Introduction
Ephrin receptors, the largest family of receptor-tyrosine kinases, are subdivided in A- and B-class receptors. In mammals there are nine EphA receptors (EphA1-EphA8, EphA10) that interact with five ephrin-A ligands (ephrin-A1–ephrin-A5) and five EphB receptors (EphB1–EphB4, EphB6) that interact with three ephrin-B ligands (ephrin-B1–ephrin-B3) (
1- Pitulescu M.E.
- Adams R.H.
Eph/ephrin molecules: a hub for signaling and endocytosis.
). There is some interclass promiscuity, as EphA4 can also interact with ephrin-B ligands, whereas EphB2 also interacts with ephrin-A5 (
2- Bowden T.A.
- Aricescu A.R.
- Nettleship J.E.
- Siebold C.
- Rahman-Huq N.
- Owens R.J.
- Stuart D.I.
- Jones E.Y.
Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling.
,
3- Himanen J.P.
- Chumley M.J.
- Lackmann M.
- Li C.
- Barton W.A.
- Jeffrey P.D.
- Vearing C.
- Geleick D.
- Feldheim D.A.
- Boyd A.W.
- Henkemeyer M.
- Nikolov D.B.
Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling.
4- Qin H.
- Noberini R.
- Huan X.
- Shi J.
- Pasquale E.B.
- Song J.
Structural characterization of the EphA4-Ephrin-B2 complex reveals new features enabling Eph-ephrin binding promiscuity.
).
The ephrin system plays a major role in a variety of cell-cell interactions. In the developing nervous system it is pivotal as an axonal guidance system, whereas in the adult brain it is involved in synaptic plasticity and long-term potentiation (
5- Boyd A.W.
- Bartlett P.F.
- Lackmann M.
Therapeutic targeting of EPH receptors and their ligands.
,
6Eph-ephrin bidirectional signaling in physiology and disease.
). Importantly, the ephrin system also plays a role in cancer biology and in the pathogenesis of several neurological disorders (
5- Boyd A.W.
- Bartlett P.F.
- Lackmann M.
Therapeutic targeting of EPH receptors and their ligands.
). EphA4 is up-regulated in spinal cord injury, traumatic brain injury, and stroke, and blocking of the receptor increases functional recovery in models for these conditions (
7- Frugier T.
- Conquest A.
- McLean C.
- Currie P.
- Moses D.
- Goldshmit Y.
Expression and activation of EphA4 in the human brain after traumatic injury.
,
8- Goldshmit Y.
- Galea M.P.
- Wise G.
- Bartlett P.F.
- Turnley A.M.
Axonal regeneration and lack of astrocytic gliosis in EphA4-deficient mice.
9- Lemmens R.
- Jaspers T.
- Robberecht W.
- Thijs V.N.
Modifying expression of EphA4 and its downstream targets improves functional recovery after stroke.
). Interestingly, antagonism of EphA4 improves long-term potentiation defects in a mouse model for Alzheimer's disease and improves outcome in animal models for amyotrophic lateral sclerosis (ALS)
9The abbreviations used are:
ALS
amyotrophic lateral sclerosis
LBD
ligand-binding domain
Nb(s)
Nanobody(ies)
SPR
surface plasmon resonance
SH2
Src Homology 2
BBB
blood brain barrier
Bis-Tris
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
ANOVA
analysis of variance
RU
response units
EA
enzyme activator
E
embryonic day.
and stroke (
9- Lemmens R.
- Jaspers T.
- Robberecht W.
- Thijs V.N.
Modifying expression of EphA4 and its downstream targets improves functional recovery after stroke.
10- Fu A.K.
- Hung K.W.
- Huang H.
- Gu S.
- Shen Y.
- Cheng E.Y.
- Ip F.C.
- Huang X.
- Fu W.Y.
- Ip N.Y.
Blockade of EphA4 signaling ameliorates hippocampal synaptic dysfunctions in mouse models of Alzheimer's disease.
,
11- Van Hoecke A.
- Schoonaert L.
- Lemmens R.
- Timmers M.
- Staats K.A.
- Laird A.S.
- Peeters E.
- Philips T.
- Goris A.
- Dubois B.
- Andersen P.M.
- Al-Chalabi A.
- Thijs V.
- Turnley A.M.
- van Vught P.W.
- et al.
EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans.
12- Vargas L.M.
- Leal N.
- Estrada L.D.
- González A.
- Serrano F.
- Araya K.
- Gysling K.
- Inestrosa N.C.
- Pasquale E.B.
- Alvarez A.R.
EphA4 activation of c-Abl mediates synaptic loss and LTP blockade caused by amyloid-β oligomers.
). EphA4 expression is inversely correlated with survival time in ALS patients, which suggests that EphA4 is also involved in ALS human pathology (
11- Van Hoecke A.
- Schoonaert L.
- Lemmens R.
- Timmers M.
- Staats K.A.
- Laird A.S.
- Peeters E.
- Philips T.
- Goris A.
- Dubois B.
- Andersen P.M.
- Al-Chalabi A.
- Thijs V.
- Turnley A.M.
- van Vught P.W.
- et al.
EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans.
). These findings suggest that EphA4 inhibition may have the potential for the treatment of neurological disorders.
Inhibition of EphA4 signaling can be accomplished by targeting the ATP-binding pocket in the kinase domain or by blocking the interaction with ephrin ligands (
13- Noberini R.
- Lamberto I.
- Pasquale E.B.
Targeting Eph receptors with peptides and small molecules: progress and challenges.
,
14- Tognolini M.
- Hassan-Mohamed I.
- Giorgio C.
- Zanotti I.
- Lodola A.
Therapeutic perspectives of Eph-ephrin system modulation.
). Because the ATP-binding pocket is highly conserved among tyrosine kinases it is very difficult to develop selective inhibitors (
13- Noberini R.
- Lamberto I.
- Pasquale E.B.
Targeting Eph receptors with peptides and small molecules: progress and challenges.
). Alternatively targeting the ligand-binding domain (LBD) certainly allows the development of more selective compounds, but this poses several other problems. On one hand, the protein interaction surface to be covered is large; 900 Å
2 in the case of EphA4 (
15Sizes of interface residues account for cross-class binding affinity patterns in Eph receptor-ephrin families.
,
16- Lamberto I.
- Lechtenberg B.C.
- Olson E.J.
- Mace P.D.
- Dawson P.E.
- Riedl S.J.
- Pasquale E.B.
Development and structural analysis of a nanomolar cyclic peptide antagonist for the EphA4 receptor.
). On the other hand, the LBD is dynamic by nature, as EphA4 can adopt a conformation similar to other EphA receptors upon interaction with ephrin-A ligands or characteristics of EphB receptors when interacting with ephrin-B ligands (
2- Bowden T.A.
- Aricescu A.R.
- Nettleship J.E.
- Siebold C.
- Rahman-Huq N.
- Owens R.J.
- Stuart D.I.
- Jones E.Y.
Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling.
,
4- Qin H.
- Noberini R.
- Huan X.
