
Biology, Monash University, Victoria 3800, Australia2Structural
Biology Program, Memorial Sloan-Kettering Cancer Center, New York,
NY 10065, USA3Tumour Targeting Program, Ludwig Institute for Cancer
Research, Austin Hospital, Heidelberg, Victoria 3084, Australia
*These authors contributed equally to this work`Author for
correspondence ([email protected])
Accepted 13 October 2012Journal of Cell Science 125, 6084–6093�
2012. Published by The Company of Biologists Ltddoi:
10.1242/jcs.112631
SummaryThe ADAM10 transmembrane metalloprotease cleaves a
variety of cell surface proteins that are important in disease,
including ligandsfor receptor tyrosine kinases of the erbB and Eph
families. ADAM10-mediated cleavage of ephrins, the ligands for Eph
receptors, issuggested to control Eph/ephrin-mediated cell-cell
adhesion and segregation, important during normal developmental
processes, andimplicated in tumour neo-angiogenesis and metastasis.
We previously identified a substrate-binding pocket in the ADAM10 C
domain
that binds the EphA/ephrin-A complex thereby regulating ephrin
cleavage. We have now generated monoclonal antibodies
specificallyrecognising this region of ADAM10, which inhibit ephrin
cleavage and Eph/ephrin-mediated cell function, including
ephrin-inducedEph receptor internalisation, phosphorylation and
Eph-mediated cell segregation. Our studies confirm the important
role of ADAM10 in
cell-cell interactions mediated by both A- and B-type Eph
receptors, and suggest antibodies against the ADAM10
substrate-recognitionpocket as promising therapeutic agents, acting
by inhibiting cleavage of ephrins and potentially other ADAM10
substrates.
Key words: ADAM metalloprotease, Eph receptor, Ephrin cleavage,
Cell-cell adhesion
IntroductionProteolytic release, or ‘shedding’, of cell
surface-bound proteins
acts as an important post-translational switch that
regulates
protein function and activity. The ADAM (a disintegrin and
metalloprotease) family of transmembrane proteases are the
most
prominent shedding enzymes for membrane-anchored proteins.
ADAMs contain multiple extracellular domains, including a
distal
metalloprotease (MP) domain, followed by disintegrin (D)-
and
cysteine-rich (C) domains involved in substrate interaction, as
well
as transmembrane and variable cytoplasmic sequences (Blobel,
2005). They are important in regulating inflammatory and
growth
factor signalling, cell migration, and cell adhesion: in
particular,
two closely related, atypical ADAMs, ADAM10 (CD156C,
MADM, Kuzbanian) and 17 [CD156B, TACE (TNFa-convertingenzyme)],
shed ligands and/or receptors regulating key cytokine,
chemokine and growth factor signalling pathways important in
disease. These include erbB/EGF receptor family ligands and
receptors, Notch receptors and ligands, TNFa and TNFRI and
II,CX3CL1, IL-6R, as well as cadherins and various cellular
adhesion molecules (CAMs), and the amyloid precursor protein
(APP) (Murphy, 2008; Saftig and Reiss, 2011). ADAM10 and 17
are also overexpressed in a variety of cancers (Murphy,
2008;
Saftig and Reiss, 2011; Sanderson et al., 2006). Together
this
implies their important involvement in diseases such as
Alzheimer’s, chronic inflammatory and heart diseases, and
cancer.
ADAM10 also cleaves ligands for Eph receptors, the largest
family of receptor tyrosine kinases, which together with
their
membrane-bound ephrin ligands, control cell migration and
positioning during normal and oncogenic development
(Nievergall et al., 2012; Pasquale, 2010). In this context
ADAM10 association with A-type Eph receptors is promoted by
binding to their ephrin-A ligands on interacting cells (Janes et
al.,
2005; Salaita et al., 2010), whereupon ADAM10 cleaves
ephrin,
disrupting the Eph–ephrin tether between cells to allow de-
adhesion, or retraction (Hattori et al., 2000; Janes et al.,
2005). This
function of ADAM10 is further regulated by kinase activity
(Blobel, 2005; Hattori et al., 2000), which we found to be
mediated
through conformational changes in the Eph cytoplasmic domain
(Janes et al., 2009), such that ADAM10 acts as a switch
between
cell-cell adhesion and segregation in response to Eph
phosphorylation levels. This switch is thought to be
important
for Eph-dependent oncogenesis, where aberrant Eph receptor
expression and/or mutation contributes to tumour development
by
promoting neo-angiogenesis, invasion and metastasis
(Nievergall
et al., 2012; Pasquale, 2010). Interestingly, while
EphB/ephrin-B
cell contacts were reported to be attenuated through
protease-
independent trans-endocytosis (Marston et al., 2003; Zimmer et
al.,
2003), ADAM10 was also recently found to be required for
EphB/
ephrin-B-dependent cell sorting, where EphB2 activation
triggers
ADAM10-mediated shedding also of E-cadherin (Solanas et al.,
2011).
Despite considerable efforts to develop ADAM metalloprotease
inhibitors, to date clinical trials based on compounds blocking
the
protease catalytic site have failed due to lack of efficacy
and
specificity (DasGupta et al., 2009; Moss et al., 2001; Saftig
and
Reiss, 2011). To a large extent, this reflects similarity of the
MP
6084 Research Article
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active site to matrix metalloproteases (MMPs) (Maskos et
al.,
1998), and the mechanism of ADAM substrate specificity,
which
does not rely on a typical cleavage signature recognised by
the
protease domain, but on non-catalytic interactions between
the
substrate and the ADAM C domain (Reddy et al., 2000; Smith
et al., 2002; White, 2003). We have previously used
structure/
function studies to identify a substrate-binding pocket within
the
ADAM10 C domain, which specifically recognises the
Eph/ephrin
complex and thereby specifies cleavage of Eph-bound ephrin
(Janes et al., 2005). We therefore set out to raise
monoclonal
antibodies (mAbs) against this region and assess their ability
to
block substrate cleavage. We now describe mAbs specific for
the
ADAM10 substrate-binding pocket, which inhibit ADAM10-
mediated ephrin cleavage, Eph activity and Eph-dependent
cell
behaviour.
