The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK
* Author for correspondence (e-mail: peter.kilshaw{at}bbsrc.ac.uk)
Accepted 8 June 2005
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Summary |
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Key words: Cadherin, Salt bridge, Strand exchange, Tryptophan, N-terminus
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Introduction |
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It has long been 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 that has a conserved tryptophan 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 Asp1 in close proximity to the acidic side chain of a conserved amino acid, Glu89, in the opposing cadherin domain, suggesting that a salt bridge could form here to stabilise Trp2 docking (Boggon et al., 2002
). 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 report 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 Glu89 to Ala. The results demonstrate with striking clarity that the salt bridge plays a vital role in adhesion by stabilising Trp2 docking and that intramolecular and intermolecular docking of Trp2 are in dynamic equilibrium. When the Glu89Ala mutation and the N-terminal extension are present in opposing cadherin molecules respectively, they form a complementary pair, each preventing intramolecular docking of Trp2 but facilitating strand exchange in one direction. In these circumstances the normal equilibrium is disturbed and the strength of cadherin adhesion is greatly increased.
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Materials and Methods |
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Cadherin-mediated cell adhesion
Adhesion tests were conducted as described in previous reports (Corps et al., 2001; Harrison et al., 2005
). 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 quantified by measuring acid phosphatase activity. Assays were conducted in quadruplicate and results are expressed as the percentage of cells adhering ±s.e.m.
Bead aggregation assay
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.
Immunofluorescent staining of K562 transfectants
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 quantified by flow cytometry using a FACSCalibur (Becton Dickinson)
Cleavage of the N-cadherin prodomain
L cells expressing mouse N-cadherin with an uncleaved prodomain were a kind gift of Weisong Shan (Montreal Neurological Institute, Canada). 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 soya bean trypsin inhibitor, 0.5 mg/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).
Viewing molecular structures
Cadherin structures were displayed using Swiss PDB Viewer (http://www.expasy.org/spdbv/).
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Results |
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Affinity between the Glu89Ala and Gly-Gly extension mutants
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. Binding between wild-type N-cadherin molecules was undetectable (Fig. 3). In contrast, the interaction between the Glu89Ala mutant and the Gly-Gly extension mutant gave strong cell surface staining, approaching that seen with antibodies to cadherins. Previous studies (Baumgartner et al., 2000; Haussinger et al., 2004
) measuring affinity between normal trans-acting cadherin molecules have reported KD values in the range 103 to 105 M. Although we have not yet obtained precise measurements of affinity between the complementary cadherin mutants, the present data clearly show a large increase in affinity compared with that between wild-type molecules.
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Effect of an uncleaved prodomain
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 Glu89Ala mutant. There was strong adhesion of the transfectants to the Glu89Ala Fc fusion protein but no adhesion to wild-type N-cadherin or to the Asp134Ala negative control (Fig. 4a). A Factor Xa cleavage site introduced into the transfected N-cadherin allowed removal of the prodomain from the cell surface with either Factor Xa or trypsin. Trypsin proved to be more efficient, almost completely removing the prodomain (Fig. 4b). In these circumstances the cells acquired the ability to adhere to wild-type N-cadherin whereas adhesion to the Glu89Ala mutant was greatly reduced (Fig. 4c).
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Removing Trp2 or blocking the hydrophobic pocket
In the experiments described so far, disruption of the salt bridge prevented intramolecular docking of Trp2 in one or both components of the dimer. In an alternative strategy to prevent intramolecular docking, we removed Trp2 by introducing the mutation Trp2Gly or blocked the hydrophobic acceptor pocket with an isoleucine side chain projecting into the cavity using the mutation Ala80Ile. N-cadherin Fc fusion proteins with these mutations were then tested against our panel of K562 transfectants (Fig. 6a). In keeping with the explanation given in Fig. 5, the Trp2Gly mutant acted as a strand acceptor and therefore adhered strongly to the Glu89Ala mutant whereas the Ala80Ile mutant was a strand donor and adhered to the Gly-Gly extension mutant. To determine whether the Trp2Gly mutant and the Ala80Ile mutant adhered strongly to one another, as would be predicted, the proteins were coated separately to Dynabeads and the two types were then mixed and tested for cadherin-dependent aggregation. The mixed preparation of beads formed large clumps, aggregating more strongly than beads coated with wild-type N-cadherin (Fig. 6b). In contrast, beads coated with the Trp2Gly or Ala80Ile mutants and tested separately clustered in twos and threes, whereas beads coated with the negative control mutant, Asp134Ala, were entirely monodisperse.