- Shi J.
- Pasquale E.B.
- Song J.
Structural characterization of the EphA4-Ephrin-B2 complex reveals new features enabling Eph-ephrin binding promiscuity.
,
17- Singla N.
- Goldgur Y.
- Xu K.
- Paavilainen S.
- Nikolov D.B.
- Himanen J.P.
Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations.
).
Nevertheless several peptides and small molecules have been identified that bind the EphA4 LBD and block its interaction with ephrin ligands (
18- Han X.
- Xu Y.
- Yang Y.
- Xi J.
- Tian W.
- Duggineni S.
- Huang Z.
- An J.
Discovery and characterization of a novel cyclic peptide that effectively inhibits ephrin binding to the EphA4 receptor and displays anti-angiogenesis activity.
19- Lamberto I.
- Qin H.
- Noberini R.
- Premkumar L.
- Bourgin C.
- Riedl S.J.
- Song J.
- Pasquale E.B.
Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor.
,
20- Noberini R.
- Koolpe M.
- Peddibhotla S.
- Dahl R.
- Su Y.
- Cosford N.D.
- Roth G.P.
- Pasquale E.B.
Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors.
,
21- Wu B.
- Zhang Z.
- Noberini R.
- Barile E.
- Giulianotti M.
- Pinilla C.
- Houghten R.A.
- Pasquale E.B.
- Pellecchia M.
HTS by NMR of combinatorial libraries: a fragment-based approach to ligand discovery.
22- Olson E.J.
- Lechtenberg B.C.
- Zhao C.
- Rubio de la Torre E.
- Lamberto I.
- Riedl S.J.
- Dawson P.E.
- Pasquale E.B.
Modifications of a nanomolar cyclic peptide antagonist for the EphA4 receptor to achieve high plasma stability.
). One of these EphA4 antagonists is the KYL peptide, which has been extensively characterized and shown to be effective in several
in vitro assays as well as in
in vivo spinal cord injury and ALS models (
11- Van Hoecke A.
- Schoonaert L.
- Lemmens R.
- Timmers M.
- Staats K.A.
- Laird A.S.
- Peeters E.
- Philips T.
- Goris A.
- Dubois B.
- Andersen P.M.
- Al-Chalabi A.
- Thijs V.
- Turnley A.M.
- van Vught P.W.
- et al.
EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans.
,
19- Lamberto I.
- Qin H.
- Noberini R.
- Premkumar L.
- Bourgin C.
- Riedl S.J.
- Song J.
- Pasquale E.B.
Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor.
,
23- Fabes J.
- Anderson P.
- Brennan C.
- Bolsover S.
Regeneration-enhancing effects of EphA4 blocking peptide following corticospinal tract injury in adult rat spinal cord.
,
24- Murai K.K.
- Nguyen L.N.
- Koolpe M.
- McLennan R.
- Krull C.E.
- Pasquale E.B.
Targeting the EphA4 receptor in the nervous system with biologically active peptides.
), suggesting the potential of an EphA4-based therapeutic approach.
The aim of this study was to develop highly selective and potent EphA4 inhibitors. To achieve this goal, we took advantage of the Nanobody technology (
25- De Meyer T.
- Muyldermans S.
- Depicker A.
Nanobody-based products as research and diagnostic tools.
,
26- Hamers-Casterman C.
- Atarhouch T.
- Muyldermans S.
- Robinson G.
- Hamers C.
- Songa E.B.
- Bendahman N.
- Hamers R.
Naturally occurring antibodies devoid of light chains.
27Nanobodies: natural single-domain antibodies.
). Nanobodies (Nbs) or VHHs are small antigen-binding fragments derived from camelid heavy-chain-only antibodies that are devoid of light chains. They are superior to conventional antibodies in terms of stability, solubility, and immunogenicity (
27Nanobodies: natural single-domain antibodies.
). Furthermore, they are much smaller than conventional antibodies (12–15 kDa
versus 150–160 kDa) and can penetrate small clefts and cavities (
28Nanobody stabilization of G protein-coupled receptor conformational states.
).
We were able to generate Nbs against the LBD of the EphA4 receptor. Two of these Nbs specifically bind the EphA4 receptor with nanomolar affinities and block ephrin-induced EphA4 phosphorylation and EphA4-mediated actin remodeling in a growth-cone collapse assay. These results demonstrate the potential of Nbs to selectively target the LBD of the EphA4 receptor. These Nbs may be useful as a therapeutic strategy in disorders in which EphA4 plays a pathogenic role.
Discussion
In view of EphA4's role in normal physiology as well as in cancer and neurodegeneration, the generation of potent and selective antagonists against this receptor is of great interest. The EphA4 LBD may be a better target than its tyrosine kinase moiety because of the difficulty to obtain selective Eph tyrosine kinase inhibitors and because of the need for such molecules to enter the cell. Here we show for the first time the generation of single domain antibodies targeting the LBD of EphA4.
Nbs are in many ways superior to small molecules when targeting the LBD of Eph receptor proteins, as high affinity binding of the LBD requires the coverage of the protein interaction surface (900 Å2 for EphA4) to block the interaction with EphA4 ligands. Both Nb 39 and Nb 53 were able to block the interaction of EphA4 with all ephrin ligands and to inhibit ephrin-induced phosphorylation of EphA4 and growth-cone collapse triggered by ephrin-B3 binding to endogenous EphA4.
In previous studies Nbs were used to fix receptors in one conformation (
28Nanobody stabilization of G protein-coupled receptor conformational states.
,
37- Rasmussen S.G.
- Choi H.J.
- Fung J.J.
- Pardon E.
- Casarosa P.
- Chae P.S.
- Devree B.T.
- Rosenbaum D.M.
- Thian F.S.
- Kobilka T.S.
- Schnapp A.
- Konetzki I.
- Sunahara R.K.
- Gellman S.H.
- Pautsch A.
- Steyaert J.
- Weis W.I.
- Kobilka B.K.
Structure of a Nanobody-stabilized active state of the β2-adrenoceptor.
). As a consequence Nbs are an ideal probe to target dynamic structures such as the EphA4 LBD (
2- Bowden T.A.
- Aricescu A.R.
- Nettleship J.E.
- Siebold C.
- Rahman-Huq N.
- Owens R.J.
- Stuart D.I.
- Jones E.Y.
Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling.
,
4- Qin H.
- Noberini R.
- Huan X.
- Shi J.
- Pasquale E.B.
- Song J.
Structural characterization of the EphA4-Ephrin-B2 complex reveals new features enabling Eph-ephrin binding promiscuity.
,
17- Singla N.
- Goldgur Y.
- Xu K.
- Paavilainen S.
- Nikolov D.B.
- Himanen J.P.
Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations.
). Four different conformations have been reported for the EphA4 LBD: a conformation similar to other EphA receptors bound with ephrin-A ligands (
2- Bowden T.A.
- Aricescu A.R.
- Nettleship J.E.
- Siebold C.
- Rahman-Huq N.
- Owens R.J.
- Stuart D.I.