ResultsGeneration of monoclonal antibodies recognising
ADAM10 in the context of Eph/ephrin signalling complexes
To generate mAbs that selectively bind the substrate
recognition
pocket within the C domain of the native ADAM10
extracellular
domain, we sequentially immunised and boosted mice with
ADAM10/EphA3+ve human embryonic kidney (HEK) 293 cells
and recombinant ADAM10 extracellular domain (ECD)
fragments, respectively. In particular, we used a protein
fragment spanning residues 214–646 of recombinant bovine
ADAM10 ECD (Janes et al., 2005), in keeping with the notion
that the lower homology to mouse sequences within the C
domain
(92.7%, compared to 94.8% homology for human; Fig. 1A), may
increase its immunogenicity and bias the mouse immune
response
against this domain. Screening of hybridoma cell lines
generated
from these mice, using ELISA and Plasmon Resonance analysis
to
test binding to recombinant bovine ADAM10 ECD, and
immunoprecipitation of endogenous human ADAM10 from
human embryonic kidney (HEK) 293 cell lysates, yielded three
monoclonal antibodies: 3A8 and 8C7, recognising bovine and
human ADAM10 (Fig. 1B,C), as well as 4G1, binding only to
bovine ADAM10 (Fig. 1B; supplementary material Fig. S1). 8C7
also bound to mouse ADAM10 present in lysates of wild-type
(Wt) mouse embryonic fibroblasts (MEFs), but not in
immunoprecipitates from ADAM2/2 MEFs (Hartmann et al.,2002),
indicating its specificity for ADAM10 (Fig. 1D).
We previously reported that association of EphA3 and ADAM10
is increased by ligand-induced receptor clustering (Janes et
al.,
2005), a finding since confirmed for other Eph receptors
(Salaita
et al., 2010; Solanas et al., 2011). To investigate if our mAbs
would
recognise cell surface ADAM10 in the context of associated
EphA3,
we analysed EphA3/HEK293 cells expressing abundant
endogenous
ADAM10 (above) by confocal microscopy. Staining of the cells
with Alexa647-conjugated 8C7 (Alexa647–8C7), when performed
on
ice to prevent endocytosis, was faint (Fig. 2A, top row),
consistent
with the reported tightly controlled levels of cell surface
ADAM10
(Marcello et al., 2010). Interestingly, incubation with 8C7 at
37 C̊
resulted in prominent punctuate staining and notable
internalisation
of stained complexes (Fig. 2A, 60 min, 2nd row), suggesting
antibody-induced clustering and its endocytosis together
with
ADAM10-containing protein complexes.
We next sought to test if the 8C7 antibody also recognises
ADAM10 that is associated with EphA3 during activation, by
incubating cells with 8C7 and with ephrin-A5-Fc, which when
cross-linked with anti (a)-human Fc antibodies causes
EphA3activation and internalisation (Lawrenson et al., 2002;
Vearing
et al., 2005). Again, we added the Alexa647–8C7 mAb and
Alexa488–ephrin-A5-Fc to cells on ice, in order to prevent
endocytosis and internalisation of ADAM10-bound 8C7 prior to
stimulation. Cells that had been fixed immediately (0 min,
ephrin-A5), and those treated for indicated times with
a-humanIgG at 37 C̊ to cross-link bound ephrin-A5-Fc, displayed
closely
matching 8C7 and ephrin-A5-staining patterns, suggesting
that
8C7 effectively binds to ADAM10 that is associated with Eph/
Fig. 1. Specificity of a-ADAM10 monoclonal antibodies. (A)
Alignment of mouse, human and bovine ADAM10 cysteine-rich domain
sequences (AA 551–
646). In the human and bovine sequences, only residues not
homologous to mouse are shown. (B) Comparison of binding of mouse
hybridoma (fusion) and
isolated cell clone supernatants to serially diluted,
immobilised bovADAM10 ECD by ELISA. Binding of non-immunised mouse
serum (control) is shown for
comparison. (C) Binding of endogenous huADAM10 by a-ADAM10
hybridoma clones, or the R&D ADAM10 mAb 1427, was compared by
immunoprecipitation
from equivalent HEK293 cell lysates and western blotting with an
a-ADAM10 pAb; u, unprocessed; p, processed ADAM10. (D) The
specificity of 8C7 for
ADAM10 was tested by immunoprecipitation from lysates of ADAM10
knockout (2/2) and Wt (+/+) mouse embryonic fibroblasts (MEFs), and
a-ADAM10
pAb western blot.
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ephrin signalling complexes (Fig. 2B). This is particularly
evident after (cross-linked) ephrin-A5-Fc-mediated EphA3
clustering and endocytosis, revealing pronounced uptake of
8C7 into EphA3-containing endocytic vesicles (Fig. 2B, 15,
60 min).
Unique mAb specificity for the ADAM10 substrate-
recognition module
To assess the epitope specificity of our ADAM10 mAbs, we
tested their ability to recognise isolated human (hu)-ADAM10
protein fragments, corresponding either to the full length
ECD
(lacking the Pro domain), or the disintegrin and
cysteine-rich
domains, expressed together or separately. Western blot
analysis
revealed that both 3A8 and 8C7 recognise recombinant protein
fragments containing the cysteine-rich domain, but not the
isolated disintegrin domain alone (supplementary material
Fig.
S2A). In comparison, 8C7 does not bind the isolated D+C
domains from either ADAM17 or ADAM19 (supplementary
material Fig. S2B), confirming its specificity. Importantly,
BIAcore analysis verified binding of the isolated ADAM10 C
domain to 8C7 conjugated to the sensor surface, while in
comparison the R&D a-ADAM10 mAb (clone 1427) recognisesonly
the full length ECD, but not the isolated C domain (Table 1).