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Discussion |
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By analogy with other 3D domain swap systems, it is likely that the activation energy for strand exchange can be reduced by means other than mutating the Glu89 salt bridge or blocking the hydrophobic acceptor pocket. In other proteins, altering the length of the hinge loop or relieving backbone tension imposed by proline residues in this region has increased the propensity to dimerize by many orders of magnitude (Green et al., 1995; Rousseau et al., 2003
; Rousseau et al., 2001
). It is not known whether similar changes to the ßA strand in cadherins would modulate intramolecular docking of Trp2 and alter the free energy balance in favour of strand exchange.
Our results were obtained with N-cadherin, a classical cadherin. But multiple alignment of amino acid sequences of non-classical cadherins and structural modelling by our laboratory and others (May et al., 2005) suggests 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
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. It is possible that variations of the strand swap model apply throughout the whole cadherin family.
Three important issues remain unresolved by the present report. First, the controversial matter of cadherin type-specificity. Conclusions about specificity have been subject to the particular tests used and the pairs of cadherins investigated (Foty and Steinberg, 2005; Klingelhofer et al., 2000
; Niessen and Gumbiner, 2002
; Nose et al., 1990
). In cell aggregation assays, cells usually segregate according to cadherin type. This behaviour has been attributed to structural features in domain 1 (Nose et al., 1990
) or, alternatively, to subtle quantitative differences in the level of cell surface cadherin expressed by different cell types in the mixed population (Foty and Steinberg, 2005
). Experiments in our laboratory (not shown) have demonstrated clear homophilic preference by N- and E-cadherins in the adhesion assay described here. To explain specificity by the strand exchange mechanism, we reasoned 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 Trp2 and its pocket. The former would be affected by the electrostatic environment in the vicinity of Glu89, and the latter by non-conserved amino acids lining the hydrophobic pocket. Our preliminary experiments, so far, are consistent with the hypothesis of energy balance and implicate both factors in the specificity displayed by N- and E-cadherins. A second question concerns the inhibitory effects of peptides that contain the motif HAV, which is conserved in classical cadherins, or related motifs present in other cadherins (Makagiansar et al., 2001
; Noe et al., 1999
; Runswick et al., 2001
; Williams et al., 2000
). The HAV motif is not essential for adhesion (Renaud-Young and Gallin, 2002
) and the mechanism of action of these peptides is unknown. Our experiments do not enlighten the issue. HAV peptides may block other putative interaction sites in domain 1 observed in crystal structures (Boggon et al., 2002
; Shapiro et al., 1995
) or, perhaps, cause local conformational changes that interfere with strand exchange. Finally, it is still unclear whether cadherin adhesion involves domains 1 and 2 only (Shan et al., 2004
) or whether contacts involving inner domains also play a role (Chappuis-Flament et al., 2001
; Perret et al., 2004
; Zhu et al., 2003
). The second domain, EC2, is required for correct coordination of calcium in the junction between EC1 and EC2 and the disruptive effect of the Asp134Ala mutation in the present experiments demonstrates that, in this respect, EC2 is essential for strand exchange. But our results do not rule out the possibility that EC3 or EC4 could provide additional contact sites or be involved in other ways. It is important to emphasise that assays used in different 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 report 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 Trp2Gly mutant and the Ala80Ile mutant which, individually, could not undergo strand exchange by tryptophan docking showed weak but detectable aggregation when tested separately. The result may reflect the presence of one or more additional contact sites. It is consistent with very weak adhesion seen in other circumstances where strand exchange cannot occur, for example the interaction between E-cadherin lacking domain 1 and the wild-type molecule (Renaud-Young and Gallin, 2002
). It is pertinent to observe that formation of the salt bridge reported here 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 (Boggon et al., 2002
) could be significant in this respect.
The present report 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 a rational basis for designing alternative strategies for modulating cadherin adhesion.
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Acknowledgments |
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