- Jones E.Y.
Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling.
,
38- Xu K.
- Tzvetkova-Robev D.
- Xu Y.
- Goldgur Y.
- Chan Y.P.
- Himanen J.P.
- Nikolov D.B.
Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5.
), a conformation similar to EphB receptors bound with ephrin-B ligands (
2- Bowden T.A.
- Aricescu A.R.
- Nettleship J.E.
- Siebold C.
- Rahman-Huq N.
- Owens R.J.
- Stuart D.I.
- Jones E.Y.
Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling.
,
4- Qin H.
- Noberini R.
- Huan X.
- Shi J.
- Pasquale E.B.
- Song J.
Structural characterization of the EphA4-Ephrin-B2 complex reveals new features enabling Eph-ephrin binding promiscuity.
,
15Sizes of interface residues account for cross-class binding affinity patterns in Eph receptor-ephrin families.
), and two unbound conformations when free in solution (
17- Singla N.
- Goldgur Y.
- Xu K.
- Paavilainen S.
- Nikolov D.B.
- Himanen J.P.
Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations.
). Fixing the EphA4 LBD in a specific conformation might limit the accessibility for all or a specific class of ephrin ligands, thereby blocking the interaction of those ligands with the LBD.
Nb 39 and Nb 53 were most selective for EphA4 but still showed residual binding to EphA7, suggesting that they target a region that is not highly conserved in all Eph receptors. However, concentrations of Nb 39 and Nb 53 that strongly inhibit EphA4 binding to ephrin ligands only partially reduced the interaction between EphA7 and ephrin-A5. Interestingly, EphA7 KO mice as well as rats treated with EphA7 antisense oligonucleotides showed enhanced recovery after spinal cord injury compared with control mice (
39- Figueroa J.D.
- Benton R.L.
- Velazquez I.
- Torrado A.I.
- Ortiz C.M.
- Hernandez C.M.
- Diaz J.J.
- Magnuson D.S.
- Whittemore S.R.
- Miranda J.D.
Inhibition of EphA7 up-regulation after spinal cord injury reduces apoptosis and promotes locomotor recovery.
), similar to the effects described for EphA4 antagonists (
24- Murai K.K.
- Nguyen L.N.
- Koolpe M.
- McLennan R.
- Krull C.E.
- Pasquale E.B.
Targeting the EphA4 receptor in the nervous system with biologically active peptides.
). Therefore, if some binding to EphA7 would occur in addition to EphA4, this could potentially be of benefit rather than detrimental.
In the last years extensive work has been performed aiming to develop EphA4 antagonists with high affinity, specificity, and with good pharmacokinetic profiles (
14- Tognolini M.
- Hassan-Mohamed I.
- Giorgio C.
- Zanotti I.
- Lodola A.
Therapeutic perspectives of Eph-ephrin system modulation.
). One EphA4 inhibitor, the KYL peptide, has been studied extensively in models of neurological disorders, such as acute injuries including spinal cord injury and stroke and neurodegenerative disorders such as ALS and Alzheimer's disease (
10- Fu A.K.
- Hung K.W.
- Huang H.
- Gu S.
- Shen Y.
- Cheng E.Y.
- Ip F.C.
- Huang X.
- Fu W.Y.
- Ip N.Y.
Blockade of EphA4 signaling ameliorates hippocampal synaptic dysfunctions in mouse models of Alzheimer's disease.
,
11- Van Hoecke A.
- Schoonaert L.
- Lemmens R.
- Timmers M.
- Staats K.A.
- Laird A.S.
- Peeters E.
- Philips T.
- Goris A.
- Dubois B.
- Andersen P.M.
- Al-Chalabi A.
- Thijs V.
- Turnley A.M.
- van Vught P.W.
- et al.
EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans.
). Unfortunately, this peptide has a
KD value of ∼1 μ
m as determined with isothermal titration calorimetry and a very short half-life in serum (11 min in mouse serum) (
19- Lamberto I.
- Qin H.
- Noberini R.
- Premkumar L.
- Bourgin C.
- Riedl S.J.
- Song J.
- Pasquale E.B.
Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor.
,
24- Murai K.K.
- Nguyen L.N.
- Koolpe M.
- McLennan R.
- Krull C.E.
- Pasquale E.B.
Targeting the EphA4 receptor in the nervous system with biologically active peptides.
). Interestingly, a recent study reported the generation of highly selective EphA4 antagonists with higher potency, the cyclic peptide APY derivatives, APY-d3 and APY-d4 (
16- Lamberto I.
- Lechtenberg B.C.
- Olson E.J.
- Mace P.D.
- Dawson P.E.
- Riedl S.J.
- Pasquale E.B.
Development and structural analysis of a nanomolar cyclic peptide antagonist for the EphA4 receptor.
,
22- Olson E.J.
- Lechtenberg B.C.
- Zhao C.
- Rubio de la Torre E.
- Lamberto I.
- Riedl S.J.
- Dawson P.E.
- Pasquale E.B.
Modifications of a nanomolar cyclic peptide antagonist for the EphA4 receptor to achieve high plasma stability.
). APY-d3 and APY-d4 have
KD values of 30 n
m and 20 n
m as determined with isothermal titration calorimetry and inhibit ephrin-A5-induced phosphorylation of EphA4 with an IC
50 of 240 n
m and 310 n
m. Both cyclic peptides are stable in mouse plasma for >72 h and are currently the best available EphA4 inhibitors (
22- Olson E.J.
- Lechtenberg B.C.
- Zhao C.
- Rubio de la Torre E.
- Lamberto I.
- Riedl S.J.
- Dawson P.E.
- Pasquale E.B.
Modifications of a nanomolar cyclic peptide antagonist for the EphA4 receptor to achieve high plasma stability.
). Nb 39 and 53 are more potent than the KYL peptide but similarly potent as APY-d3 and APY-d4, with
KD values in the nanomolar range, as measured with SPR. Moreover, both Nbs could inhibit EphA4 phosphorylation with an IC
50 of 170 n
m and 261 n
m, respectively. Similar to APY-d3 and APY-d4, Nb 39 and Nb 53 were stable in mouse serum for >72 h and maintained their full capability of binding EphA4 LBD. The affinity of Nbs could still be further increased through error-prone PCR mutagenesis and/or making bispecific Nbs (
40- Saerens D.
- Ghassabeh G.H.
- Muyldermans S.
Single-domain antibodies as building blocks for novel therapeutics.
,
41- Sheedy C.
- MacKenzie C.R.
- Hall J.C.
Isolation and affinity maturation of hapten-specific antibodies.
). Bivalent or bispecific Nbs can be obtained by connecting two identical or different Nbs with a linker, thereby improving the avidity. However, this strategy requires caution in the setting of developing EphA4 antagonists, as dimerization of Eph receptors may induce clustering and subsequently activation, similar to what is obtained with a preclustered antibody (
42- Vearing C.
- Lee F.T.
- Wimmer-Kleikamp S.
- Spirkoska V.
- To C.
- Stylianou C.
- Spanevello M.