Interestingly, 8C7 seems to bind with six- to seven-fold
higher
affinity to the isolated C domain (,14 nM) as compared to
theentire ECD (,93 nM); this is mainly due to a substantiallyhigher
association rate, suggesting this interaction might be
conformation dependent, with the epitope partially masked in
the
full length molecule.
We next sought to test if our mAbs selectively target the
substrate-binding pocket within the ADAM10 C domain. We
previously identified three glutamic acid residues at
positions
573, 578 and 579 which contribute to the negative charge of
the
pocket and are essential for efficient binding of the
ephrin/Eph
complex, as shown by Glu/Ala substitution at these sites
(ADAM10 3E-A mutant) (Janes et al., 2005). We therefore
compared binding of aADAM10 mAbs to Wt huADAM10–GFPand the
ADAM10 3E-A mutant, by immunoprecipitation from
lysates of transfected ADAM102/2 MEFs. When compared to
the R&D a-ADAM10 mAb, which binds equally well to Wt
andmutant ADAM10 (supplementary material Fig. S3A), both 8C7
and 3A8 showed significantly attenuated recognition of the
substrate-binding pocket mutant, indicating their specificity
for
the Wt form of this region (Fig. 3B). Similarly, Ala
substitutions
at residues 617 and 618, which are part of the lip of the
binding
pocket (Fig. 3A), also decreased binding of our mAbs, while
8C7
binding to an unrelated, catalytic domain ADAM10 mutant with
a Glu384 to Ala substitution was equivalent to the Wt
protein
(supplementary material Fig. S3B).
Fig. 2. Co-staining of cells with ADAM10 mAb 8C7 and
ephrin-A5-Fc
reveals colocalisation and co-internalisation with EphA3. (A)
EphA3/
HEK293 cells were incubated on ice with Alexa647–8C7 mAb and
fixed for
imaging (0 min) or first allowed to warm to 37 C̊ for 60 min.
(B) Cells were
labelled with Alexa647–8C7 and with Alexa488–ephrin-A5-Fc and
fixed
immediately (0 min) or incubated at 37 C̊ with a-humanFc to
cluster ephrin-
A5-Fc for the indicated time periods before fixation. The insets
are enlarged
images of the regions within the dotted lines. Cells incubated
for 60 min with
Alexa488–ephrin-A5-Fc alone are shown as a control in the bottom
panels.
Scale bars: 25 mm.
Table 1. BIAcore affinity measurements of ADAM10 mAb binding to
immobilised human ADAM10 ECD or isolated C domain
Analysed protein
8C7 mAb R&D mAb 1427
KD (M) ka (M/s) kd (s21) x2 KD (M) ka (M/s) kd (s
21) x2
ADAM 10 ECD 9.361028 2.36104 2.261023 14.3 2.061028 1.66105
3.361023 31.9ADAM10 C domain 1.461028 4.66105 6.461023 16.1 No
binding
The shown affinity (rate) constants are for the interaction
between the recombinant soluble ADAM10 extracellular domain
proteins binding to BIAcore sensorchip surfaces conjugated with
mouse 8C7 or with R&D mAb 1427, determined using a linear
kinetic model provided in the BIAcore evaluation
software(vers.3.1).
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mAbs against the ADAM10 substrate recognition domain
block ephrin cleavage and Eph activation
These data, suggesting that our mAbs may specifically bind,
and
thereby block access to the substrate recognition motif
(ephrin/
Eph-binding pocket) of ADAM10, prompted us to test their
effect
on ADAM-mediated ephrin cleavage, and its uptake into Eph-
expressing HEK293 cells. For this we monitored cleavage of
ephrin from ephrin-A5-Fc-coated tissue culture plates by
HEK293 cells stably transfected with either EphA3 or with
EphB2, which also binds and is activated by ephrin-A5
(Himanen
et al., 2004). Cells that had been treated with increasing doses
of
8C7 were plated onto wells pre-coated with Alexa594-labelled
ephrin-A5-Fc. After 30 min the cells were recovered and
analysed for internalised Alexa594–ephrin-A5 by microscopy
(Fig. 4A). In similar experiments we analysed by flow
cytometry
ephrin shedding and uptake by cells treated with 8C7 alone,
or
with anti-mouse IgG cross-linked 8C7, with enhanced binding
avidity (Fig. 4B). Both image and flow analysis showed
robust
uptake of ephrin-A5 by the cells, and its significant, dose-
dependent inhibition by pre-incubation of cells with 8C7
mAb,
which was most pronounced with cross-linked 8C7.
To test this inhibition of ADAM10 in a physiologically more
relevant model, we assessed ephrin cleavage and uptake in
co-
cultures of EphA3/HEK293 cells and GFP–ephrin-A5/HEK293
cells (Janes et al., 2005), by monitoring co-localised
GFP–ephrin
within Cell-Tracker-Red-labelled EphA3 cells (Fig. 4C,
cont.,
arrows). Pre-incubation of cell cultures with 8C7
significantly
inhibited ephrin uptake. Despite a more modest effect,
likely
reflecting the lesser sensitivity of this assay, inhibition by
8C7
was significantly stronger than that achieved by treatment
with
the broad MMP/ADAM metalloprotease inhibitors GM6001 or
TAPI1 (Fig. 4C, graph). Inhibition of cleavage was
particularly
evident in images in which the GFP-tagged ephrin persisted
at
cell-cell junctions, rather than being cleaved and internalised
by
the EphA3-expressing cells (Fig. 4C, 8C7, ).
Receptor endocytosis is an important modulator of
signalling,
not only in signal attenuation, but importantly by
facilitating
cytosolic signalling on endosomes acting as signalling
platforms
(Dobrowolski and De Robertis, 2012). We therefore tested the
effect of 8C7 treatment on Eph receptor tyrosine
phosphorylation
in co-cultures of EphA3- and ephrin-A5-expressing HEK293
cells.
