Patent Application
20080274950
The present invention relates to cadherin molecule adhesion.
Cadherins are a family of cell surface adhesion molecules that are essential for maintaining the structural integrity of all vertebrate solid tissues. The cadherin family includes classical (“Type I”) cadherins, non-classical (“Type II”), desmosomal cadherins and protocadherins (reviewed in Patell et al., 2003). Cadherins determine cell-cell recognition during morphogenesis and have signalling functions which influence cell migration and differentiation (Cavallaro and Christofori, 2004; Hirano et al., 2003; Thiery, 2003; Wheelock and Johnson, 2003). Indeed, cadherins provide the principal adhesion mechanism for maintaining the integrity of all solid tissues and, in addition, play a major role in controlling segregation of cells during organ formation in embryonic development. Cadherins are often found to malfunction in cancer. In metastatic carcinomas, expression of E-cadherin is often down-regulated or the molecule has suffered a functional mutation. In contrast, N-cadherin is frequently upregulated and through its cell signalling capacity stimulates invasive behaviour. Lack of cadherin-mediated adhesion is a major cause of cancer metastasis.
Cadherin molecules usually stick to their own type, i.e. E-cadherin sticks to another E-cadherin molecule but not as well to an N-cadherin molecule. Cadherins engage each other at their tip ends and the interaction between individual molecules has a low affinity but, cumulatively, they provide strong adhesion between cells. Cadherin-cadherin contacts, as well as providing an ‘intercellular glue’, also convey signals to the cell and modulate signalling by growth factors. In the case of N-cadherin these signals promote cell survival and cell migration.
Adhesive interactions by cadherins are mostly, but not exclusively, homophilic and cadherin type-specific. Classical cadherins comprise five extracellular β-barrel-like domains (domains “EC1” to “EC5”, also known as “ectodomains”), a transmembrane domain and a cytoplasmic domain. Each of the extracellular domains contains seven β strands and, in most cases, calcium binding sites. Adhesion requires the presence of calcium bound in the interdomain junctions of the extracellular domains and it is known that this rigidifies the cadherin molecule into a curved rod-like structure projecting from the cell (Boggon et al., 2002; He et al., 2003; Miyaguchi, 2000; Pokutta et al., 1994). Despite more than a decade of research, the mechanism by which cadherin extracellular domains form adhesive contacts remains controversial.
Insights into the process of adhesion have come mainly from four experimental strategies: observations of the effects of point mutations or domain deletions on cell adhesion, co-immunoprecipitation of epitope-tagged cadherin molecules in adhesive complexes between cells, structural studies of cadherins by NMR or X-ray crystallography, and physical studies, including measurements of intermolecular forces between cadherin molecules and direct observation of cadherins by electron microscopy. Cumulatively, these techniques have led to several alternative models for adhesion.
Amino acids which co-ordinate calcium in the junction between the first and second domains, EC1 and EC2 (also known as “ECD1” and “ECD2”, respectively), have been shown to play an essential role in adhesion (Corps et al., 2001; Klingelhofer et al., 2002) and structural studies have suggested that calcium will instigate dimerisation of the recombinant protein EC1-EC2 via contact surfaces in the domain junction and EC1 (Haussinger et al., 2002; Pertz et al., 1999). This effect of calcium has been demonstrated by physical measurements and electron microscopy (Alattia et al., 1997). Scanning mutagenesis in the N-terminal domain (EC1) has shown that tryptophan 2 (Trp2), the second amino acid of the mature cadherin molecule, and amino acids lining an adjacent hydrophobic pocket are also indispensable for adhesion (Kitagawa et al., 2000; Tamura et al., 1998). The importance of Trp2 has been confirmed by immunoprecipitation studies which have demonstrated that this residue is required for the formation of both adhesive (trans) dimers and lateral (cis) dimers (Laur et al., 2002; Ozawa, 2002). A possible explanation for the significance of Trp2 has been provided by three X-ray crystallography studies which have revealed a mechanism for dimerisation in which Trp2 in strand A of EC1 docks into a hydrophobic pocket in EC1 of its neighbour, a mutual process which holds the two EC1 protomers together (Boggon et al., 2002; Haussinger et al., 2004; Shapiro et al., 1995). In principle this interaction (strand exchange) could mediate dimerisation in either cis- or trans-alignment. A recent immunoprecipitation study which was designed to discriminate between strand exchange and a calcium-mediated mechanism for dimerisation is consistent with the strand exchange model (Troyanovsky et al., 2003).
A different perspective has emerged from measurements of intermolecular forces between recombinant cadherin molecules. That data suggest that contact surfaces on two or more cadherin domains are required for adhesion and that opposing cadherin molecules can engage in several alternative anti-parallel alignments (Chappuis-Flament et al., 2001; Sivasankar et al., 2001; Zhu et al., 2003). That idea is at variance with direct observation, by electron microscopy, of purified recombinant cadherin molecules and cadherins in junctional complexes. Those images suggest that both cis- and transdimerisation takes place exclusively via EC1 (Ahrens et al., 2003; Ahrens et al., 2002; He et al., 2003; Pertz et al., 1999). A central issue in those conflicting models is whether Trp2 serves only to stabilise an adhesive contact surface in domain 1 or whether strand exchange is the primary event in adhesion.
The potential role of so called ‘cell adhesion recognition motifs’ (CARs) in cadherin adhesion has been emphasised. A principal CAR in cadherins is the amino acid sequence HAV in domain 1 (EC1). EC1 is the most N-terminal domain of a mature cadherin molecule obtained after the prodomain or precursor sequence of amino acids has been removed by normal cellular processing. Cyclic peptides which include the HAV sequence have been shown to inhibit cadherin-mediated adhesion and in some circumstances to trigger apoptosis. The potential use of HAV-type peptides as pharmaceutical agents to inhibit cell adhesion in a wide range of therapeutic applications or to stimulate cadherin-mediated signalling has been appreciated by companies such as Adherex Inc., Ottawa. Adherex patent documents cover many potential clinical applications for peptide mimetics of CARs, antibodies which recognise CARs or other CAR-binding agents. Their lead product, Exherin, is an HAV cyclic peptide which inhibits N-cadherin function.
Due to the importance of cadherin binding in biological process, there remains a need to develop effective ways of manipulating cadherin adhesion both for in vivo and potentially in vitro applications.
According to a first aspect of the present invention, there is provided a pair of cadherin molecules modified to enhance intermolecular adhesion (i.e. adhesion or binding between the pair of cadherin molecules) compared with corresponding unmodified cadherin molecules.
The present inventors show definitive evidence for the primary mechanism of cadherin-mediated adhesion. Our data (see below) shows that the so-called ‘strand-exchange’ model is correct. It is a further example of so called ‘3D domain swapping,’ one of several mechanisms that cause proteins to dimerise or polymerise. This mechanism does not depend on a cadherin CAR—we now have evidence that the primary and crucial molecular contact in cadherin-mediated adhesion does not involve HAV or any CAR—and is quite distinct from the idea which forms the scientific basis for the Adherex strategy. It is a novel and unexpected finding that cadherin molecules as modified herein have modulated adhesion (or altered adhesive) properties of the type disclosed. In particular, an increase in intermolecular adhesion between complementary pairs of cadherin molecules compared with that between normal cadherin molecules is novel and unpredicted. This modulating effect has several uses and benefits, as elaborated herein.
In the present invention, intermolecular adhesion between the cadherin molecules may be enhanced by reducing or eliminating intramolecular binding within each cadherin molecule. For example, intramolecular binding may be reduced or eliminated by diminishing or preventing intramolecular binding of an N-terminal binding strand of each cadherin molecule with a binding strand acceptor pocket of each cadherin molecule. For example, the N-terminal binding strand may be derived from or equivalent to the βA strand (with tryptophan at amino acid position 2) of the EC1 domain of mature wild-type human N-cadherin (or a function equivalent thereof; see below). For example, the binding strand acceptor pocket may be derived from or equivalent to the hydrophobic Trp 2 acceptor pocket in EC1 which accepts insertion of tryptophan at amino acid position 2 of mature human N-cadherin (or a function equivalent thereof; see below). Intramolecular binding may be prevented or eliminated or diminished by substituting Trp2 with an alternative amino acid, for example glycine, and/or by obstructing the hydrophobic Trp 2 acceptor pocket, for example by introducing the mutation Ala80Ile (with reference to alanine at amino acid position 80 of mature wild-type human N-cadherin or a functional equivalent thereof see below).
Additionally or alternatively, the intramolecular binding may be reduced or eliminated by diminishing or preventing the formation of an intramolecular ionic bond (for example, a salt bridge) between the NH2 terminus of each cadherin molecule with a contact acidic amino acid residue (for example, glutamic acid, aspartate, asparagine or glutamine) of each cadherin molecule. The contact acidic amino acid residue may, for example, be derived from or equivalent to glutamic acid at amino acid position 89 of mature N-cadherin (or a function equivalent thereof; see below).
In accordance with the findings of the present inventors, intermolecular adhesion may be facilitated by an ionic bond between a contact acidic amino acid residue of one cadherin molecule and the NH2 terminus of the other cadherin molecule. Intermolecular adhesion may also be facilitated by binding of an N-terminal binding strand of one cadherin molecule with a binding strand acceptor domain of the other cadherin molecule. The features of the cadherin molecules contributing to intermolecular adhesion are as mentioned herein for intramolecular binding.
As used herein, “intermolecular adhesion” means adhesion or binding between two (or more) cadherin molecules. Intermolecular adhesion may include insertion or “docking” of the N-terminal binding strand of a first cadherin molecule with a binding strand acceptor pocket of a second cadherin molecule (for example the docking or insertion of Trp2 of a first mature N-cadherin molecule or a modified version thereof into the hydrophobic Trp2 acceptor pocket in the EC1 domain of a second mature N-cadherin molecule or a modified version thereof), and/or formation of an intermolecular ionic bond between NH2 terminus of a first cadherin molecule and the contact amino acid residue of a second cadherin molecule (for example, the formation of a salt bridge between the NH2 terminus of a first mature N-cadherin molecule or modified version thereof and Glu89 of a second mature N-cadherin molecule or a modified version thereof).