- Brechbiel M.
- Boyd A.W.
- Scott A.M.
- Lackmann M.
Concurrent binding of anti-EphA3 antibody and ephrin-A5 amplifies EphA3 signaling and downstream responses: potential as EphA3-specific tumor-targeting reagents.
).
EphA4 has been found to play a role in cancer biology and in the pathogenesis of several neurological disorders (
5- Boyd A.W.
- Bartlett P.F.
- Lackmann M.
Therapeutic targeting of EPH receptors and their ligands.
). In the central nervous system, EphA4 is highly expressed in cell bodies, dendritic spines, and axons of neurons in various brain regions (
43- Martone M.E.
- Holash J.A.
- Bayardo A.
- Pasquale E.B.
- Ellisman M.H.
Immunolocalization of the receptor tyrosine kinase EphA4 in the adult rat central nervous system.
,
44- Tremblay M.E.
- Riad M.
- Bouvier D.
- Murai K.K.
- Pasquale E.B.
- Descarries L.
- Doucet G.
Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus.
). EphA4 expression is up-regulated in axonal stumps after injury (
45- Fabes J.
- Anderson P.
- Yáñez-Muñoz R.J.
- Thrasher A.
- Brennan C.
- Bolsover S.
Accumulation of the inhibitory receptor EphA4 may prevent regeneration of corticospinal tract axons following lesion.
) and in sprouting neurons of aged mice after stroke, which contributes to reduced recovery (
46- Li S.
- Overman J.J.
- Katsman D.
- Kozlov S.V.
- Donnelly C.J.
- Twiss J.L.
- Giger R.J.
- Coppola G.
- Geschwind D.H.
- Carmichael S.T.
An age-related sprouting transcriptome provides molecular control of axonal sprouting after stroke.
). Furthermore, blocking EphA4 induced more sprouting after spinal cord injury (
8- Goldshmit Y.
- Galea M.P.
- Wise G.
- Bartlett P.F.
- Turnley A.M.
Axonal regeneration and lack of astrocytic gliosis in EphA4-deficient mice.
). EphA4's negative effect on axonal regeneration is mediated through the interaction of the receptor with ephrin ligands on surrounding cells such as muscle cells, astrocytes, microglia, and oligodendrocytes (
47- Ilieva H.
- Polymenidou M.
- Cleveland D.W.
Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond.
). Neuronal EphA4 may interact with ephrin-A5 and ephrin-B2, of which the expression is highly up-regulated in reactive astrocytes after injury (
48- Bundesen L.Q.
- Scheel T.A.
- Bregman B.S.
- Kromer L.F.
Ephrin-B2 and EphB2 regulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in adult rats.
,
49- Overman J.J.
- Clarkson A.N.
- Wanner I.B.
- Overman W.T.
- Eckstein I.
- Maguire J.L.
- Dinov I.D.
- Toga A.W.
- Carmichael S.T.
A role for ephrin-A5 in axonal sprouting, recovery, and activity-dependent plasticity after stroke.
50- Ren Z.
- Chen X.
- Yang J.
- Kress B.T.
- Tong J.
- Liu H.
- Takano T.
- Zhao Y.
- Nedergaard M.
Improved axonal regeneration after spinal cord injury in mice with conditional deletion of ephrin B2 under the GFAP promoter.
). This high astrocytic ephrin-A5 expression inhibited axonal sprouting and motor recovery after stroke (
49- Overman J.J.
- Clarkson A.N.
- Wanner I.B.
- Overman W.T.
- Eckstein I.
- Maguire J.L.
- Dinov I.D.
- Toga A.W.
- Carmichael S.T.
A role for ephrin-A5 in axonal sprouting, recovery, and activity-dependent plasticity after stroke.
). Also, ephrin-B2 expression decreased axonal sprouting, as deleting ephrin-B2 from reactive astrocytes reduced glial scar formation and improved recovery after spinal cord injury, and this was correlated with an increased regenerative capacity of sprouting spinal cord axons (
45- Fabes J.
- Anderson P.
- Yáñez-Muñoz R.J.
- Thrasher A.
- Brennan C.
- Bolsover S.
Accumulation of the inhibitory receptor EphA4 may prevent regeneration of corticospinal tract axons following lesion.
,
48- Bundesen L.Q.
- Scheel T.A.
- Bregman B.S.
- Kromer L.F.
Ephrin-B2 and EphB2 regulation of astrocyte-meningeal fibroblast interactions in response to spinal cord lesions in adult rats.
,
50- Ren Z.
- Chen X.
- Yang J.
- Kress B.T.
- Tong J.
- Liu H.
- Takano T.
- Zhao Y.
- Nedergaard M.
Improved axonal regeneration after spinal cord injury in mice with conditional deletion of ephrin B2 under the GFAP promoter.
). Oligodendrocytes precursor cells are glial cells responsible for remyelination during neuronal injury or degeneration, thereby inhibiting axonal outgrowth (
51Oligodendrocytes: biology and pathology.
). Neuronal EphA4 may interact with ephrin-A1, ephrin-A3, ephrin-A5, ephrin-B1, and ephrin-B2, which have been identified in oligodendrocyte precursor cells and in mature oligodendrocytes (
52- Benson M.D.
- Romero M.I.
- Lush M.E.
- Lu Q.R.
- Henkemeyer M.
- Parada L.F.
Ephrin-B3 is a myelin-based inhibitor of neurite outgrowth.
,
53- Linneberg C.
- Harboe M.
- Laursen L.S.
Axo-glia interaction preceding CNS myelination is regulated by bidirectional Eph-Ephrin signaling.
54- Prestoz L.
- Chatzopoulou E.
- Lemkine G.
- Spassky N.
- Lebras B.
- Kagawa T.
- Ikenaka K.
- Zalc B.
- Thomas J.L.
Control of axonophilic migration of oligodendrocyte precursor cells by Eph-ephrin interaction.
). In addition, enhanced functional recovery was observed in ephrin-B3 KO mice after spinal cord injury, and blocking ephrin-B3 promotes remyelination
in vivo in a rat model of remyelination (
55- Duffy P.
- Wang X.
- Siegel C.S.
- Seigel C.S.
- Tu N.
- Henkemeyer M.
- Cafferty W.B.
- Strittmatter S.M.
Myelin-derived ephrinB3 restricts axonal regeneration and recovery after adult CNS injury.
,
56- Syed Y.A.
- Zhao C.
- Mahad D.
- Möbius W.
- Altmann F.
- Foss F.
- Sentürk A.
- Acker-Palmer A.
- Lubec G.
- Lilley K.
- Franklin R.J.M.
- Nave K.A.
- Kotter M.R.N.
Antibody-mediated neutralization of myelin-associated EphrinB3 accelerates CNS remyelination.
). The relative importance of the different surrounding cells and different ligands is not yet known. However, as EphA4 is a promiscuous receptor, targeting EphA4 by blocking the interaction with all ephrin ligands might be the most efficient therapeutic strategy against neurological diseases.