Pre-incubation of the cells with increasing concentrations of
8C7
caused a reproducible, dose-dependent decrease in EphA3
phosphorylation (Fig. 5), particularly at later time points
(.10 min), consistent with its likely effects on
receptorendocytosis. In contrast, 8C7 treatment had no effect
on
activation of EphA3 by soluble clustered ephrin-A5-Fc
(Fig. 5C), which causes EphA3 activation and internalisation
independent of ADAM activity, in keeping with 8C7 acting via
inhibition of ephrin cleavage.
a-ADAM10 mAb 8C7 blocks Eph/ephrin-mediated cell
repulsion and cell segregation
High or low levels of Eph kinase activity correlate with
cell-cell
segregation or adhesion, respectively (Holmberg et al., 2000;
Janes
et al., 2011; Nievergall et al., 2012; Poliakov et al., 2008).
Also,
ADAM10-mediated cleavage of ephrin (Hattori et al., 2000;
Janes
et al., 2005) and cadherins (Solanas et al., 2011) during
Eph/
ephrin-mediated cell-cell interactions suggests essential roles
in
severing the bond between cells that allows cell segregation
between the ephrin- and Eph-expressing populations to occur.
We
analysed effects of our ADAM10 mAbs specifically on cleavage
of
ephrins during Eph/ephrin-mediated cell repulsion, using a
modified stripe assay (Knöll et al., 2007; Walter et al.,
1987), in
which Eph-expressing HEK293 cells are plated onto coverslips
pre-coated with stripes of fluorescently labelled ephrin, in our
case
Alexa594–ephrinA5-Fc and EphB2/HEK293 cells. As a positive
control for Eph-dependent cell adhesion, we tested cells
expressing
a truncated, signalling defective, dominant negative mutant
EphB2
(DICD), which cannot segregate in response to ephrin (Janes et
al.,2011). EphB2/293 cells were found to actively avoid stripes
of
high ephrin density, with only 20% remaining on stripes
after
overnight incubation (Fig. 6A, top row). In comparison,
EphB2
DICD cells displayed marked adhesion to the stripes (Fig.
6A,bottom row), consistent with the Eph cytoplasmic domain
being
required for Eph-mediated cell repulsion (Janes et al.,
2011;
Fig. 3. Site-directed mutagenesis of the ADAM10
substrate-binding pocket disrupts mAb binding. (A) Structure of the
bovine ADAM10 D and C domains
showing the location of key residues targeted by site-directed
mutagenesis. (B) Comparison of aADAM10 mAb binding to Wt and
substrate-binding pocket
mutant huADAM10. Alanine substitutions at Glu 573, 578 and 579
(3EA) or at residues 617 and 618 (617AA) were made in huADAM10-GFP,
and Wt and
mutant constructs were transfected into ADAM102/2 MEFs (control:
untransfected). Binding of a-ADAM10 mAbs was assessed by
immunoprecipitation from
equivalent cell lysates, and western blotting with a-ADAM10 pAb
(non-relevant lanes removed; the altered molecular mass pattern
reflects the GFP-tagged
huADAM10). The graph shows binding of 8C7 and 3A8 relative to
the R&D mAb, determined by densitometry (one-way ANOVA;
**P,0.01 compared to R&D
sample; n.s., not significant; n53).
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Mellitzer et al., 1999) and validating the assay. Interestingly,
pre-
treatment of EphB2/293 cells with increasing concentrations
of
8C7 caused significant, dose-dependent inhibition of
repulsion
from ephrinA5-Fc stripes, leading to a more random
distribution,
similar to the effect of the metalloprotease inhibitor
GM6001
(Fig. 6A,B), confirming specific ADAM10 inhibition as
effective
in blocking Eph-mediated repulsion. We confirmed that,
underlying this inhibition of cell retraction, 8C7 inhibits
EphB2
phosphorylation that is induced with cell-expressed ephrin-A5
to
stimulate its kinase activity: as expected and consistent with
its
effects on ephrin-A5/EphB2-mediated repulsion, we observed
marked, dose-dependent inhibition of EphB2 phosphorylation
by
8C7 (Fig. 6C).
We also tested effects of 8C7-inhibited ADAM10 activity on
the
segregation, or sorting, between Eph- and ephrin-expressing
cells,
a process previously shown to be dependent on Eph signalling
Fig. 4. 8C7 a-ADAM mAb blocks ephrin shedding and
internalisation. (A) Ephrin internalisation from
Alexa594–ephrin-A5-coated tissue culture surfaces.
EphA3/293 cells were pre-incubated with 0, 100 or 400 mg/ml 8C7
for 1 h before plating onto Alexa594–ephrin-A5-Fc-coated tissue
culture surfaces. Cells weredetached and plated onto
fibronectin-coated glass coverslips for fluorescence microscopy.
Representative images from two independent experiments are
shown,
and internalised ephrin fluorescence/cell quantified from a
minimum of 10 fields of view, using ImageJ software. (B) Cells
treated with non-clustered or pre-
clustered (X-lk) 8C7 at the indicated concentrations were plated
onto Alexa594–ephrin-A5-Fc as in A and detached cells were analysed
by flow cytometry.
Histograms of the different cell populations show the mean and
s.e.m. (normalised to the non-treated sample with a value of 1)
from three independent
experiments. (C) Ephrin internalisation of GFP–ephrin-A5 cells.
GFP–ephrin-A5/293 cells and Cell-Tracker-red-labelled EphA3/293
cells were pre-incubated
with 0, 100, 200 or 400 mg/ml 8C7 mAb, or with metalloprotease
inhibitors GM6001 (GM; 50 mM) or TAPI1 (50 mM), then co-cultured
for 1 h before fixationand nuclear staining with Hoechst (blue).
Images were taken by confocal microscopy; examples are shown from
control and treated cultures (8C7, 400 mg/ml).Arrows: internalised
GFP–ephrin in red-labelled EphA3 cells, bar ( ) indicates blockade
of ephrin cleavage at cell-cell junctions. Ephrin internalisation
was
calculated using colocalisation of green (ephrin) with red
(Eph/293); values are means 6 s.e.m., n55. *P,0.05, ***P,0.001:
significant difference from the
untreated or indicated sample. Scale bars: 20 mm.