As used herein, “intramolecular binding” means binding (or self-docking or adhesion) within a cadherin molecule to form a closed or partially closed monomeric cadherin molecule. Intramolecular binding may include insertion or “docking” of the N-terminal binding strand of each cadherin molecule with a binding strand acceptor pocket of each cadherin molecule (for example the docking or insertion of Trp2 into the hydrophobic Trp2 acceptor pocket in the EC1 domain of mature wild-type human N-cadherin or a modified version thereof) and/or formation of an intramolecular ionic bond between NH2 terminus and the contact amino acid residue of the cadherin molecule (for example, the formation of a salt bridge between the NH2 terminus and Glu89 of mature wild-type human N-cadherin or a modified version thereof).
The present invention is based, in part, on the finding that cadherin adhesion depends on a dynamic equilibrium between intramolecular binding and intermolecular adhesion. The dynamic equilibrium means that structural features which bring about adhesion can be manipulated to favour intramolecular binding or intermolecular adhesion. These structural features include the NH2 terminus, the contact amino acid residue, the N-terminal binding strand and the binding strand acceptor pocket, of each cadherin molecule (or polypeptide). Intramolecular binding occurs when the N-terminal binding strand on one cadherin molecule binds with the binding strand acceptor pocket of the same molecule, a reaction that is stabilised by the formation of an ionic bond (for example, a salt bridge) between the NH2 terminus of the cadherin molecule and the contact amino acid residue of the same molecule. Intermolecular adhesion occurs when the NH2 terminus of a first cadherin molecule forms an ionic bond (for example, a salt bridge) with the contact amino acid residue of a second cadherin molecule, and/or when the N-terminal binding strand on the first cadherin molecule binds with the binding strand acceptor pocket of the second cadherin molecule.
In one aspect of the present invention, the cadherin molecules may be modified by altering the primary structure of each cadherin molecule.
For example, the following pairs of cadherin molecules may be used according to the present invention:
(i) a first cadherin molecule in which the N-terminus is extended by addition of one or more amino acids to a mature (processed) cadherin molecule (for example mature N-cadherin), and/or in which the correct processing of the cadherin prodomain or precursor sequence has been prevented, in each case preventing the formation of an intramolecular ionic bond; and a second cadherin molecule in which the acidic acid residue is mutated to remove functionality, thereby preventing formation of an intramolecular ionic bond (for example, Glu89 of mature N-cadherin mutated to Ala89), and/or in which binding of the N-terminal binding strand of one cadherin molecule (for example the βA strand of mature N-cadherin with tryptophan at amino acid position 2) is prevented from binding into the binding strand acceptor pocket (for example, by mutation of alanine at amino acid position 80 of mature N-cadherin to isoleucine, or an equivalent mutation, to block tryptophan docking into the hydrophobic acceptor pocket); and
(ii) a first cadherin molecule in which the N-terminal binding strand of one cadherin molecule (for example the EC1 domain βA strand of mature N-cadherin with tryptophan at amino acid position 2) has been functionally mutated, for example by removal or replacement of tryptophan at amino acid position 2 of mature N-cadherin; and a second cadherin molecule as the second cadherin molecule in (i) above.
Additionally or alternatively, the cadherin molecules may, for example, be modified by one or more mutations to the βA strand of domain EC1 which remove, add or substitute one or more amino acids so as to inhibit or diminish intramolecular binding and/or to enhance or facilitate intermolecular adhesion.
Additionally or alternatively, the cadherin molecules may be modified by contacting one or both cadherin molecules with one or more substances which enhance intermolecular adhesion and/or reduce or eliminate intramolecular binding. The substance may be an organic molecule, preferably a small organic molecule, a drug, a peptide, a peptidometic, an antibody and/or a modified cadherin molecule that contacts each cadherin molecule. For example, the substance may bind to the cadherin molecules such that intramolecular binding is reduced or inhibited by diminishing or preventing the formation of the intramolecular ionic bond between the NH2 terminus of each cadherin molecule with the contact acidic amino acid residue of each cadherin molecule, and/or by preventing or diminishing intramolecular binding of an N-terminal binding strand of each cadherin molecule with a binding strand acceptor pocket of each cadherin molecule, such that intermolecular adhesion is enhanced.
Further provided according to the present invention is a pair of polypeptides which adhere to each other with an affinity greater than that between wild-type human N-cadherin molecules. Here, the polypeptides may be chemically synthesised using techniques well known to the person skilled in the art, for example. The structure of the polypeptides may be based on cadherin molecules (or functional fragments thereof) with desired intermolecular adhesion properties, for example the mutated cadherin molecules as described herein.
Also provided according to the present invention is a method of adhering a pair of polypeptides such as cadherin molecules by intermolecular adhesion, comprising contacting the polypeptides or cadherin molecules as defined herein, thereby allowing intermolecular adhesion.
Further provided is a method of increasing adhesion between two cadherin molecules, comprising reducing or eliminating intramolecular binding within each cadherin molecule and allowing formation of an ionic bond between an acidic amino acid residue of one cadherin molecule and the NH2 terminus of the other cadherin molecule. Here, intermolecular adhesion may be facilitated by binding of an N-terminal binding strand of one cadherin molecule with a binding strand acceptor domain of the other cadherin molecule.
In a further aspect of the invention there is provided a substance (see above) which modulates intramolecular binding of one or more cadherin molecules by reducing or enhancing intermolecular adhesion between the molecules, wherein the substance excludes antibodies.
Also provided according to the present invention is the use of a substance (including antibodies and substances elaborated above) which modulates intramolecular binding of one or more cadherin molecules in order to modulate intermolecular adhesion between the molecules.
In another aspect there is provided a method for screening a candidate compound for the ability to modulate cadherin-mediated cell adhesion, comprising contacting the pair of cadherin molecules or the pair of polypeptides as defined herein in the presence and absence of the candidate compound and thereby evaluating the ability of the candidate compound to modulate cadherin-mediated cell adhesion.
There is also provided a method of increasing adhesion between a first cell and a second cell, comprising contacting the pair of cadherin molecules or the pair of polypeptides as defined herein, when one of the pair is attached to the first cell and the other of the pair is attached to the second cell.
The invention further provides an isolated nucleic acid molecule encoding the pair of cadherin molecules or the pair of polypeptides as defined herein. Alternatively, the invention provides a pair of isolated nucleic acid molecules in which each nucleic acid molecule encodes one of the pair of cadherin molecules or one the pair of polypeptides as defined herein. Also provided is an isolated nucleic acid molecule which hybridises under low stringent conditions, moderately stringent conditions or highly stringent conditions with the above-mentioned isolated nucleic acid molecule. The phrase “low stringency conditions” as used herein refers to hybridisation in 10% formamide, 5× Denhart's solution, 6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at 50° C. As used herein, the phrase “moderately stringent conditions” refer to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the target DNA, with greater than about 90% identity to target-DNA being especially preferred. Preferably, moderately stringent conditions are conditions equivalent to hybridisation in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C. The phrase “high stringency conditions” are conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridisation in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. For solutions and methods, see for example Sambrook et al. (1989).
In another aspect of the invention there is provided a host cell comprising any of the group consisting of: the cadherin molecules (for example, one or both of the pair of cadherin molecules), the polypeptides (for example, one or both of the pair of polypeptides), the isolated nucleic acid molecule(s), and one or both of the pair of isolated nucleic acid molecules, as defined herein.
In alternative aspects of the invention, there is provided a pair of cadherin molecules modified to reduce or eliminate intermolecular adhesion compared with corresponding unmodified cadherin molecules, a pair of polypeptides which adhere to each other with an affinity lower than that between wild-type human N-cadherin molecules, a method of decreasing adhesion between tow cadherin molecules, and a method of decreasing adhesion between a first cell and a second cell. In these alternative aspects, the strategy as outlined for increasing adhesion is reversed, so as to favour intramolecular binding within the respective cadherin molecules or polypeptides.
The invention further provides a kit comprising any of the group consisting of: the cadherin molecules (for example, the pair of cadherin molecules), the polypeptides (for example, the pair of polypeptides), the isolated nucleic acid molecule(s), the pair of isolated nucleic acid molecules, and the host cell, as defined herein.
There is also provided according to the present invention a method for adhering two cadherin molecules, comprising the steps of:
(i) providing a first cadherin molecule with a binding domain comprising an N-terminus and a bridge amino acid residue (or “contact acidic amino acid residue” as described herein) at a site remote from the N-terminus and corresponding to residue Glu89 of mature wild-type human N-cadherin, in which the N-terminus of the first cadherin molecule is disrupted or prevented from forming an intramolecular ionic bond with the bridge amino acid of the first cadherin molecule;
(ii) providing a second cadherin molecule with a binding domain comprising an N-terminus and a bridge amino acid residue at a site remote from the N-terminus and corresponding to residue Glu89 of mature wild-type human N-cadherin, in which the bridge amino acid of the second cadherin molecule is disrupted or prevented from forming an intramolecular ionic bond with the N-terminus of the second cadherin molecule; and
(iii) contacting the first and second cadherin molecules.
Also provided is a method for adhering two cadherin molecules, comprising the steps of:
(i) providing a first cadherin molecule with a binding domain (or “N-terminal binding strand” as described herein) comprising a ligand amino acid residue corresponding to residue Trp2 of mature wild-type human N-cadherin and a ligand-acceptor hydrophobic pocket (or “binding strand acceptor pocket” as described herein) corresponding to the Trp2-acceptor hydrophobic pocket of mature wild-type human N-cadherin, in which the ligand amino acid of the first cadherin molecule is disrupted or prevented from intramolecular docking between (or into) the ligand-acceptor hydrophobic pocket of the first cadherin molecule;
(ii) providing a second cadherin molecule with a binding domain comprising a ligand amino acid residue corresponding to residue Trp2 of mature wild-type human N-cadherin and a ligand-acceptor hydrophobic pocket corresponding to the Trp2-acceptor hydrophobic pocket of mature wild-type human N-cadherin, in which the ligand amino acid of the second cadherin molecule is disrupted or prevented from intramolecular docking between (or into) the ligand-acceptor hydrophobic pocket of the second cadherin molecule; and
(iii) contacting the first and second cadherin molecules.