Treating neurological disorders by targeting EphA4 implies using substances that are able to penetrate the blood brain barrier (BBB) to reach their target. In acute injuries, this barrier is impaired. In neurodegenerative diseases such as ALS, getting drugs across the BBB remains a major challenge despite the fact that the BBB is abnormal in this disease (
57- Garbuzova-Davis S.
- Sanberg P.R.
Blood-CNS Barrier impairment in ALS patients versus an animal model.
,
58- Garbuzova-Davis S.
- Saporta S.
- Haller E.
- Kolomey I.
- Bennett S.P.
- Potter H.
- Sanberg P.R.
Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS.
59- Miyazaki K.
- Ohta Y.
- Nagai M.
- Morimoto N.
- Kurata T.
- Takehisa Y.
- Ikeda Y.
- Matsuura T.
- Abe K.
Disruption of neurovascular unit prior to motor neuron degeneration in amyotrophic lateral sclerosis.
). Several strategies to get biologics across the BBB have been developed, such as transcytosis through clathrin vesicles and receptor-mediated transcytosis by targeting the low-density lipoprotein receptor, the transferrin receptor, or the insulin receptor (
60- Abulrob A.
- Sprong H.
- Van Bergen en Henegouwen P.
- Stanimirovic D.
The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells.
61- Boado R.J.
- Hui E.K.
- Lu J.Z.
- Pardridge W.M.
Glycemic control and chronic dosing of rhesus monkeys with a fusion protein of iduronidase and a monoclonal antibody against the human insulin receptor.
,
62- Farrington G.K.
- Caram-Salas N.
- Haqqani A.S.
- Brunette E.
- Eldredge J.
- Pepinsky B.
- Antognetti G.
- Baumann E.
- Ding W.
- Garber E.
- Jiang S.
- Delaney C.
- Boileau E.
- Sisk W.P.
- Stanimirovic D.B.
A novel platform for engineering blood-brain barrier-crossing bispecific biologics.
,
63- Rissiek B.
- Koch-Nolte F.
- Magnus T.
Nanobodies as modulators of inflammation: potential applications for acute brain injury.
,
64- Wang D.
- El-Amouri S.S.
- Dai M.
- Kuan C.Y.
- Hui D.Y.
- Brady R.O.
- Pan D.
Engineering a lysosomal enzyme with a derivative of receptor-binding domain of apoE enables delivery across the blood-brain barrier.
65Receptor-mediated endocytosis and brain delivery of therapeutic biologics.
). Interestingly, one study showed that Nbs with a high isoelectric point can cross the BBB spontaneously (
66- Li T.
- Bourgeois J.P.
- Celli S.
- Glacial F.
- Le Sourd A.M.
- Mecheri S.
- Weksler B.
- Romero I.
- Couraud P.O.
- Rougeon F.
- Lafaye P.
Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging.
).
Nbs also have some drawbacks. Although Nb 39 and Nb 53 remained stable in mouse plasma for >72 h, the small size of Nbs limits their half-life to ∼1.5 h after
in vivo administration (
67- Coppieters K.
- Dreier T.
- Silence K.
- de Haard H.
- Lauwereys M.
- Casteels P.
- Beirnaert E.
- Jonckheere H.
- Van de Wiele C.
- Staelens L.
- Hostens J.
- Revets H.
- Remaut E.
- Elewaut D.
- Rottiers P.
Formatted anti-tumor necrosis factor alpha VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis.
). Due to their small size they are rapidly cleared from blood via the kidney (
27Nanobodies: natural single-domain antibodies.
). This low half-life can be overcome by linking the Nb to serum albumin, which can increase the half-life to 20–30 h in mice (
68- Dennis M.S.
- Zhang M.
- Meng Y.G.
- Kadkhodayan M.
- Kirchhofer D.
- Combs D.
- Damico L.A.
Albumin binding as a general strategy for improving the pharmacokinetics of proteins.
). In humans, this approach extended the half-life of Nbs to 19 days (
68- Dennis M.S.
- Zhang M.
- Meng Y.G.
- Kadkhodayan M.
- Kirchhofer D.
- Combs D.
- Damico L.A.
Albumin binding as a general strategy for improving the pharmacokinetics of proteins.
,
69- Dixon F.J.
- Maurer P.H.
- Deichmiller M.P.
Half-lives of homologous serum albumins in several species.
). Another strategy to increase the half-life of Nbs is coupling polyethylene glycol (PEG) groups to the Nbs (
70The impact of PEGylation on biological therapies.
). The addition of PEG groups increases the apparent molecular weight above the glomerular filtration limit, avoiding renal clearance and/or evades cellular clearance mechanisms (
71PEGylated antibodies and antibody fragments for improved therapy: a review.
).
In summary, we describe for the first time the development of Nbs that target the EphA4 LBD. We identified two Nbs that were specific for EphA4 with residual EphA7 binding and with KD and IC50 values in the nanomolar range. Both Nbs were able to block the interaction of EphA4 with all ephrin ligands and inhibit EphA4 phosphorylation and the growth-cone collapse mediated by EphA4 activation upon ephrin-B3 interaction. These Nbs may be useful tools to study the role of the EphA4 receptor in preclinical cancer or neurodegeneration models. Furthermore, they may represent a starting point for an EphA4-based therapeutic approach using Nbs.
Experimental procedures
Expression and purification of the EphA4 ligand-binding domain
The EphA4 LBD (amino acids 22–203; Ref.
17- Singla N.
- Goldgur Y.
- Xu K.
- Paavilainen S.
- Nikolov D.B.
- Himanen J.P.
Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations.
) was cloned from the EphA4 Human cDNA ORF clone (Origene) and expressed in the
E. coli strain BL21 codon + pICA2 transformed with the pLH36Epha plasmid. Expression was induced by isopropyl β-
d-1-thiogalactopyranoside under control of a pL-promotor developed by the Protein Service Facility of VIB. The pLH36 plasmid was provided with a His
6 tag followed by a murine caspase-3 site. The murine caspase-3 site can be used to remove the His
6 tag attached at the N terminus of the protein of interest during purification. The transformed bacteria were grown in Luria Bertani medium supplemented with ampicillin (100 μg/ml) and kanamycin (50 μg/ml) overnight at 28 °C inoculation (1/100) in a 20-liter fermenter provided with Luria Bertani medium supplemented with ampicillin (100 μg/ml) and 1% glycerol. The initial stirring and airflow was 200 rpm and 1.5 liters/min, respectively. Furthermore, this was automatically adapted to keep the
pO
2 at 30%. The temperature was kept at 28 °C. The cells were grown to an optical density of
A600 nm = 1.0 and transferred at 20 °C, and expression was induced by the addition of 1 m
m isopropyl β-
d-1-thiogalactopyranoside overnight. Cells were then harvested and frozen at −20 °C. After thawing, the cells were resuspended at 3 ml/g in 20 m
m NaH
2PO
4, pH 7.5, 500 m
m NaCl, 20 m
m imidazole, and 1 m
m phenylmethylsulfonyl fluoride. The cytoplasmic fraction was prepared by sonication of the cells followed by centrifugation at 18,000 ×
g for 30 min. All steps were conducted at 4 °C. The clear supernatant was applied to a 74-ml Ni
2+-Sepharose 6 FF column (GE Healthcare), equilibrated with 20 m
m NaH
2PO
4, pH 7.5, 500 m
m NaCl, 20 m
m imidazole, and 0.1% CHAPS. The column was eluted with 20 m
m NaH
2PO
4, pH 7.4, 20 m
m NaCl, 400 m
m imidazole, and 0.1% CHAPS after an extra wash step with 50 m
m imidazole. The elution fraction was diluted
with 20 m
m Tris, pH 8.0, and 0.1% CHAPS and loaded on a 20-ml Source 15Q column (GE Healthcare) to remove contaminants. After equilibration, the protein of interest was eluted by a linear gradient over 20 column volumes of NaCl from 0 to 1
m in 20 m
m Tris, pH, 8.0 and 0.1% CHAPS. To the EphA4-containing fractions, activated murine caspase-3 (1/100% murine caspase-3/Epha4) with 10 m
m DTT was added to remove the His
6 tag. After a 1-h incubation at 37 °C, the reaction solution was injected on a HiLoad 26/60 Superdex 75 prep grade with PBS as the running solution for formulation and removal of minor contaminants, His
6 tag and murine caspase-3. The obtained fractions were analyzed by SDS-PAGE, and the concentration was determined using the Micro-BCA assay (Thermo Fisher Scientific, Ghent, Belgium).