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(Poliakov et al., 2008) and on ADAM10 (Solanas et al., 2011).
In
co-cultures EphB2/293 cells, co-expressing membrane-targeted
GFP, were found to segregate from ephrin-A5/293 cells, such
that
the GFP-labelled EphB2 cells formed tight clusters of
increased
fluorescence intensity compared to mono-cultures, as
previously
observed when co-cultured with ephrin-B1/293 cells (Janes et
al.,
2011; Poliakov et al., 2004). Co-incubation with 8C7 alone,
or
cross-linked with anti-mouse antibody, clearly affected this
segregation process, reducing the number of bright
segregated
colonies, as determined by image analysis of
intensity-thresholded
images (Fig. 7A–C). Inhibition was most pronounced using
cross-
linked 8C7, consistent with its greater inhibition of ephrin
cleavage
(Fig. 4), and comparable to effects of the less selective
inhibitor
TAPI1. In separate experiments, cross-linked 8C7 treatment
also
dose-dependently inhibited segregation of EphB2- and
ephrin-B1-
293 cells (Fig. 7D), and segregation of U251 glioma and
ephrin-A-
expressing HEK293 cells (Fig. 7E), a co-culture model we
previously used as a cancer relevant model of Eph/ephrin-A-
dependent cell sorting (Nievergall et al., 2010).
DiscussionInhibitors of ADAM10 and its closest relative ADAM17
(TACE)
have been actively sought over the last decade. While inhibition
of
TNFa shedding by TACE was the initial target for treatment
ofinflammatory diseases, including rheumatoid arthritis, the role
of
both ADAM10 and 17 in shedding a range of disease relevant
targets has broadened interest in raising ADAM-targeted
therapeutics. This is particularly true in cancer, in which
growth
factor/RTK signalling is frequently de-regulated, and where
ADAM-mediated shedding of ligands for the EGF receptor
(erbB, HER) family underlies autocrine feedback and
transactivation of EGFR signalling by stress and G-protein-
coupled receptor signalling (Blobel, 2005; Fischer et al.,
2003).
ADAM10- and -17-mediated cleavage of HER2 and HER4,
respectively, also contribute to their signalling and
underminereceptor-targeted therapies (Liu et al., 2006; Rio et al.,
2000; Sardi
et al., 2006). In addition, ADAM10-mediated shedding
regulates
cell-cell interactions important for tumour development and
invasion, in particular via targeting cadherins (Solanas et
al.,2011) and ephrins (Hattori et al., 2000), which modulate
cell-cell
adhesion and segregation during multiple stages of cancer
development (Nievergall et al., 2012; Pasquale, 2010).
Despite this interest, and the validation of ADAM inhibition
in
vitro, no inhibitors targeting the metalloprotease catalytic
site
have yet successfully passed phase II clinical trials, due to
lack of
specificity, stemming from the structural similarity between
theproteolytic cleft of ADAMs and matrix metalloproteases
(MMPs)
(DasGupta et al., 2009; Saftig and Reiss, 2011). However,
the
Fig. 5. ADAM10 mAb 8C7 inhibits EphA3 phosphorylation in
response to
stimulation by cell-bound ephrin. (A) 293/EphA3 cells were
pretreated with
0, 10 and 100 mg/ml of 8C7 mAb for 2 h and stimulated for the
indicated times.a-EphA3 immunoprecipitates from the cell lysates
were analysed by western
blot with a-phosphotyrosine (pY) and a-EphA3 antibodies as
indicated. A
representative image from four experiments is shown. (B)
EphA3
phosphorylation relative to EphA3 protein levels was calculated
from replicate
experiments as described in A, using densitometry analysis.
Graph shows
means 6 s.e.m., n54. (C) 8C7 does not inhibit EphA3
phosphorylation induced
by soluble clustered ephrin-A5. EphA3/293 cells, pre-incubated
with or without
8C7 (100 mg/ml) for 2 hours, were stimulated for 20 min with
pre-clusteredephrin-A5-Fc, or left unstimulated, as indicated.
EphA3 immunoprecipitates
from cell lysates were analysed by western blotting as in A.
Fig. 6. ADAM10 mAb 8C7 blocks Eph/ephrin-mediated cell
repulsion.
(A) EphB2/HEK293 cells labelled with Cell Tracker Green were
pre-treated
with vehicle (Cont), 8C7 (50, 200 or 400 mg/ml), or with GM6001
(GM,50 mM), and plated onto coverslips pre-coated with fibronectin
and stripes ofalexa594-labelled ephrin-A5-Fc. As a comparison,
cells expressing a
signalling-deficient EphB2 mutant (DICD) were also used. After
18 hours the
cells were imaged by fluorescence microscopy, from which
examples are
shown (8C7, 400 mg/ml). Scale bar: 250 mm. (B) The percentage of
cellsadhering to ephrin stripes was calculated from ,20 images for
eachtreatment; the graph shows the averages 6 s.e.m. from three
experiments.
(C) 8C7 inhibits ephrin-A5-induced EphB2 phosphorylation.
Effects of 8C7
treatment on activation of EphB2/HEK293 cells by
ephrin-A5/HEK293 cells
was assessed as in Fig. 5A, following stimulating for 40
minutes.
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specificity of ADAM function is mediated via
enzyme-substrateinteractions outside the protease domain, within
the Cys-rich
domain, and we previously elucidated the structure of theADAM10
Cys-rich domain and identified a binding pocket thatconfers
selectivity for the EphA/ephrin-A complex formed at
cell-cell junctions (Janes et al., 2005). We thus surmised
thatantibodies raised against this pocket might potentially
blocksubstrate binding and more specifically inhibit
ADAM10-mediated proteolysis.