Also provided is a method for adhering two cadherin molecules, comprising the steps of:
(i) providing a first cadherin molecule with a binding domain which is disrupted or prevented from forming an intramolecular ionic bond between an N-terminus of the first cadherin molecule and a bridge amino acid residue of the first cadherin molecule at a site remote from the N-terminus and corresponding to residue Glu89 of mature wild-type human N-cadherin;
(ii) providing a second cadherin molecule with a binding domain which is disrupted or prevented from intramolecular docking between (or of) a ligand amino acid residue of the second cadherin molecule corresponding to residue Trp2 of mature wild-type human N-cadherin and (or into) a ligand-acceptor hydrophobic pocket of the second cadherin molecule corresponding to the Trp2-acceptor hydrophobic pocket of mature wild-type human N-cadherin; and
(iii) contacting the first and second cadherin molecules.
Step (iii) of the above methods may allow formation of an intermolecular ionic bond between a bridge amino acid residue on one cadherin molecule at a site remote from an N-terminus corresponding to residue Glu89 of mature wild-type human N-cadherin of the cadherin molecule and an N-terminus of the other cadherin molecule. Step (iii) of the above methods may additionally or alternatively allow intermolecular docking between a ligand amino acid on one cadherin molecule corresponding to residue Trp2 of mature wild-type human N-cadherin and a ligand-acceptor hydrophobic pocket of the other cadherin molecule corresponding to the Trp2-acceptor hydrophobic pocket of mature wild-type human N-cadherin.
Adhesion between the two cadherin molecules may be increased, for example compared to adhesion between two mature wild-type human N-cadherin molecules.
The intramolecular ionic bond may comprise or consist of a salt bridge.
The N-terminus and/or bridge amino acid may be prevented or disrupted from forming an intramolecular ionic bond by one or more of the following: an N-terminal extension of the binding domain from a distal amino acid residue corresponding to residue Asp1 of mature wild-type human N-cadherin (for example, an N-terminal extension comprising the amino acids GG or MDP); a molecule (for example a peptide, a peptidometic or an antibody) which binds to or near the distal amino acid residue; a molecule (for example a peptide, a peptidometic or an antibody) which binds to or near the bridge amino acid residue; and a functional mutation in the bridge amino acid residue (for example E89A).
The ligand amino acid residue and/or ligand-acceptor hydrophobic pocket may be disrupted or prevented from intramolecular docking by one or more of the following: a functional mutation in the ligand amino acid residue (for example W2G); a molecule (for example a peptide, a peptidometic or an antibody) which binds to or near the ligand amino acid residue; a molecule (for example an antibody, a peptide or a peptidometic) which binds to or near the ligand-acceptor hydrophobic pocket; a functional mutation in the ligand-acceptor hydrophobic pocket (for example A80I); and a peptide, peptidometic, drug or antibody which binds at or near to the base of the βA strand (or “N-terminal binding strand”) of one cadherin molecule.
Also provided is the use of the first cadherin molecule as defined above for binding to the second cadherin molecule as defined above, comprising contacting the first cadherin molecule with the second molecule.
The present invention provides in a further aspect a method of modifying the binding domain of a first cadherin molecule to modulate its binding with a complementary binding domain of a second cadherin molecule by ablating or reducing intramolecular docking within the binding domain and complementary binding domain, thereby making the binding domain of the first cadherin molecule available for intermolecular binding with the complementary binding domain of the second cadherin molecule, for example making the βA strand (or “N-terminal binding strand”) of the binding domain of the first cadherin molecule available for intermolecular binding with the complementary binding domain of the second cadherin molecule.
As used herein, the βA strand of cadherin is a strand corresponding to approximately the first ten to twelve N-terminal amino acid residues of mature wild-type cadherins, or a functional homologue thereof. In a preferred embodiment, the “N-terminal binding strand” of the cadherin molecule as described herein comprises or consists of the βA strand of cadherin. For mature wild-type human N-cadherin and most classical cadherins, Trp2 forms the second amino acid in the βA strand from the N terminus.
The invention also provides a method for modulating (for example, increasing) adhesion between two or more cadherin molecules comprising the step of contacting a first cadherin molecule with a complementary second cadherin molecule such that the N-terminus of only one of the cadherin molecules forms an intermolecular ionic bond (such as a salt bridge) with an amino acid residue (preferably an acidic amino acid residue) corresponding to residue Glu89 of mature wild-type human N-cadherin on the other cadherin molecule or an alternative acidic amino acid in the immediate vicinity (for example, within 1, 2, 3, 4, 5, 6, 10 or more amino acids from Glu89). Furthermore, an intermolecular bond between a ligand amino acid residue corresponding to residue Trp2 of mature wild-type human N-cadherin on one cadherin molecule may be formed with a ligand-acceptor hydrophobic pocket corresponding to the Trp2-acceptor hydrophobic pocket of mature wild-type human N-cadherin on the other cadherin molecule.
Also provided is a pair of cadherin molecules with complementary extracellular domains, wherein the N-terminus of only one of the cadherin molecules forms an intermolecular ionic bond (such as a salt bridge) with an amino acid residue (preferably an acidic amino acid residue) on the other cadherin molecule corresponding to residue Glu89 of mature wild-type human N-cadherin when the molecules are contacted. The pair of cadherin molecules when contacted may also form an intermolecular bond between a ligand amino acid residue corresponding to residue Trp2 of mature wild-type human N-cadherin on one cadherin molecule with a ligand-acceptor hydrophobic pocket corresponding to the Trp2-acceptor hydrophobic pocket of mature wild-type human N-cadherin on the other cadherin molecule.
In one embodiment of the invention, the base (or hinge) of a βA strand (or “N-terminal binding strand”) of a cadherin molecule is modified to place the βA strand into a position where intramolecular binding (or adhesion) is prohibited or reduced. The βA strand (or “N-terminal binding strand”) of the cadherin molecule may thus be able to bind to a complementary cadherin molecule by intermolecular binding (such as an intermolecular ionic bond and/or a ligand-acceptor hydrophobic bond of the type herein described). The molecule may be modified for example by a drug, a peptide, peptidometic or an antibody which targets the base of the βA strand (or “N-terminal binding strand”) or by substituting one or more amino acids in the βA strand with alternative amino acids or by adding or removing one or more amino acids from the βA strand without substitution. A modified cadherin molecule or pair of modified cadherin molecules thus formed are also within the scope of the present invention.
The βA strand (or “N-terminal binding strand”) of each of two cadherin molecules may form an intermolecular ionic bond (for example, a salt bridge) of the type herein described.
The invention encompasses the situation in which intramolecular binding or binding of the βA strand (or “N-terminal binding strand”) of each of two cadherin molecules is prevented while their intermolecular interaction is permitted.
The cell adhesion modulating agent of the present invention may be one or more cadherin molecules or polypeptide as described herein, or an agent that effects increased or decreased adhesion of cadherin molecules as described herein (for example, the substance as described herein). The cell adhesion modulating agent may also be a candidate compound detected by the method as described herein.
In another aspect of the invention, there is provided a method of stabilising adhesion between two cadherin molecules, comprising forming one or more thiol (for example, disulphide) bonds between amino acid residues, preferably cysteine residues, which are in close apposition during cadherin adhesion. For example, each cadherin molecule may comprise cysteine residues corresponding to amino acid positions 1 and/or 27 of wild-type mature human N-cadherin, which may be achieved for example by mutating Asp1 and Asp27 to Cys1 and Cys27, respectively. The invention also covers one or a pair of cadherin molecules comprising a structure modified for stabilising according to the above method.
The cadherin molecule or polypeptide of the present invention may comprise a modified mature wild-type human classical (type I) cadherin (for example N-cadherin, R-cadherin, E-cadherin, C-cadherin, P-cadherin, M-cadherin or T-cadherin), non-classical (type II) cadherin (for example VE-cadherin), desmosomal cadherin (for example desmocollin-1, desmocollin-2, desmocollin-3, desmoglein-1, desmoglein-2 or desmoglein-3), or protocadherin, or a functional homologue or functional fragment thereof (for example, modified extracellular domains 1 and 2 of wild-type human N-cadherin).
Each of or both cadherin molecules or polypeptides may comprise a modified N-cadherin-Fc fusion protein.
Each of or both cadherin molecules or polypeptides may be attached to one or more or the following: a cell; a surface (for example an assay surface such as a plastic plate); a magnetic bead; a non-magnetic bead; a solid matrix; and a semi-solid matrix.
Mature wild-type human N-cadherin has the amino acid sequence:SEQ ID No 1
The mature wild-type human N-cadherin sequence corresponds to residues 160 to 906 of precursor Neural-cadherin (N-cadherin) provided in Genbank accession No. P19022.
As used herein, unless otherwise stated, the term “cadherin” or “cadherin molecule” encompasses a functional fragment, homologue or variant thereof. The terms “cadherin molecule(s)” or “polypeptide(s)” as used in the present invention also include within their scope a functional fragment, equivalent, homologue or variant of wild-type mature human N-cadherin (see below). Each cadherin molecule or each polypeptide may have at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or greater homology with wild-type mature human N-cadherin or a functional fragment, equivalent, homologue or variant of wild-type human N-cadherin. Alternatively, each cadherin molecule or each polypeptide may have at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80, 85, 90, 95% or greater homology with wild-type mature human N-cadherin or a functional fragment, equivalent, homologue or variant of wild-type human N-cadherin excluding the transmembrane and/or cytoplasmic domains of wild-type human N-cadherin.