Construction of a VHH library
Nbs targeting the EphA4 LBD were obtained in collaboration with the VIB Nanobody Service Facility. An alpaca was injected subcutaneously on days 0, 7, 14, 21, 28, and 35, each time with 250 μg of human EphA4 LBD. On day 39 anticoagulated blood was collected for lymphocyte preparation. A VHH library was constructed as previously described (
72- Els Conrath K.
- Lauwereys M.
- Wyns L.
- Muyldermans S.
Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs.
,
73- Saerens D.
- Kinne J.
- Bosmans E.
- Wernery U.
- Muyldermans S.
- Conrath K.
Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen.
74- Steeland S.
- Puimège L.
- Vandenbroucke R.E.
- Van Hauwermeiren F.
- Haustraete J.
- Devoogdt N.
- Hulpiau P.
- Leroux-Roels G.
- Laukens D.
- Meuleman P.
- De Vos M.
- Libert C.
Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.
) and screened for the presence of antigen-specific Nbs. In brief, total RNA from peripheral blood lymphocytes was used as the template for first-strand cDNA synthesis with the oligo(dT) primer. Using this cDNA, the VHH-encoding sequences were amplified by PCR, digested with PstI and NotI, and cloned into the PstI and NotI sites of the phagemid vector pMECS. A VHH library of ∼2 × 10
8 independent transformants was obtained.
Isolation of hEphA4 LBD-specific Nbs
To screen for the presence of EphA4-specific Nbs, the library was subjected to four consecutive rounds of panning, performed on solid-phase-coated EphA4 LBD (concentration: ∼200 μg/ml, ∼20 μg/well). The enrichment for antigen-specific phages after each round of panning was assessed by comparing the number of phages eluted from antigen-coated wells with the number of phages eluted from negative control (only-blocked) wells. These experiments suggested that the phage population was enriched for antigen-specific phages after the 3rd and 4th round of panning. In total, 190 individual colonies from the 3rd and 4th round (95 from each round) were randomly selected and analyzed by ELISA for the presence of antigen-specific Nbs in their periplasmic extracts (ELISA using crude periplasmic extracts including soluble Nbs). Of 190 colonies, 41 colonies (14 and 27 from the 3rd and 4th round, respectively) scored positive in this assay. The VHH genes of the selected genes were sequenced to identify the different Nbs.
Expression and purification of recombinant Nbs
pMECS vectors harboring Nb genes were transformed into WK6 E. coli cells, and the transformed cells were grown in Luria Bertani medium supplemented with ampicillin (100 μg/ml) and 0.1% glucose at 37 °C overnight. Subsequently, the cultures were inoculated 1/100 to have 1-liter productions in Terrific Broth medium supplemented with ampicillin (100 μg/ml) and 0.1% glucose in baffles shake flasks. The temperature was kept at 37 °C. The cells were grown to an optical density of A600 nm = 1.0 and transferred at 28 °C, and expression was induced by the addition of 1 mm isopropyl β-d-1-thiogalactopyranoside overnight. Cells were then harvested and frozen at −20 °C. The expressed Nbs were extracted from the periplasm by osmotic shock and purified using His GraviTrap (GE Healthcare) in parallel, equilibrated with 20 mm NaH2PO4, pH 7.5, 300 mm NaCl, 20 mm imidazole, and 1 mm PMSF. After loading, the columns were washed with 20 column volumes of the same buffer. The Nbs were first eluted with 20 mm NaH2PO4, pH 7.5, 20 mm NaCl, 50 mm imidazole, 1 mm PMSF and then with 400 mm imidazole in the same buffer. Finally, the Nbs were desalted to PBS on Sephadex G25 (GE Healthcare). The obtained fractions were analyzed with Coomassie-stained SDS-polyacrylamide gels. Protein concentration was measured by the Micro-BCA assay (Thermo Fisher Scientific).
Western blot
For immunoprecipitation experiments, 1 μg of EphA4 LBD, hEphA4-Fc (R&D Systems, Abingdon, UK), and mEphA4-Fc (R&D Systems) were boiled for 10 min in reducing sample buffer (Thermo Fisher Scientific), and proteins were separated in a 4–12% Bis-Tris SDS-PAGE gel (Thermo Fisher Scientific). After SDS-PAGE, the gel was transferred to Immobilon-P membrane (Merck Millipore, Overijse, Belgium) and subsequently blocked with 10% Blotting-grade blocker (Bio-Rad) for 1 h at room temperature. One μg Nb and mouse anti-HA antibody 1/1000 (clone 16B12, MMS-101P-200, lot D13FF01646, Covance) were used to detect EphA4. To determine Nb stability, 10 ng of Nbs were denatured with reducing sample buffer (Thermo Fisher Scientific) and separated in a 12% Tris-glycine SDS-PAGE gel. The gel was transferred to Immobilon-P membrane, which was afterward blocked as described above. To detect the Nbs, 1/1000 anti-HA antibody (clone C29F4, 3724S, lot 8, Cell Signaling) was used.