We now describe a-ADAM10 mAbs, which recognise the Cdomain and
appear to specifically target the substrate-bindingpocket, as
suggested by loss of binding following mutagenesis of
key exposed residues within this region.
Interestingly,considerably higher affinity of these mAbs for the
isolated Cdomain compared to the full-length extracellular
domain,
principally due to a higher association rate, suggests
thebinding epitope may be masked in much of the full-lengthprotein,
and thus dependent on the conformation of the ADAM10ECD. Although
the structure of the full ADAM10 ECD is yet to
be solved, functional studies of the close relative
ADAM17suggest activity-dependent conformation changes (Wang et
al.,2009; Willems et al., 2010), and structures of related
snake
venom metalloproteases also suggest distinct open and
closedconformations that are suggested to regulate substrate access
tothe C domain (Guan et al., 2010), a notion which would be
consistent with the binding behaviour of our mAbs to this
region.
Our a-ADAM10 antibodies also show specific binding toADAM10 on
cells, where staining with 8C7 colocalises with
ephrin-A5-Fc-bound EphA3, consistent with the previousreported
interactions of ADAM10 with EphA receptors (Janeset al., 2005;
Salaita et al., 2010; Solanas et al., 2011). Moreover,
8C7 is effectively co-internalised following EphA3 receptor
stimulation with soluble ephrin, suggesting a close
functionalassociation of ADAM10 and EphA3 in cells and the
effective
targeting of this complex by 8C7. Importantly, assays
withrecombinant or cell-expressed ephrin show that 8C7 blocks
cleavage and internalisation of ephrin-/Eph complexes, but
alsoinhibits receptor phosphorylation and ensuing biological
responses. Thus, repulsion of EphB2-expressing HEK293 cellsfrom
stripes of immobilised ephrin-A5-Fc was inhibited, as was
segregation of cells expressing various combinations of A-
andB-type Ephs and ephrins. The inhibitory effect of 8C7 on
EphB2-
mediated ephrin shedding, cell repulsion and cell-cell
segregation
in our assays indicates that in addition to EphA/ephrin-A
clusters(Janes et al., 2005; Salaita et al., 2010), ADAM10 also
effectively
targets ephrins bound to EphB2: It confirms the
recently-reportedinteraction between ADAM10 and EphBs (Solanas et
al., 2011),
and demonstrates that 8C7 is effective in blocking the
ADAM10-facilitated function of both Eph subtypes.
Interestingly, 8C7 also inhibited Eph receptor phosphorylationin
EphA3- and EphB2-expressing HEK293 cells in response to
ephrin-A5/HEK293 cells, but did not affect EphA3 stimulationwith
soluble, pre-clustered ephrin-A5-Fc. We argue that rather
than directly affecting ephrin-induced Eph activation,
8C7inhibition of ADAM10 prevents ephrin cleavage and thus
endocytosis of ligated Ephs, resulting in persisting
Eph/ephrinclusters at cell-cell junctions (Fig. 4B), highly
reminiscent of
those observed in cells overexpressing dominant-negative
ADAM10 (Nievergall et al., 2010). However, the
increasedresidency at the cell membrane does not result in a
prolonged
activation of EphA3, but in agreement with the lower Ephactivity
commonly observed under conditions of Eph-mediated
Fig. 7. 8C7 blocks Eph/ephrin-mediated cell segregation.
(A–C) HEK293 cells expressing EphB2 and membrane-
targeted GFP were co-cultured with ephrin-A5/HEK293 cells
in the presence of 0.4 mg/ml 8C7, with or without
crosslinking
with anti-mouse IgG. Wells treated with anti-mouse IgG alone
or with TAPI1 served as negative and positive controls,
respectively. Confluent cultures were analysed by
fluorescence
microscopy for GFP, and Hoechst staining of nuclei to show
the total cell population, and representative images are
shown
in A. Segregated cell clusters were counted in whole well
images by counting areas of thresholded intensity above a
set
size equating to roughly 40–50 cells (Solanas et al., 2011)
(B), and mean numbers (n54) were calculated with standard
errors (C). (D) Segregation assay performed with EphB2–GFP-
and ephrin-B1-expressing HEK293 cells showing key
representative examples and quantification. (E) Segregation
assay performed with Cell-Tracker-Green-labelled EphA3/B2-
expressing U251 glioma cells and HEK293 cells. *P,0.05,
**P,0.01 relative to control (cont).
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cell-cell adhesion (Holmberg et al., 2000; Janes et al.,
2011;
Nievergall et al., 2012; Poliakov et al., 2008) leads to an
overall
reduced phosphorylation. In agreement, in other studies we
have also noted that inhibition of EphA3 endocytosis by
overexpression of the clathrin coat-associated protein AP180
decreases ligand-induced receptor phosphorylation (C.
Stegmeyer, P.W.J., M.L., unpublished data). The underlying
relationship between reduced EphA3 endocytosis and
phosphorylation is also consistent with the inhibition of
EphA3
endocytosis observed upon overexpression of the protein
tyrosine
phosphatase (PTP) 1B, which regulates EphA3 function and
trafficking by controlling its phosphorylation and kinase
activity
(Nievergall et al., 2010). Reduction of EphA3 phosphorylation
by
8C7 was particularly evident at later time points (after 10
min)
coincident with EphA3 endocytosis (Wimmer-Kleikamp et al.,
2004)
which would imply it has reduced tyrosine-dependent signalling
from
endosomes. This is in keeping with the notion that endosomes,
rather
than merely downregulating RTK signalling, compartmentalise
and
thus facilitate signals that are prevented at the plasma
membrane
(Dobrowolski and De Robertis, 2012; Scita and Di Fiore, 2010).