In specific embodiments described below, the extracellular region (or “ectodomain”) of N-cadherin is fused to human IgG Fc to form a N-cadherin-Fc fragment which is used in binding assays. In such specific embodiments, it is the cadherin ectodomain which exhibits binding function. The term “cadherin” or “cadherin molecule” may thus encompass a molecule (for example, a polypeptide) comprising the cadherin ectodomain but lacking other cadherin domains. Similarly, the term “cadherin” or “cadherin molecule” encompasses a molecule (for example, a polypeptide) which is a functional fragment, homologue or variant of at least one EC domain of a cadherin molecule. The molecule or polypeptide in one embodiment comprises the EC1 and EC2 domains of cadherin (for example, of mature human N-cadherin as defined above) but lacks other cadherin domains.
The invention provides in one aspect a method for enhancing delivery of a drug to a cell (for example a tumour cell or a central nervous system cell), comprising administering to the cell: (a) a cell adhesion modulating agent as described herein that modulates cadherin-mediated cell adhesion; and (b) a drug; and thereby enhancing the delivery of the drug to the cell.
Also provided according to the invention is a method for inhibiting the development of cancer in an animal (for example a mammal such as a human), comprising administering to the animal a cell adhesion modulating agent as described herein that modulates cadherin-mediated cell adhesion, thereby inhibiting development of cancer in the animal. A cell adhesion modulating agent which functions as an anticancer compound is further provided according to present invention. Such anticancer compounds may be used for example in combination with classical anti-cancer therapy (for example, irradiation) to prevent tumour cells from migration.
In another aspect there is provided a method for inhibiting angiogenesis in an animal (for example a mammal such as a human), comprising administering to the animal a cell adhesion modulating agent as described herein that modulates cadherin-mediated cell adhesion, thereby inhibiting angiogenesis in the animal. Angiogenesis is the growth of blood vessels that occur in both normal and diseased cells. In cancer cells there is uncontrolled cellular growth sustained by hyper active angiogenesis. Therapeutics that can specifically target the blood supply of cancer cells are known as angiolytics and have been widely studied since the 1990s which led to the first approved anti-angiogenic, Avastin, appearing in early 2004. Angiolytic drugs are also known as vascular targeting agents (“VTAs”) and are a new class of drug designed to cause structural damage to the cells of blood vessels which in turn limits blood flow to a vascularised tumour cell causing cell death. The present invention allows for the process of angiogenesis to be inhibited by using a cell adhesion modulating agent as described herein that modulates cadherin-mediated cell adhesion.
In a further aspect there is provided a method for enhancing wound healing in an animal (for example a mammal such as a human), comprising administering to the animal a cell adhesion modulating agent as described herein that modulates cadherin-mediated cell adhesion, thereby enhancing wound healing in the animal.
The invention further provides a method for enhancing adhesion of foreign tissue implanted within an animal (for example a mammal such as a human), comprising administering to the animal a cell adhesion modulating agent as described herein that modulates cadherin-mediated cell adhesion, thereby enhancing adhesion of foreign tissue implanted within the animal.
Also provided is a method for modulating the immune system of an animal (for example a mammal such as a human), comprising administering to the animal a cell adhesion modulating agent as described herein that modulates cadherin-mediated cell adhesion, thereby modulating the immune system of the animal.
In another aspect, there is provided a method for modulating vasopermeability in an animal (for example a mammal such as a human), comprising administering to the animal a cell adhesion modulating agent as described herein that modulates cadherin-mediated cell adhesion, thereby modulating vasopermeability in the animal.
Also provided is a method for treating a demyelinating neurological disease in an animal (for example a mammal such as a human), comprising administering to the animal: (a) a cell adhesion modulating agent as described herein that inhibits cadherin-mediated cell adhesion; and (b) one or more cells capable of replenishing an oligodendrocyte population; and thereby treating a demyelinating neurological disease in the animal.
Further provided is a method for facilitating migration of an N-cadherin expressing cell on astrocytes, comprising contacting an N-cadherin expressing cell with: (a) a cell adhesion modulating agent as described herein that inhibits cadherin-mediated cell adhesion; and (b) one or more astrocytes; and thereby facilitating migration of the N-cadherin expressing cell on the astrocytes.
The invention also provides a method for inhibiting synaptic stability in an animal (for example a mammal such as a human), comprising administering to the animal a cell adhesion modulating agent as described herein that inhibits cadherin-mediated cell adhesion, and thereby inhibiting synaptic stability in the animal.
Further provided is a method for modulating neurite outgrowth, comprising contacting a neuron with a modulating agent of the type herein described, and thereby modulating neurite outgrowth.
Also provided is a method for treating spinal cord injuries in an animal (for example a mammal such as a human) comprising administering to the animal a cell adhesion modulating agent as described herein that enhances neurite outgrowth, and thereby treating a spinal cord injury in the animal.
Additionally provided is a method for treating macular degeneration in an animal (for example a mammal such as a human), comprising administering to the animal a cell adhesion modulating agent as described herein that enhances classical cadherin-mediated cell adhesion, and thereby treating macular degeneration in the animal.
Also provided is a method for reducing unwanted cellular adhesion in an animal (for example a mammal such as a human), comprising administering to the animal with unwanted cellular adhesion a modulating agent as described herein and thereby reducing unwanted cellular adhesion, wherein the modulating agent inhibits cadherin mediated cell adhesion.
Further provided is a method for enhancing the delivery of a pharmaceutical active substance through the skin of an animal (for example a mammal such as a human), comprising contacting epithelial cells of the animal with a pharmaceutical active substance and a modulating agent as described herein and thereby enhancing the delivery of the substance through the skin, wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of the substance across the epithelial cells, and wherein the modulating agent inhibits cadherin mediated cell adhesion.
Additionally provided is a method for inducing apoptosis in a cadherin-expressing cell, comprising contacting a cadherin-expressing cell with a modulating agent as described herein and thereby inducing apoptosis in the cell, wherein the modulating agent inhibits cadherin mediated cell adhesion.
Also provided is a method for preventing pregnancy in an animal (for example a mammal such as a human), comprising administering to the animal a modulating agent as described herein and thereby preventing pregnancy in the animal, wherein the modulating agent inhibits cadherin mediated cell adhesion.
Further provided is a method for facilitating blood sampling in a mammal, comprising contacting epithelial cells of a mammal with a cell adhesion modulating agent as described herein and thereby facilitating blood sampling in the mammal, wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of one or more blood components across the epithelial cells, wherein the modulating agent inhibits cadherin mediated cell adhesion.
Additionally provided is a method for stimulating blood vessel regression, comprising administering to an animal (for example a mammal such as a human) a cell adhesion modulating agent as described herein and thereby stimulating blood vessel regression, wherein the modulating agent inhibits cadherin mediated cell adhesion.
The invention further provides a method for inhibiting endometriosis in a mammal, comprising administering to the mammal a cell adhesion modulating agent as described herein, wherein the modulating agent inhibits cadherin mediated cell adhesion.
Also provided is a method for enhancing inhaled compound delivery in an animal (for example a mammal such as a human), comprising contacting lung epithelial cells of the animal with a cell adhesion modulating agent as described herein and thereby enhancing inhaled compound delivery, wherein the modulating agent inhibits cadherin mediated cell adhesion.
Local disruption of cell-cell junctions in the skin or endothelial lining of blood vessels would increase permeability of the barrier and therefore improve access of drugs to the underlying tissues. E-cadherin, VE-cadherin and N-cadherin may be considered as principal targets here. The strategy would apply to topical application of drugs to the skin or oral mucosa or to anti-cancer drugs injected into tumour vasculature. The same logic applies to the blood-brain barrier (BBB) where control of permeability to drugs, especially in treating brain tumours, is a major problem. Cadherins make an important contribution to the BBB and its development. The current strategy used to increase permeability of the BBB, hyertonic shock, could be supplemented or replaced by a drug that disrupts cadherin-mediated adhesion via the salt bridge.
In addition to increasing vascular permeability for drug delivery to solid tumours, disruption of cadherin junctions in endothelial cells would also be expected to cause apoptosis. Not only do cadherin-cadherin interactions in endothelial cells maintain the integrity of the barrier, they also impart survival signals to endothelial cells. A cadherin antagonist injected locally into solid tumour vasculature would therefore disrupt the blood supply and cause tumour shrinkage.
A hallmark of malignant tumours is a propensity for invasiveness and metastatic spread. Adhesion by the principal epithelial cadherin, E-cadherin, is usually reduced in cancer and this is a consequence of mutational damage to the molecule or to decreased cell surface expression. Reduction in E-cadherin function is often accompanied by an increase in expression of N-cadherin which triggers signalling pathways which cause epithelial/mesenchymal transition and invasive behaviour. The present disclosure points the way to the rational design of drugs which would increase the affinity of E-cadherin-mediated cell adhesion by a large factor. An ideal strategy would be to combine this with an specific inhibitor of N-cadherin function. A drug to increase the affinity of E-cadherin-mediated adhesion may be designed to prevent intramolecular docking of Trp2 in domain 1 and formation of the intramolecular salt bridge, but would permit intermolecular βA strand exchange and formation of the salt bridge in trans. We have demonstrated the feasibility of this approach with our experimental cadherin constructs (see below). Our understanding of the strand exchange mechanism encourages optimism that drugs could be used to increase affinity of E-cadherin adhesion by several orders of magnitude. This effect is likely to counteract the deficiencies in cell adhesion which cause metastasis. The strategy could very useful in pre-cancerous conditions, particularly where topical application of the drug could be used to avoid possible adverse systemic effects. For example, in the mouth many oral cancers arise in pre-existing white patches (leukoplakia). In some cases these white patches are very widespread and management is a problem because it is not possible to remove them and it is also very difficult to determine whether they have become malignant. It would be feasible to apply a drug topically to these patches to increase epithelial adhesion and prevent invasion and cancer development. A similar strategy would be appropriate also for cervical cancer and for some skin cancers, e.g. basal cell carcinomas. These are specific examples but potential applications could be much wider, as would be appreciated by a person skilled in the art.