Immunoprecipitation experiments
1.5 mg of protein G magnetic Dynabeads (Thermo Fisher Scientific) was preblocked with 1% BSA for 1 h at room temperature, washed 4 times with PBS, and incubated with 2.5 μg of recombinant mouse or human EphA4 protein (R&D Systems) for 10 min at room temperature. After washing 4 times with PBS the beads were incubated with 1 μg of Nb overnight at 4 °C. The beads were washed 4 times with PBS and boiled for 10 min in reducing sample buffer (Thermo Fisher Scientific), and proteins were separated in a 4–12% Bis-Tris SDS-PAGE gel (Thermo Fisher Scientific). After SDS-PAGE, the gel was transferred to Immobilon-P membrane (Merck Millipore) and subsequently blocked with 10% Blotting-grade blocker (Bio-Rad) for 1 h at room temperature. Mouse N-terminal anti-EphA4 antibody 1/1000 (EM2801, lot 1, ECM Biosciences) and mouse anti-HA antibody 1/1000 (clone 16B12, MMS-101P-200, lot D13FF01646, Covance) were used to detect EphA4 and Nb, respectively. To capture EphA4 with Nb anti-HA magnetic Dynabeads (Thermo Fisher Scientific) were used, and all the following steps were performed as described above.
Stability experiments
To determine the Nb stability in plasma, Nbs were incubated at 37 °C at a concentration of 10 ng/μl in sodium heparin not-filtered C57BL/6 mouse plasma (BioreclamationIVT). At every time point an aliquot was collected and diluted
in PBS for further Western blot analysis. As a control, Nbs were incubated for the same time and at the same concentration at 37 °C in PBS. Nb stability in all samples was next determined with Western blot as described above. Statistical analysis was performed using the two-way ANOVA followed by Sidak's multiple comparisons post hoc test as indicated in the figure legends. A 95% confidence interval was used, and values of p < 0.05 were considered as statistically significant.
To determine functional stability of Nbs, they were first incubated at 37 °C in sodium heparin not-filtered C57BL/6 mouse plasma at 500 nm concentration. At every time point and for a maximum of 72 h, an aliquot was collected and stored for further analysis with Alphascreen technology.
Surface plasmon resonance
The equilibrium dissociation constant (KD) and the association (ka1 and ka2) and dissociation rates (kd and kd2) were determined using surface plasmon resonance detection on a BIACore T200 (GE Healthcare). Two approaches were used. First, the extracellular N-terminal domain of human EphA4 was immobilized directly onto a CM5 S series sensor chip (GE Healthcare) using standard amine coupling. After activation of the carboxyl moieties on the matrix on the chip surface with a 7-min injection of a 1:1 mixture of 0.4 m EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 0.1 m NHS, the N-terminal domain of human EphA4 (50 μg/ml in 10 mm acetate, pH 4.5) was immobilized to a predefined level of 300 response units (RU). A flow channel activated with EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide)/NHS and immediately afterward blocked by ethanolamine served as reference channel. Throughout the analysis 10 mm Hepes, 150 mm NaCl, 3 mm EDTA, and 0.01 mm Surfactant P-20, pH 7.35, was used as the running buffer. 20 mm glycine, pH 2.5, was used to regenerate the channels (remove all bound proteins). The second approach used the anti-human Fc capture kit as described by the manufacturer. Briefly, ∼8000 RU anti-human Fc antibody was captured on the surface of the channels using amine coupling as described above. In a next step, EphA4-hFc (both human and mouse EphA4) (R&D Systems) fusion protein (2 μg/ml diluted in running buffer) was injected (10 μl/min for 6 min) over the channel resulting in the capture of ∼500 RU of EphA4-hFc fusion protein on the surface of the channel. In this approach, a channel with only anti-human Fc antibody served as the reference. 3 m MgCl2 was used as regeneration buffer. The Nbs were diluted to the indicated concentrations in running buffer and injected (60 μl/min) over the channel with immobilized EphA4 and the reference channel. After correction of the response using the responses from the reference channel and a blank injection of running buffer over the Eph4A-immobilized channel (double referencing), kinetic parameters were determined using Biacore T200 evaluation software (GE Healthcare). Interactions of Nbs with different EphA4 recombinant proteins were calculated with a 1:1 binding model or a two-state model. The latter model was used to calculate all Nb interactions with EphA4 LBD and interactions of Nb 34, 31, and 50 with human and mouse EphA4.
AlphaScreen
To test the specificity of the Nbs for the different Eph receptors, all Nbs were biotinylated with a five times molar excess of EZ link NHS biotin (Thermo Fisher Scientific). The Nbs were incubated with the biotin for 2 h on ice allowing the interaction of the biotin with the primary amines on the surface of the protein. To remove unbound biotin, dialysis was performed with PBS in the Slide-A-Lyzer mini dialysis device (10-kDa cutoff, Thermo Fisher Scientific). Ten microliters of biotinylated Nbs (100 nm) were incubated with 10 μl of a subhooking concentration of mouse recombinant Eph receptor (Fc-tagged; R&D Systems) for 1 h in standard buffer (50 mm Hepes, 100 mm NaCl, 0.1% Triton, and 0.1% BSA) in white opaque 384-well microplates (PerkinElmer Life Sciences) to avoid the hooking effect (oversaturation of donor and acceptor beads inhibiting their association). The determined subhooking concentrations were 10 nm for EphA2, A3, A4, A6, B2, and B6, 30 nm for EphA7 and B3, B4, and 100 nm for EphA8. Subsequently we incubated first 1 h with 10 μl of anti-IgG AlphaLISA acceptor beads (20 μg/ml, PerkinElmer Life Sciences) and then incubated an additional 30 min with 10 μl of streptavidin donor beads (20 μg/ml, PerkinElmer Life Sciences). Incubation steps were performed at room temperature and protected from light.
To test the inhibition of interaction between EphA4 and its different ligands, the EphA4 LBD (2 mg/ml) was biotinylated with a 2.5× molar excess of EZ link NHS biotin (Thermo Fisher Scientific) for 2 h on ice, allowing the interaction of the biotin with primary amines on the surface of the protein. To remove unbound biotin, dialysis was performed with PBS in the Slide-A-Lyzer mini dialysis device (10-kDa cutoff, Thermo Fisher Scientific). Five microliters of a subhooking concentration of biotinylated EphA4 LBD (10 nm) was incubated with 5 μl of different concentrations of Nb for 1 h in standard buffer (50 mm Hepes, 100 mm NaCl, 0.1% Triton, and 0.1% BSA). Subsequently, 5 μl of a subhooking concentration of recombinant ephrin ligand (Fc-tagged; R&D Systems) was added and incubated for 1 h at room temperature. The determined subhooking concentrations were 3 nm for ephrin-A1 and ephrin-A4 and 10 nm for ephrin-A2/3, ephrin-A5, and ephrin-B1–3. Next, 5 μl of anti-IgG AlphaLISA acceptor beads (20 μg/ml; PerkinElmer Life Sciences) and 5 μl of streptavidin donor beads (10 μg/ml; PerkinElmer Life Sciences) were added and incubated for 1 h and 30 min, respectively, at room temperature, protected from light. A Nb targeting superoxide dismutase 1 (SOD1) was used as negative control in all Eph-binding assays.