A
prominent mechanism, known to regulate endocytic signalling
of
several RTKs including receptors for EGF, PDGF and Insulin, is
via
RTK-initiated, localised production of reactive oxygen
species
(ROS), which accumulate in endosomes (Ushio-Fukai, 2009),
and
transiently inactivate regulatory PTPs to enhance RTK
phosphorylation and signalling activity (Oakley et al., 2009;
Rhee,
2006; Tiganis, 2011). While such a mechanism remains to be
confirmed for Ephs, similarities in the regulation of their
signalling
activity by regulatory PTPs and endocytosis (Nievergall et al.,
2012;
Pasquale, 2010) would suggest a similar concept controlling
Eph
signalling.
In conclusion, our novel monoclonal antibodies against
ADAM10, by targeting the substrate recognition pocket,
effectively inhibit its ability to cleave ephrin from cell
surfaces
and thus facilitate Eph function. Considering that ADAM10 has
a
variety of substrates in addition to ephrins, many of which
are
critically implicated in a range of chronic diseases,
including
inflammatory, heart and neurodegenerative diseases as well
as
cancer, it will be of considerable interest to examine the
efficacy
of our antibodies to inhibit shedding of these other ADAM10
substrates in cell and animal-based disease models.
Importantly,
the high specificity of our mAbs for ADAM10 will likely
provide
a significant advantage over current, less selective,
protease-
targeted inhibitors. We expect that highly specific
inhibition
of ADAM10 activity in pro-inflammatory and/or oncogenic
signalling pathways will provide a basis for the development
of
our antibodies as potential therapeutics.
Materials and MethodsExpression constructs
The sequences encoding the extracellular domain [amino acids
(AA) 214–646,lacking the prodomain] from bovine (Howard et al.,
1996) and human (OriGene)ADAM10, as well as isolated disintegrin
(AA 455–550) and cysteine-rich domains(AA 551–646), or D+C domains
combined (AA 455–646), were subcloned as Fcfusions into pcDNA3.1
vector (Invitrogen), including an N-terminal prolactinsignal
sequence and a C-terminal thrombin cleavage site followed by the
Fcdomain of human IgG, for expression in human embryonic kidney 293
(HEK293)cells. Following initial purification on
Protein-A–Sepharose (Amersham), the Fctag was removed by thrombin
cleavage, and ADAM10 proteins were furtherpurified to homogeneity
by gel filtration chromatography.
Point mutations to human ADAM10-turboGFP (OriGene) were
introduced bysite-directed mutagenesis (Quickchange XL,
Stratagene). Human ADAM10 3EAand 617AA mutants were generated by
introducing Alanines at G573,578,579, or atRH617,618,
respectively.
Generation of monoclonal antibodies against ADAM10
Mice were immunised with ADAM10/EphA3-expressing HEK293 cells,
followedby subsequent injections with bovine ADAM10 extracellular
domain and isolatedD+C fragments. Subsequently, B cells from mouse
spleens were fused withmyeloma cells in order to generate
hybridomas which were selected formonoclonal antibody production.
Hybridoma supernatants from pooled fusionsand from isolated clones
were collected and screened for binding to ADAM10using
enzyme-linked immunosorbant assay (ELISA), followed by
IP/westernblotting, immunofluorescence and surface plasmon
resonance (BIAcore) analysis,as described in the text. Antibodies
were purified from supernatants on Protein G.
Surface plasmon resonance
Analysis of protein interactions by surface plasmon resonance
was carried out on aBIAcore 3000 biosensor (BIAcore) as described
previously for other antigenantibody interactions (Lackmann et al.,
1996). Monoclonal antibodies wereimmobilized onto BIAcore CM5
sensorchips using NHS chemistry and binding ofADAM10 proteins,
diluted between 50 and 0.08 nM into running buffer (10 mMHEPES, pH
7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20), was performedon
sensor-chip surfaces derivatised on parallel channels with
non-relevant protein.The binding kinetics was derived from the
plasmon resonance sensorgrams aftersubtraction of baseline
responses (measured on the control channel) by globalanalysis using
the BIA Evaluation software (version 3.02, BIAcore). The surface
ofthe chip was regenerated after each injection of sample with 3 M
MgCl2, 0.075 MHEPES/NaOH, 25% ethylene glycol, pH 7.2, followed by
two washes with runningbuffer.
Cell lines and culture
HEK293 cells stably expressing, membrane-targeted GFP and EphB2,
orEphB2DICD, were kindly provided by David Wilkinson and Alexei
Poliakov(NIMR, London) and Claus Jorgensen (ICR, London) and were
previously described(Janes et al., 2011; Jørgensen et al., 2009;
Poliakov et al., 2008). EphA3/HEK293 cellswere also previously
described (Lawrenson et al., 2002). Mouse embryonic
fibroblasts(MEFs) from Wt or ADAM102/2 mice (Hartmann et al., 2002)
were kindly providedby Dr Saftig (Kiel). Cell lines were cultured
in DMEM supplemented with 10% fetalcalf serum and transiently
transfected using Fugene HD (Roche). Metalloproteaseinhibitors
GM6001 and TAPI1 were from Calbiochem.
Immunoprecipitation and western blotting
Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS
(Lawrenson et al.,2002) were immunoprecipitated with antibodies
against ADAM10 (R&D systemsmAb 1427, or mAbs 3A8 or 8C7) or
turboGFP (OriGene) followed by protein A–Sepharose, or EphA3 (mAb
IIIA4 (Lackmann et al., 1996) conjugated toMinileakTM beads).
Western blots were performed using rabbit polyclonalantibodies
against ADAM10 (Abcam pAb 39177), turboGFP (OriGene) or
PTyr(Invitrogen), or a-EphA3 sheep pAb (Nievergall et al., 2010) or
a-EphB2 mousemAb (R&D systems). Where indicated cells were
stimulated with 1.5 mg/mlephrin-A5-Fc [produced as described
previously (Lawrenson et al., 2002)] pre-clustered with 0.75 mg/ml
a-human IgG (Jackson Immunoresearch). Densitometricanalysis was
with ImageQuant software.