In another aspect, the invention may be used to prevent cancer (for example tumoural) cells from detaching and invading other tissue (anti-metastatic).
Pemphigus is a group of potentially life-threatening autoimmune diseases characterised by cutaneous and mucosal blistering. Pemphigus vulgaris is caused by failure in desmosomal cadherin-mediated adhesion due to the presence of autoantibodies against Dsg3 and/or Dsg1. Current treatment is centered on the use of immunosuppressive drugs and steroids to reduce antibody levels. A drug to enhance the affinity of the adhesive interaction between cadherins, both E-cadherin and/or desmosomal cadherins, would be very beneficial in maintaining integrity of the epidermal barrier. Similar logic applies to the treatment of other exfoliative skin diseases, e.g. Staphylococcal scalded skin syndrome and bullous impetigo.
The mutant cadherins described herein that increase adhesion (for example E89A and the N-terminal extension Gly Gly; see also for example
In one biotechnological application, a first modified cadherin molecule according to the invention and a second modified cadherin molecule according to the invention are provided such that the molecules exhibit enhanced intermolecular adhesion. These first and second molecules are termed “SuperCadh-1” and “SuperCadh-2”, respectively, in the following two embodiments.
In the first embodiment, SuperCadh-1 and SuperCadh-2 may be used to isolate, quantify, identify and/or characterise one or more cells or molecules. SuperCadh-1 may be chemically linked to a binding substance such as streptavidin. This will provide a means to attach SuperCadh-1 into a SuperCadh-1-complex including an antibody adapted to bind with the binding substance, for example a biotinylated antibody which binds to SuperCadh-1 linked to streptavidin through the interaction of streptavidin with biotin. The antibody may be cross-reactive with the cell or molecule. SuperCadh-2 may be attached to a surface such as a plate (such as a plastic dish or well), matrix, bead or column. When placed into contact with each other in the presence of a medium containing calcium ions, SuperCadh-2 will adhere to SuperCadh-1 thereby allowing retention on the surface of the SuperCadh-1-complex including the cell or molecule. After optional washing steps, release of the SuperCadh-1-complex from the surface may be effected by removing calcium ions from the medium, for example using a chelating agent such as EGTA (ethylene glycol-bis[β-aminoethyl ether]-N,N,N′,N′-tetraacetic acid). In one example, CD4+ lymphocytes are isolated from whole blood using this method. Positively selected cells are recovered by adding EGTA and cell number and viability are then assessed.
In the second embodiment, a variation of the first embodiment above, there is provided a method for isolating, quantifying, identifying or characterising one or more cells which are genetically modified following insertion of a nucleotide sequence (for example, a gene or an RNAi molecule) of interest. Here, the one or more cells may be genetically modified also to express SuperCadh-1 on the surface of the one or more cells. In one example, the nucleotide sequence of interest and SuperCadh-1 may be co-expressed or expressed simultaneously by the cell (for example under the control of the same promoter). SuperCadh-1 expressed by the one or more cells may then be allowed to adhere to SuperCadh-2 in the presence of a medium containing calcium ions, thereby allowing retention of the one or more cells onto a surface such as a matrix. In this way, one or more cells which have been successfully genetically modified may be selected, isolated, characterised and/or purified. After optional washing steps, release of the cells expressing SuperCadh-1 from the surface may be effected by removing calcium ions from the medium, for example using a chelating agent such as EGTA. In one example, a vector encoding SuperCadh-1 and green fluorescent protein (GFP) is transfected into a variety of mammalian cells lines using standard techniques. Cell surface expression of SuperCadh-1 is used to select cells transfected with GFP by adhesion to a matrix coated with SuperCadh-2. Cell number and viability are assessed after release from the matrix by calcium removal using EGTA.
In each of the first and second embodiments described above, a simple, fast, non-aggressive and non-stressful method for cell or molecule recovery is provided. The simple addition of a chelating agent, for example, reverses adhesion between SuperCadh-1 and SuperCadh-2, allowing cell or molecule collection without the need for scraping, sorting by cytofluorometry, or addition of large quantities of peptides.
The invention also provides a method for reducing aggregation of cultured cells, comprising contacting cultured stem cells with a cell adhesion modulating agent as described herein and thereby reducing aggregation of stem cells, wherein the modulating agent inhibits cadherin mediated cell adhesion.
So that the manner in which the above-recited features, advantages and objects of the invention, as well as others which will become apparent, are attained and can be understood in detail, more particular description of the invention summarised above may be had by reference to the embodiments thereof illustrated in the accompanying drawings which form a part of this specification. It is to be noted, however, that the accompanying drawings illustrate only specific embodiments of the invention and are therefore not to be considered limiting of its scope as the invention may admit to other equally effective embodiments.
In the drawings:
a)-(c)—are graphs showing the binding of peptide-specific antibody K7 to wild type and mutant N-cadherin-Fc fusion protein.
a)-(i) are graphs showing binding of mAb GC4 to dimeric N-cadherin-Fc mutants in the presence or absence of calcium.
a) & (b) are graphs showing the effect of cysteine point mutations on the adhesive capacity of monomeric N-cadherin-Fc.
a)-(c) show expression of cell surface N-cadherin by K562 transfectants and by DX3 melanoma cells.
a) & (b) are Western blots showing the formation of ‘reporter’ disulphide bonds during cadherin-mediated cell adhesion.
a) is a graphic and (b) shows immunofluorescent staining results, pertaining to the salt bridge between Asp1 and Glu 89 of N-cadherin.
a)-(c) are graphs showing the effect of the salt bridge on cadherin-mediated cell adhesion.
a), (c) are graphs and (b) is a western blot, relating to effect of the N-cadherin prodomain on adhesion.
a) shows graphs and (b) shows photographs, demonstrating the effect on binding of removing Trp2 or blocking the hydrophobic acceptor pocket of N-cadherin.
The figure legends in more detail are as follows:
In
In
In
a) shows binding of mAb GC4 to dimeric N-cadherin Fc bearing point mutations, in the presence of 1.25 mM calcium. In
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In
In
In
In
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a) shows adhesion of N-cadherin transfectants to wild type or mutant N-cadherin Fc fusion proteins coated at 1 g/ml. Extension of the N-terminus by two amino acids, GG, or the mutation E89A inhibited adhesion to wild type N-cadherin but, when present in opposing molecules, they formed a complementary pair resulting in enhanced adhesion. The mutation D134A prevents co-ordination of calcium in the junction between domains 1 and 2 and served as a negative control.
In
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In the present example we have used antibodies to detect conformational changes in EC1 of N-cadherin, prepared as an Fc-fusion protein, to investigate the effect of Trp2 and Ca2+ on the stability of this domain. In addition we have investigated the effect of calcium on the propensity of Trp2 to dock into a hydrophobic pocket in its own domain. Finally, we provide persuasive evidence for strand exchange as the primary event in adhesion. A novel strategy has been used involving the formation of a ‘reporter’ disulphide bond which captures mutant cadherin molecules in trans-alignment as cells undergo adhesion. This bond can form only if the molecules are orientated by strand exchange.
A polyclonal sheep antiserum was prepared by standard methods against the synthetic peptide PQELVRIRSDRDK SEQ ID No 2, which spans the MB strand of chicken N-cadherin. The peptide was conjugated to keyhole limpet haemocyanin for immunization. Preliminary experiments established that this antibody did not react with wild type N-cadherin-Fc in its native conformation but gave a strongly positive result with N-cadherin-Fc that had been partially denatured by direct adsorption to a plastic surface. The rat mAb NCD-2, specific for an epitope in the BC loop of domain 1 of chicken N-cadherin, was obtained from R & D Systems (code BTA6). Mouse mAb 8C11, specific for domain 4 of human N-cadherin, was a gift from Dr M J Wheelock (Puch et al., 2001) and mouse mAb GC4 (also known as GB-9) was obtained from Sigma (code C2542). A rabbit pan anticadherin antiserum specific for a conserved sequence of 24 amino acids in the cytoplasmic domain (Sigma, code C3678) was used for immunoblotting.
Antibody binding to N-cadherin-Fc fusion proteins was detected by enzyme-linked immunosorbent assay (ELISA) based on a previously described method (Corps et al., 2001). Briefly, assay plates were coated with varying levels of monomeric or dimeric Ncadherin Fc-fusion proteins via rabbit or goat anti-human Fc. Assay plates were then pre-equilibrated for 7 minutes with varying levels of calcium chloride added to calcium-free Hanks balanced salt solution (HBSS), containing 0.075% Tween 20. Antiserum K7 (1:75) or mAb GC4 (2 μg/ml) was then added in IBSS containing the appropriate level of calcium and incubated for 1 hour at room temperature. Antibody binding was detected with anti-sheep or anti-mouse HRP-labelled secondary antibody. Assays were conducted in duplicate or triplicate and results are presented as mean +/−sem.
Predictions of disulphide bond formation were based on the strand exchange structure PDB 1NCI (Shapiro et al., 1995). It was viewed and manipulated using Swiss PDB Viewer (http://www.expasy.org/spdbv/). Two extra amino acids at the N-terminus of this structure were removed to give the correct sequence. Alternative pairs of mutations, D1C,R25C or D1C,D27C, were introduced so that either pair would form a disulphide bond between the two domain 1 protomers; numbering refers to the mature cadherin protein. Formation of the C1-C25 disulphide bond required torsion, within Ramachandran limits, of psi angles in the α-carbon backbone of the βA strand in the vicinity of Val3, while maintaining the side chain of Trp2 in an unchanged position. Formation of the C1-C27 disulphide bond required only rotation of the side chain of C1.
Adjustments were also made to the side chains of C25 and C27. After energy minimisation, the beta carbon atoms of the paired cysteines for both bonds were within 4A, which is optimal for disulphide bond formation. There were no amino acid clashes in either case.