To test the inhibition of EphA7 and ephrin-A5 interaction, His-tagged human ephrin-A5 (Sino Biological, Beijing, China) was biotinylated with a five-times molar excess of EZ link NHS biotin (Thermo Fisher Scientific) as is described for the Nbs. Briefly, ephrin-A5 was incubated with the biotin for 2 h on ice and dialyzed in PBS with the Slide-A-Lyzer mini dialysis device (10-kDa cutoff, Thermo Fisher Scientific) to remove unbound biotin. Five microliters of a subhooking concentration of human EphA7 (3 nm) was incubated with 5 μl of different concentrations of Nb for 1 h in standard buffer (50 mm Hepes, 100 mm NaCl, 0.1% Triton, and 0.1% BSA). Next, 5 μl of subhooking concentration of biotinylated ephrin-A5 ligand (30 nm) was added and incubated for 1 h at room temperature. Finally, 5 μl of anti-IgG AlphaLISA acceptor beads (20 μg/ml; PerkinElmer Life Sciences) and 5 μl of streptavidin donor beads (20 μg/ml; PerkinElmer Life Sciences) were added and incubated for 1 h and 30 min, respectively, at room temperature, protected from light. A control Nb targeting chicken lysozyme was used as negative control, and untagged ephrin-A5 was used as positive control.
To determine the capability of Nbs to bind EphA4 after plasma incubation, 500 nm Nbs preincubated in plasma were first diluted in PBS at the time of the Alphascreen assay to reach a final subhooking Nb concentration of 10 nm. Five microliters of a subhooking concentration of biotinylated EphA4 LBD (10 nm) was incubated with 5 μl of 10 nm Nb 39 and Nb 53 for 1 h in standard buffer (50 mm Hepes, 100 mm NaCl, 0.1% Triton, and 0.1% BSA). Next, 5 μl of anti-HA AlphaLISA acceptor beads (20 μg/ml; PerkinElmer Life Sciences) and 5 μl of streptavidin donor beads (10 μg/ml; PerkinElmer Life Sciences) were added and incubated for 1 h and 30 min, respectively, at room temperature, protected from light. As a control, to assess the specificity of the binding, 300 nm ephrin-B2 ligand (Fc-tagged; R&D Systems) was incubated with EphA4 LBD for 1 h before the addition of the Nbs.
Plates were read on the Envision Multilabel Reader (PerkinElmer Life Sciences).
Phosphorylation assay
The amount of phosphorylation of EphA4 was determined using the PathHunter assay (DiscoveRx Corp., Birmingham, UK) with U2OS cells adapted for the EphA4 receptor according to manufacturer's instructions. In short, a small peptide epitope is expressed recombinantly on the intracellular C terminus of the EphA4 receptor-tyrosine kinase. An interaction partner containing SH2 domains is co-expressed with a larger sequence, termed enzyme activator (EA). Activation with recombinant human ephrin-A1-Fc causes EphA4 receptor dimerization, leading to cross-phosphorylation of tyrosine residues on the cytoplasmic domain of the receptor. The SH2-EA fusion protein binds the phosphorylated receptor enabling the complementation of EA and the peptide epitope, yielding an active β-galactosidase enzyme. This interaction can be visualized with a chemiluminescent substrate. Increasing concentrations of the Nbs were added to the medium before ephrin-A1-Fc stimulation. 20 μl of 5000 EphA4-expressing U2OS cells were plated in 384-well plates and incubated for 24 h at 37 °C 5% CO2. Five-μl Nb dilutions or vehicle were added per well followed by incubation for 1 h at 37 °C. Five μl of ephrin-A1-Fc (1.2 μg/ml) or vehicle were added to each well followed by incubation for 3 h at room temperature. Twelve μl of detection reagent (Galacton Star, Emerald II solution, PathHunter cell assay buffer in relative volumes of 1:5:19, respectively) was added, incubated for 1 h at room temperature, and read on a Pherastar (BMG Labtech, Ortenberg, Germany). Percentage activity was calculated as (signal − non-stimulated control (no ephrin added))/(ephrin-stimulated condition − non-stimulated control)) × 100. Positive and negative controls were Dasatinib and a Nb targeting Superoxide Dismutase 1 (SOD1), respectively.
Growth-cone collapse
The cortex of E17.5 mouse embryos was dissociated by trypsinization and trituration. Cortical neurons were cultured on 13-mm-diameter coverslips coated with poly-l-lysine, at a density of 15,000 cells/coverslip in minimum Eagle's medium (MEM) with Earle's salt and l-glutamine and supplemented with 10% heat-inactivated horse serum and penicillin and streptomycin (Thermo Fisher Scientific). Cultures were kept in a 5% CO2 humidified incubator at 37 °C. Medium was replaced for Neurobasal medium supplemented with B27, 500 μm l-glutamine and penicillin and streptomycin (Thermo Fisher Scientific) 4 h after plating. 24 h after plating the cultures were incubated for 30 min with KYL peptide at a concentration of 1 μm or 30 μm or Nb 53 or Nb 39 at a concentration of 1 μm. Cultures were next stimulated with 1 μg/ml mouse preclustered ephrin-B3-Fc, or Fc (R&D Systems) as a control and for 30 min in the presence of the KYL peptide or the Nbs. Cortical neurons were fixed for 20 min in 4% paraformaldehyde (Thermo Fisher Scientific), permeabilized in 0.2% Triton X-100 in PBS, and stained with Alexa Fluor 555-conjugated phalloidin (Thermo Fisher Scientific). Growth-cone collapse was scored for each condition under a Zeiss Axioimager M1 epifluorescence and brightfield upright microscope with a Zeiss A-Plan 40×/0.65 ∞/0.17 objective (Carl Zeiss, Oberkochen, Germany). Growth-cone collapse was considered when no lamellipodia or filopodia were present at the tip of the longest neurite of every scored neuron. Growth-cone collapse was scored in a blinded manner for 40–160 neurons in every condition and experiment. Images were obtained with Axiocam MRm monochrome digital camera and Zeiss AxioVision V 4.8.2.0 software (Carl Zeiss) at the same magnification. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test as indicated in the figure legends. A 95% confidence interval was used, and values of p < 0.05 were considered as statistically significant.
Article info
Publication history
Published online: May 19, 2017
Received in revised form:
May 16,
2017
Received:
January 3,
2017
Edited by Paul E. Fraser
Footnotes
This work was supported by grants from VIB, the KU Leuven-University of Leuven (GOA/11/014), Vertex Pharmaceuticals Inc., Research Foundation Flanders (FWO-Vlaanderen; G.0996.14N), the ALS League Belgium, the Thierry Latran Foundation, Geneeskundige Stichting Koningin Elisabeth (G.S.K.E.), Laevers Fund for ALS Research, and the Fund “Een Hart voor ALS.” This work was also supported by the European Community's Health Seventh Framework Programme (FP7/2007–2013) under Grant 259867, the European Research Council, and the European's Seventh Framework Programme (FP7/2007–2013)/ERC Grant 340429 and structural funding for medium-scale research infrastructure (HERCULES, AUGE/09/040). The authors declare that they have no conflicts of interest with the contents of this article. The views expressed are those of the authors and not necessarily those of the NHS (National Health Service), the NIHR (National Institute for Health Research), or the Department of Health.
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© 2017 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.