Microscopy
Antibodies or ephrin-A5-Fc proteins were coupled to Alexa dyes
(488, 594, 647;Invitrogen) as per the manufacturer’s instructions,
and incubated with cells for 1 houror as indicated followed by
washing with PBS and fixing with 4% PFA in PBS for30 mins at room
temperature. Samples were mounted in Moviol and
coverslipped.Confocal microscopy was on a Leica SP5 TCS SP5 II or
Nikon C1 confocalmicroscope equipped with 405, 488, 561 and 633 nm
lasers. Stripe and segregationassays were imaged on a Leica
AF6000LX Live Cell Imaging workstation. Imageswere processed using
Leica TCS, ImageJ and Adobe Photoshop and InDesign software.
Ephrin cleavage/internalisation assay
Alexa647–ephrin-A5-Fc was bound to 24-well tissue culture plates
(BD Falcon,USA) as follows: to increase binding capacity, plates
were pretreated withnitrocellulose dissolved in methanol (Lemmon et
al., 1989) under a laminar hood,followed by 100 mg/ml protein A/G
(Pierce) treatment for 1 hour at 37 C̊.Unbound protein A/G was
removed by two PBS washes. Alexa647–ephrin-A5-Fcwas bound to the
protein A/G layer at 5 mg/ml for 1 hour at 37 C̊ followed by twoPBS
washes.
293/EphA3 cells and EphB2/GFP 293 cells were treated in
suspension with 8C7at the indicated concentrations, with or without
pre-cross-linking with a-mouseIgG (Jackson Immunoresearch) at 4:1
ratio for 1.5 hours. They were then added tothe
Alexa647–ephrin-A5-Fc coated plates and incubated for 30 min at 37
C̊. Thecells adhered to the bottom of the plate were taken off with
two vigorous PBSwashes followed by another wash with 10 ml of PBS.
Cells were mounted on toslides for analysis by confocal microscopy
and fixed with 2% PFA/PBS for20 min, or fixed in suspension with 2%
PFA in FACS buffer (1% FCS, 1 mMEDTA in PBS) for flow cytometry.
Flow cytometry analysis was performed using
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BD LSR II flow cytometer (BD Biosciences) under HeNe laser (18
mW at633 nm). Further analysis was done on FlowJo 7.6.1 software
package (Tree StarInc., USA). Only single cells were gated for
expression of Alexa647–ephrin-A5-Fc.Histograms were generated for
every sample indicating the shift of fluorescence.Mean of the
fluorescence distribution was calculated using FlowJo.
For assaying ephrin cleavage from cells, 293/EphA3 cells were
stained with CellTracker Red (Invitrogen) as described in the
manufacturer’s protocol, allowed toadhere onto eight-well chamber
slides (BD falcon), and pretreated with vehicle, 8C7,GM6001 or
TAPI1 at the indicated concentrations. 293/ephrinA5 GFP cells
werethen added for 40 min at RT followed by fixing with 4% PFA, and
nuclear stainingwith DAPI. Cells were mounted and imaged using a
Nikon upright C1 confocalmicroscope and 1006 oil objective. Ephrin
internalisation was estimated from theMander’s coefficient of
colocalisation of green (ephrin) with red (EphA3 cells).
Stripe assay
A silicon matrix (obtained from Prof. Bastmeyer, Karlsruhe
Institute ofTechnology, Germany) with channels was placed on a
22622 mm glasscoverslip, and protein A/G (Pierce, USA) solution (20
mg/ml) resuspended inHanks Buffered Salt Solution (HBSS,
Invitrogen) was applied to the channels asdescribed (Knöll et al.,
2007). Coverslips were then coated with fibronectin(Sigma) at 10
mg/ml in PBS. EphrinA5-Fc labelled with Alexa594 (Invitrogen) at16
mg/ml in HBSS was added to the coverslips for 30 mins at 37 C̊ and
washedtwice in HBSS. EphB2/GFP 293 cells treated with or without
8C7 mAb antibodyor GM6001 at the indicated concentrations, or
EphB2DICD 293 cells pre-labelledwith Cell Tracker Green, were
incubated on the coverslips at 37 C̊ overnightbefore imaging by
fluorescence microscopy. To determine the percentage ofmigrated
cells in each image the number of cells on the stripes (counted
manually)was taken as a percentage of the total number of cells
(estimated using the imageanalysis software Cell-Profiler, Broad
Institute).
Cell segregation assays
EphB2-expressing HEK293 cells co-expressing membrane-targeted
GFP (Poliakovet al., 2008), or U251 cells labelled with Cell
Tracker Green (Invitrogen) were co-cultured with ephrin-expressing
HEK293 cells in 96-well plates seeded with20,000 cells per well,
for 48 h or until confluent. Cells were fixed and nucleistained
with Hoechst before imaging on a Leica AF6000LX
fluorescencemicroscope, using the tile scanning function to image
the entire well. Theimages were then analysed with ImageJ software
by thresholding areas offluorescence intensity above that seen in
monocultures. Cell clusters greater than aset area corresponding to
roughly 40–50 cells (Solanas et al., 2011) were thencounted by
particle count of thresholded areas. Each treatment was performed
onfour replicate wells, and repeated in three or more
experiments.
AcknowledgementsWe thank Momchil Kolev (Sloan-Kettering),
Chanley Chheang andJulien Robinson (Monash) for technical
assistance, Monash MicroImaging for microscopy assistance, and Kaye
Wycherley,Monoclonal Antibody Facility, The Walter and Eliza Hall
Instituteof Medical Research, for invaluable help with generating
monoclonalantibodies.
FundingThis work was supported by the National Health and
MedicalResearch Council of Australia [grant numbers 384242 to
M.L.,P.W.J. and D.B.N., 487922 to M.L., and R. D. Wright, and
SeniorResearch Fellowships to P.W.J. (334085) and M.L.
(384113)].Operational Infrastructure funding from the Victorian
Government[to A.M.S.]; National Institutes of Health [grant number
NS38486 toD.B.N.]; and the New York State Spinal Cord Injury
researchprogram [grant number C-022047 to N.S.].
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.112631/-/DC1
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