Full length chicken N-cadherin cDNA in pcDNA3.1 was obtained from Prof. P Doherty, King's College, London. The point mutations D1C, R25C and D27C were introduced using a QuikChange mutagenesis kit (Stratagene). Mutant and wild type constructs were stably transfected into the human myeloid leukemic cell line K562 by electroporation and selection in G418 (1 mg/ml G418 in DMEM+10% FCS). Clonal cell lines were obtained by limiting dilution and were matched for equal expression of N-cadherin by cell surface immunofluorescent staining and flow cytometry. Chicken N-cadherin-Fc fusion protein linked by disulphide bonds at the Ig hinge region to form a dimer was prepared as follows: cDNA for the five extracellular domains of N-cadherin, coding up to the amino acid sequence GLGT, was isolated by PCR and cloned into the vector pIgSig (R&D Systems). This vector provides a signal sequence from CD33 and adds the CH2 and CH3 domains and the hinge region of human IgG1 heavy chain. The construct was modified to produce monomeric N-cadherin-Fc by introducing the mutations F405A and Y407A into the CH3 domain of Fc (Dall'Acqua et al., 1998) which prevent dimerisation of this domain. The fidelity of all DNA constructs was verified by sequencing.
Soluble N-cadherin-Fc fusion protein was obtained by transient transfection of Cos7 Cells as previously described (Corps et al., 2003). Wild type monomeric N-cadherin-Fc and the double mutant W2G,D134A were checked for molecular size by gel filtration on a Superdex-200 PC3.2/30 Column equilibrated with 50 mM Tris.Cl, pH 7.4, 150 mM NaCl, 1 mM CaCl2 and were shown to be monodisperse and monomeric. Soluble N-cadherin-Fc was routinely quantitated using an ELISA for Fc, standardised against purified cadherin-Fc fusion proteins.
96 well immunoassay plates (Costar) were coated overnight with affinity-purified goat anti-human Fc (Sigma, code 12136) at 5 μg/ml in PBS, then blocked with 1% BSA for 2 hours at room temperature. Monomeric or dimeric N-cadherin-Fc fusion protein in Cos cell supernatants was added as described for the ELISA assay. DX3 Cells, a human melanoma cell line which expresses N-cadherin, were dissociated with Cell Dissociation Solution (Sigma, code C5789), resuspended in HBSS with 2% FCS and assessed for adhesion to wild type or mutant N-cadherin-Fc as previously described (Corps et al., 2001). Microscopic examination established that the cells were present as a single cell suspension as they settled onto the plate. For assays conducted in reducing conditions, 10 mM DTT was present during the adhesion and washing steps. Determinations were conducted in triplicate and results are presented as mean +/−sem.
Monomeric N-cadherin-Fc (1 μg/ml) bearing the three alternative cysteine point mutations, D1C, R25C or D27C, was immobilised on 96-well plates with goat antihuman IgG Fe as previously described for E-cadherin-Fc (Corps et al., 2001). Unbound cadherin was removed by washing with HBSS, 0.1% BSA, followed by HBSS alone. The plates were not blocked with additional protein. K562 transfectants expressing N-cadherin bearing complementary or non-complementary point mutations (6×104 Cells in 100 μl HBSS containing 10 mM DTT), in single cell suspension, were added to the coated wells which contained 100 μl HBSS. The final concentration of DTT was therefore 5 mM DTT during the adhesion stage. The cells were allowed to settle for 10 min at room temperature before incubation at 37° C. for 30 minutes to complete adhesion. Microscopic examination of the wells before washing showed that the cells were not clumped but remained as a carpet of single cells on the surface of the plate. Non-adherent cells were then removed by washing with HBSS without DTT to restore oxidising conditions (4-6 washes over 15 to 20 minutes). Adherent cells from a pool of 4 wells for each experimental condition were then solubilised in sample buffer for SDS-PAGE and analysed on NuPAGE Novex gradient gels, 3-8% or 4-12%, (Invitrogen), under nonreducing or reducing conditions. Cellular cadherin was detected by immunoblotting using rabbit pan anti-cadherin antiserum specific for the cytoplasmic domain. Alternatively, N-cadherin-Fc fusion protein was detected with rabbit anti-human IgG, Fc-specific (Pierce, code 31142). The secondary antibody for both was peroxidase-conjugated AffinPure goat anti-rabbit IgG, F(ab′)2 fragment-specific (Jackson ImmunoResearch Labs, code 111-035-006).
In an alternative protocol, Dynabeads (Dynal) coupled to Protein A were coated with dimeric N-cadherin-Fc (1 μg/ml), bearing the mutation D27C, for 1 hour at room temperature in the presence of 0.1% Tween 20 and 4 mM EGTA (to prevent aggregation). The beads were then washed with HBSS. K562 transfectants expressing cell surface Ncadherin with the mutations D1C, R25C or D27C, were treated with 10 mM DTT for 15 minutes at 37° C., then washed and resuspended in HBSS without DTT. Cells (2.4×105) were mixed with 3 μl of beads coated with mutant N-cadherin-Fc in a final volume of 100 μl HBSS. Cells and beads were incubated together at room temperature for 2 hours with slow rotation to allow adhesion. Approximately five beads became attached to each cell and there was some clumping of attached and unattached beads. Iodoacetamide, 2 μl of 1.0M solution, was then added to alkylate free sulphydryl groups. The samples were then spun down at 1500 g and the cells were lysed in HBSS, 0.075% SDS, 1% NP40, 0.2 mM AEBSF, for 4 minutes on ice. Beads were then isolated with a magnet and washed twice with lysis buffer. Disulphide-bonded complexes between cellular cadherin and the Fc-fusion protein were analysed by SDS-PAGE and immunoblotting for cadherin cytoplasmic domain as described above. To ensure equal loading, membranes were stripped and re-assayed with anti-Human Fc.
N-cadherin-Fc was purified using Protein A Sepharose as previously described (Corps et al., 2001). Samples were centrifuged at 100,000 g for 45 minutes to remove any aggregates and diluted to 1 μg/ml in 5 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH7.5, supplemented with 5 mM NiCl2. A volume of 50 μl was pipetted onto freshly cleaved mica (Goodfellow, Huntingdon, UK) and incubated at room temperature for 10 minutes. Unattached protein was then washed away with the same buffer. The protein molecules were then examined in the presence of 30 μl fresh buffer. AFM imaging was performed using a Nanoscope IIIa Multimode atomic force microscope (Veeco/Digital Instruments, Santa Barbara, Calif.) equipped with a J scanner. The N-cadherin-Fc molecules were imaged using oxide-sharpened silicon nitride probes (DNP-S; Digital Instruments) with a spring constant of 0.32 N/m operating in tapping mode at a drive frequency of ˜7-9 kHz.
Electron microscopic examination of cadherin molecules as pentamers fused to cartilage oligomeric matrix protein (COMP) or as dimers linked to immunoglobulin-Fc, has revealed ‘ring’ and ‘spectacle-like’ structures deemed to represent cis- and trans-(adhesive) dimerisation, respectively (Ahrens et al., 2003; Pertz et al., 1999). As a first step in the current series of experiments, we examined our preparation of N-cadherin-Fc protein by atomic force microscopy (AFM) in the presence of 5 mM calcium to see whether similar structures were detectable. All wild type molecules had a Y-shaped form with the cadherin domains curving away from each other and the Fc region clearly distinguishable as a structure forming the ‘stem’ of the Y (
A polyclonal antibody, K7, was prepared against a synthetic linear peptide in the βB strand of N-cadherin domain 1 (
The titrations were closely similar to those in
The epitope for a hitherto uncharacterized commercial N-cadherin antibody, GC4 (Volk and Geiger, 1984), was mapped using a panel of N-cadherin-Fc fusion proteins bearing point mutations (
The result is consistent with impaired Trp2 docking. In contrast, when calcium was removed, both wild type and the A78M mutant gave equally high binding (
An extension would be expected to have a negative effect on integration of Trp2 into the domain (Haussinger et al., 2004) but, again, would not necessarily preclude docking (Pertz et al., 1999; Schubert et al., 2002). In keeping with results for the A78M mutation, the MDP mutation strongly inhibited binding of GC4 in the presence of calcium (
The result was similar with the MDP extension mutant (
Elegant structural studies have demonstrated cadherin dimerisation by strand exchange (Boggon et al., 2002; Haussinger et al., 2004; Shapiro et al., 1995). The question remains whether this happens in a physiological context between opposing cadherin molecules during cell adhesion. Strand exchange would orientate the molecules so that specific amino acids near Trp2 and its hydrophobic pocket are brought into close apposition. We reasoned that if complementary cysteine point mutations were located in these positions they should generate a ‘reporter’ disulphide bond during cell adhesion, if strand exchange occurs. Using the strand exchange structure PDB 1NCI (Shapiro et al., 1995), we modelled formation of two alternative disulphide bonds in these circumstances using the complementary mutations D1C-D27C (
To test for the formation of disulphide bonds during cell adhesion, K562 Cells were transfected with N-cadherin bearing a single cysteine point mutation and allowed to adhere, in reducing conditions, to an assay plate coated with monomeric N-cadherin-Fc bearing the complementary mutation. Oxidising conditions were then restored and the formation of a disulphide bond between cellular cadherin and cadherin-Fc was detected by immunoblotting. Initially, essential parameters of the experimental strategy were validated by testing adhesion of N-cadherin+ve DX3 melanoma cells to N-cadherin-Fc molecules bearing the cysteine point mutations.
Monomeric N-cadherin-Fc, mutated to prevent Fc-Fc interaction, was used for most of our experiments in order to avoid the complication of disulphide bonded dimerisation in the hinge region of the Fc-fusion protein.
The K562 transfectants were allowed to adhere to N-cadherin-Fc monomer bearing complementary or non-complementary cysteine mutations. Trans-dimers were detected by immunoblotting, either for cellular cadherin using pan cadherin antibody to the cytoplasmic domain (
A second protocol was used to test for the formation of the disulphide bond in trans-alignment. It was designed to avoid detection of cis-dimers. In this strategy, mutant Ncadherin-Fc (the conventional fusion protein dimerised via Fc) was coated to magnetic beads which were allowed to adhere to the K562 transfectants. The cells were then lysed and the beads were separated from the lysate to extract the disulphide-bonded species in trans-alignment from the remainder of the cellular cadherin.
In this example we have used two antibodies to investigate the stability of domain 1 in relation to the roles of calcium and Trp2 and have demonstrated a major effect of both factors acting in concert. The data complements recent NMR studies (Haussinger et al., 2004) and provides a perspective that is not available from crystal structures. It is possible that antibody binding could itself direct conformational change, but this would be subject to the varying constraints imposed by calcium and Trp2 in our experiments and, therefore, would not affect our conclusions. The published crystal structures of cadherins all show a full complement of calcium atoms in the domain 1-2 junction, with or without intercalation of Trp2 into the domain structure. The α-carbon backbone is closely similar in all cases. In contrast, the original NMR structure of domain 1 of Ecadherin (Overduin et al., 1995) shows neither intercalated Trp2 nor correctly coordinated calcium atoms, and here the α-carbon trace shows significant displacement compared with that in the crystal structures. Our present data suggest that this NMR structure would be relatively unstable and the βB strand readily displaced from the domain. Results with the peptide antibody K7 show that Trp2 and calcium act in concert to stabilise domain 1, each by separate means limiting flexibility of the βB strand and constraining the overall conformation.
Binding of antibody GC4 showed an absolute requirement for Trp2, regardless of the presence or absence of calcium. Because this amino acid is located on the opposite side of the domain, 30 Å away from the GC4 epitope, the result argues persuasively that reactivity with GC4 requires Trp2 to be located in the hydrophobic cavity in domain 1. In these circumstances Trp2 would impose structural constraints on the GC4 epitope, either via the core of the domain or by limiting movement of the βA strand at its base. Our data show that reduction of calcium in the domain 1-2 junction increased GC4 binding. The effect was modest with wild type N-cadherin but greater with the mutant A78M or the N-terminally extended version MDP; each of these modifications would compromise intercalation of Trp2. The results taken together argue persuasively that calcium modulates the dynamic equilibrium between docked and undocked Trp2. Thus, at low calcium levels Trp2 is more firmly integrated than at physiological levels.
Dimerisation by strand exchange requires that Trp2 swaps from insertion in its own domain to that of its neighbour, overcoming an activation barrier (Haussinger et al., 2004). The present results are consistent with recent NMR data showing that calcium facilitates this process (Haussinger et al., 2004). Our interpretation of the effect of calcium predicts that dimerisation by strand exchange requires calcium but, once formed, the dimer can be isolated from the cell surface in buffers lacking calcium. This is consistent with empirical evidence (Chitaev and Troyanovsky, 1998; Klingelhofer et al., 2002).
Our data with GC4 reflect intramolecular docking of Trp2 rather than strand exchange because we obtained closely similar results (not shown) with monomeric N-cadherin-Fc over a wide titration range where, at lower coating levels, N-cadherin monomers would be widely spaced on the assay plate. In this example we did not test whether GC4 detects crossintercalation of Trp2, as well as intramolecular docking. It is notable that our cell adhesion experiments demonstrate that monomeric N-cadherin coated to an assay plate, over a range of concentrations, supports cell adhesion equally as efficiently as the normal fusion protein dimerised at the IgG heavy chain hinge region. This dispels a widely held view that cis-dimerisation is an obligatory stage in the formation of the adhesive complex (Brieher et al., 1996; Ozawa, 2002; Takeda et al., 1999; Tomschy et al., 1996). Recently, Troyanovsky et al. (Troyanovsky et al., 2003) used a bifunctional sulphydryl cross-linking reagent to determine the orientation of cadherin molecules in cis- and trans-dimers and concluded that a strand exchange mechanism provided the best explanation for both types of dimer. The present example addresses this issue by a more direct strategy. The formation of a disulphide bond during cell adhesion using complementary, but not identical, cysteine point mutations on opposing cadherin molecules provides compelling evidence for strand exchange. This degree of specificity in the formation of the bond demands that during adhesion Trp2 is either inserted into the hydrophobic pocket of the opposing cadherin molecule or is poised very close to it. By similar reasoning, the cis-dimers we detected between adjacent cadherin molecules bearing the same mutation, D1C or D27C, could not be formed by the mutual strand exchange mechanism depicted in current crystal structures (Boggon et al., 2002; Haussinger et al., 2004; Shapiro et al., 1995). This does not rule out the possibility that strand-exchange cis-dimers may occur on the cell surface; they would not be disulphide-linked and would escape detection on our gels. It is important to emphasise that disulphide bonded cis-dimers were formed with the D1C and D27C mutations, but not with the R25C mutation. This demonstrates that these bonds were not a consequence of random contacts between cadherin molecules. Specificity of the bond for D1C and D27C limits the possible orientations that the molecules can adopt in making the cis-contact. A favourable orientation to achieve this discrimination is for adjacent cadherin molecules to be aligned in parallel, similar to the calcium-dependent C2-symmetric E-cadherin dimer recently determined by NMR (Haussinger et al., 2002). Alternatively, cross-intercalation of one Trp2 residue, as opposed to mutual exchange, may allow sufficient rotation of the domains to bring two D27C mutations into apposition. This arrangement can be seen in a hypothetical structure (PDB 1Q5C) for the orientation of desmosomal cadherins, based on electron tomography (He et al., 2003).
The present example provides the most direct evidence so far that strand exchange is a primary event in cadherin-mediated cell adhesion. This conclusion must be reconciled with three controversial outstanding issues, viz, the questions of cadherin type-specificity (Klingelhofer et al., 2000; Niessen and Gumbiner, 2002; Nose et al., 1990), the role of the conserved HAV motif (Makagiansar et al., 2001; Renaud-Young and Gallin, 2002; Williams et al., 2000; Williams et al., 2002) and the contribution of domains 2-4 to cell adhesion (Chappuis-Flament et al., 2001; Zhu et al., 2003). We envisage that Trp2 exchange is the initial event in cadherin-mediated adhesion; the HAV motif is not directly involved and the interaction is not cadherin type-specific. Subsequently, secondary interactions that require other regions of the cadherin molecule follow. These contacts facilitate clustering, provide specificity or initiate intracellular signalling. We suggest that our present strategy of using ‘reporter’ disulphide bonds to reveal adhesive surfaces in a physiological setting may be a powerful tool to investigate these interactions.
Example 1 provides compelling evidence that the mutual “strand exchange” (also known as “βA strand exchange”) mechanism is a primary event in cadherin mediated cell adhesion. In essence, the process depends entirely on formation an ionic bond (salt bridge) between cadherin molecules on opposing cells. The bond is formed between the N-terminal NH2 group on Asp1 in domain EC1 of one cadherin molecule and the acidic side chain of Glu89 on EC1 of the opposing molecule (
It is known that correct post-translational processing of cadherin molecules is essential for adhesion (Ozawa and Kemler, 1990). Type I cadherins are synthesised with a prodomain of more than 100 amino acids which has the structure of a typical cadherin fold (Koch et al., 2004). There is an unstructured linker of approximately 30 amino acids between the prodomain and the first domain, EC1, of the mature molecule. A multi-basic recognition motif is cleaved by furin proteases to give the mature cadherin molecule which has a conserved typtophan as the second amino acid from the N-terminus. Failure to remove the prodomain prevents adhesion (Koch et al., 2004; Ozawa and Kemler, 1990). The presence of even a few additional amino acids at the N-terminus completely ablates adhesive function (Corps et al., 2001; Ozawa and Kemler, 1990). In keeping with this observation, a recent NMR study (Haussinger et al., 2004) showed that correct processing at the N-terminus was required for the strand exchange mechanism or for intramolecular docking of Trp2 into its own domain. The crystal structure of C-cadherin, in which the N-terminus is correctly processed, shows that strand exchange brings the amino group of Asp 1 in close proximity to the acidic side chain of a conserved amino acid, Glu 89, in the opposing cadherin domain, suggesting that a salt bridge could form here to stabilise Trp2 docking (Boggon et al., 2002). However, the significance of the putative salt bridge has been questionable because crystal structures of E- and N-cadherins show Trp2 integrated into the domain fold despite extension of the N-terminus and, consequently, the absence of this ionic bond (Pertz et al., 1999; Schubert et al., 2002; Shapiro et al., 1995).
In the present example we have investigated the significance of the salt bridge in cell adhesion mediated by N-cadherin. We have prevented formation of the bond in one or both components of the adhesive dimer by extending the N-terminus or by mutating Glu 89 to alanine. The results demonstrate with striking clarity that the salt bridge plays a vital role in adhesion by stabilising Trp 2 docking and that intramolecular and intermolecular docking of Trp 2 are in dynamic equilibrium. When the E89A mutation and the N-terminal extension are present in opposing cadherin molecules respectively, they form a complementary pair, each preventing intramolecular docking of Trp 2 but facilitating strand exchange in one direction. In these circumstances the normal equilibrium is disturbed and the strength of cadherin adhesion is greatly increased. Therefore, by manipulating this salt bridge by mutagenesis we can completely ablate or enhance strand exchange and hence modulate cadherin-mediated cell adhesion. The inhibitory activity of a well-known commercial antibody to mouse N-cadherin (NCD2) is now understandable because its epitope is directly adjacent to the E89 salt bridge. By targeting this salt bridge or the flexible hinge at the base of the βA strand, inhibition or enhancement of adhesion may be achieved, for example by drugs. The present invention therefore provides a rational basis for the design of drugs which will inhibit or enhance cell adhesion in all solid tissues, including skin, gut, blood vessels, organs and solid tumours. Because cadherin-mediated recognition is also central to the development of neuronal networks in the brain and to the function of the neuronal synapse, such drugs may be useful in therapy for neurodegenerative disease. It is important to note that the salt bridge mechanism which we have elucidated is more widespread in the cadherin superfamily than the HAV recognition motif mentioned above. We provide here that antibodies, proteins, peptides or natural or synthetic organic compounds could be used to interfere with the salt bridge or the flexible hinge at the base of the βA strand which regulates strand exchange.
Preparation of DNA Constructs and their Transfection into Cell Lines
Mutations were prepared in full length chicken N-cadherin cDNA in pcDNA3.1 using the QuikChange mutagenesis method (Stratagene). Constructs were stably transfected into K562 lymphomyeloid cells as described in Example 1 above. These cells have been shown to lack natural expression of cadherins (see Example 1). Clonal cell lines were obtained by limiting dilution and selected for equal expression of N-cadherin. Wild type and mutant chicken N-cadherin Fc fusion proteins containing the five extracellular domains were prepared, standardised and quantitated as described in Example 1. All cell lines were cultured in DMEM+10% FCS containing G418 at 1 mg/ml.
Adhesion tests were conducted substantially as described in Example 1. Briefly, K562 Cells or L cells transfected with wild type or mutant N-cadherin were allowed to settle for 45 minutes at 37° C. onto N-cadherin Fc fusion proteins coated at 1 μg/ml to a 96 well plate. Non-adherent cells were then washed off and residual adherent cells were quantitated by measuring acid phosphatase activity. Assays were conducted in quadruplicate and results are expressed as % cells adhering +/−SEM.
Dynabeads (Dynal Biotech) coupled to Protein A were coated with N-cadherin Fc at 1 μg/ml in calcium and magnesium-free HBSS containing 0.1% Tween 20, 1% FCS and 4 mM EDTA. Eppendorf tubes containing beads and fusion protein were rotated slowly for 1 hour at room temperature to allow binding to take place. The beads were then washed in the above assay buffer lacking EDTA and then resuspended in the same buffer supplemented with 1.25 mM CaCl2. Beads were allowed to aggregate in a volume of 100 μl for 2 hours at 37° C. by slow rotation, in an eppendorf tube, at approximately 20 rpm. Aggregation was then assessed by light microscopy.
Cells were stained for chicken N-cadherin using antibody NCD-2 (R & D Systems) at 5 μg/ml. The secondary antibody was FITC-labelled goat anti-rat IgG (Serotec, UK). For staining transfectants with N-cadherin Fc fusion proteins, the cells were treated with the fusion proteins at 5 μg/ml for 90 minutes on ice in Hanks Balanced Salt Solution (HBSS) containing 2% FCS and 0.1% sodium azide. After washing, bound fusion protein was detected with FITC-labelled goat anti-human Fc (Serotec) and quantitated by flow cytometry using a FACSCalibur (Becton Dickinson).
L cells expressing mouse N-cadherin with an uncleaved prodomain (see Koch et al., 2004) were obtained from Dr Weisong Shan (Montreal Neurological Institute). The normal furin cleavage site, RQKR, had been replaced with a Factor Xa site, IEGR, to give the correct N-terminus after digestion. Trypsin also cleaved at this position (Koch et al., 2004) and proved to be more efficient than Factor Xa. L cells suspended in HBSS containing 0.1% BSA were treated with 0.01% trypsin (Sigma, Type XI) in the presence of 2 mM Ca2+ for 10 minutes at 37° C. and the digestion was then quenched with soyabean trypsin inhibitor, 0.5 mg per ml (Sigma, Type I-S). Cells were then washed and tested for adhesion to N-cadherin Fc-fusion protein. To check for complete removal of the prodomain, the cells were lysed in SDS sample buffer and the cadherin analysed by SDS PAGE under reducing conditions on a 4-12% gradient gel. N-cadherin was identified by western blotting using rabbit anti-pan cadherin antiserum specific for the cytoplasmic domain (Sigma, code C3678) followed by affinity-purified HRP-labelled sheep anti-rabbit IgG, F(ab′)2-specific (Serotec).
Cadherin structures were displayed using Swiss PDB Viewer (http://www.expasy.org/spdbv/).
a shows the position of the two salt bridges formed during mutual strand exchange. To prevent formation of this bond, Glu 89 of N-cadherin was mutated to alanine or, alternatively, the N-terminus was extended by adding two glycine residues to Asp 1 to displace the N-terminal amino group away form the acidic side chain of Glu 89. Mutant N-cadherin proteins were expressed in K562 lymphomyeloid cells which were matched for equal cell surface expression of the respective cadherins (
To investigate whether the enhanced adhesion observed with this complementary pair reflects an increase in affinity, we tested whether soluble N-cadherin Fc fusion proteins would bind to cell surface N-cadherin using a protocol similar to immunofluorescent staining by antibodies.
In order to investigate whether an uncleaved prodomain has the same effect as a two amino acid extension to the N-terminus, we tested the ability of L cell transfectants expressing unprocessed N-cadherin to adhere to the E89A mutant.
An explanation for our results is given in
In the experiments described so far in this example, disruption of the salt bridge prevented intramolecular docking of Trp 2 in one or both components of the dimer. In an alternative strategy to prevent intramolecular docking, we removed Trp2 by introducing the mutation W2G or blocked the hydrophobic acceptor pocket with an isoleucine side chain projecting into the cavity using the mutation A801. N-cadherin Fc fusion proteins with these mutations were then tested against our panel of K562 transfectants (
Example 2 demonstrates decisively that the strand exchange mechanism of cadherin adhesion requires formation of a salt bridge between opposing cadherin molecules involving the N-terminus of one molecule and E89 of the other. The bond is a major feature of the free energy landscape that governs strand exchange. A second factor is the hydrophobic interaction between Trp 2 and its acceptor pocket. For stable adhesion, the energy contributions of both factors are required. In addition, a hydrogen bond formed between the amide nitrogen of Val 3 and the carbonyl oxygen of residue 25 may also contribute to the stability of Trp 2 intercalation (Haussinger et al., 2004). Formation of the cadherin adhesive dimer can be regarded as a relatively uncomplicated example of the 3D domain swap mechanism for protein oligomerization (Bennett and Eisenberg, 2004; Rousseau et al., 2003). A high energy barrier must be overcome as the structural component to be exchanged is released from its own domain and becomes available for exchange, but the difference in free energy between the monomer and the dimer is small. In our experiments, intramolecular docking of Trp 2 was prevented in both components of the cadherin dimer by disrupting the salt bridge. This greatly reduced the energy barrier for strand exchange. By using a complementary pair of mutations, the GG N-terminal extension on one side and E89A on the other, the energetics were changed strongly to favour exchange of one strand. These mutants behaved as “molecular Velcro™” forming a strongly adhesive complementary pair but neither adhering to its own kind. It is likely that the effect of the GG extension in this context was solely to displace the N-terminus away from the acidic side chain of E89 to prevent formation of the salt bridge. This is corroborated by our observations (data not shown) that alternative short extensions to the N-terminus, e.g. Met-Asp-Pro or a single Cys, had a similar complementary effect with the E89A mutant (see
Our results were obtained with N-cadherin, a classical cadherin. On the basis of multiple alignment of amino acid sequences of non-classical cadherins and structural modelling by the present inventors and others (see May et al., 2005), the present invention also provides that non-classical (Type II) cadherins, desmocollins and desmogleins all have a similar strand exchange mechanism dependent on a salt bridge in the position described here. In the protocadherin family, N-terminal peptide analysis suggests that protocadherins alpha also have a conserved tryptophan as the second amino acid (Gevaert et al., 2003), indicating that the same mechanism may apply in this group also. Variations of the strand swap model are therefore proposed according to the present invention to apply throughout the whole cadherin family.
To explain cadherin type-specificity by the strand exchange mechanism, we propose according to the present invention that optimal adhesion between wild type cadherins may require free energy changes accompanying mutual strand exchange to be equal on both sides of the adhesive dimer. At least two factors influence the energy landscape, the N-terminal salt bridge and the hydrophobic interaction between Trp 2 and its pocket. The former would be affected by the electrostatic environment in the vicinity of Glu 89 and the latter by non-conserved amino acids lining the hydrophobic pocket. Our experiments are consistent with the hypothesis of energy balance and implicate both factors in the specificity displayed by N- and E-cadherins.
The second cadherin domain, EC2, is required for correct co-ordination of calcium in the junction between the first cadherin domain, EC1, and EC2, and the disruptive effect of the D134A mutation in the present experiments demonstrate that, in this respect, EC2 is essential for strand exchange. Our results do not rule out the possibility that EC3 or EC4 Could provide additional contact sites or be involved in other ways. Assays used in prior art studies to test for adhesive contacts involving inner domains have varied greatly in sensitivity and results must be interpreted accordingly. The cell adhesion test in the present example is very robust and is unlikely to reveal weak interactions. In contrast, our bead aggregation assay is more sensitive and it is notable that the W2G mutant and the A80I mutant which, individually, could not undergo strand exchange by tryptophan docking showed weak but detectable aggregation when tested separately. The result reflect the presence of one or more additional contact sites, for example not located on EC1 or EC2, which are also provided according to the present invention. It is pertinent to observe that formation of the intermolecular salt bridge between E89 and the N-terminus is likely to require correct angular alignment of opposing N-terminal domains. The presence of the inner domains may facilitate optimal orientation, indeed, the curvature of the complete extracellular region may be significant in this respect.
The present invention offers new insights into the strand exchange mechanism. The observation that cadherin affinity can be greatly increased by lowering activation energy using salt bridge mutations provides in one aspect a rational basis for designing alternative strategies for modulating, for example greatly increasing, cadherin adhesion.
The foregoing examples are meant to illustrate the invention and do not limit it in any way. One of skill in the art will recognize modifications within the spirit and scope of the invention as indicated in the claims.
All references cited are incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0501216.6 | Jan 2005 | GB | national |
| 0515423.2 | Jul 2005 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2006/000173 | 1/18/2006 | WO | 00 | 4/15/2008 |
