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Synergy between Extracellular Modules of Vascular Endothelial Cadherin Promotes Homotypic Hexameric Interactions

2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês

10.1074/jbc.m111597200

1083-351X

Stéphanie Bibert, Michel Jaquinod, Evelyne Concord, Christine Ebel, E.A. Hewat, Christophe Vanbelle, Pierre Legrand, Marianne Weidenhaupt, Thierry Vernet, Danielle Gulino-Debrac,

Cancer-related gene regulation

Vascular endothelial (VE) cadherin is an endothelial specific cadherin that plays a major role in remodeling and maturation of vascular vessels. Recently, we presented evidence that the extracellular part of VE cadherin, which consists of five homologous modules, associates as a Ca2+-dependent hexamer in solution (Legrand, P., Bibert, S., Jaquinod, M., Ebel, C., Hewat, E., Vincent, F., Vanbelle, C., Concord, E., Vernet, T., and Gulino, D. (2001)J. Biol. Chem. 276, 3581–3588). In an effort to identify which extracellular modules are involved in the elaboration and stability of this hexameric structure, we expressed various VE cadherin-derived fragments overlapping individual or multiple successive modules as soluble proteins, purified each to homogeneity, and tested their propensity to self-associate. Altogether, the results demonstrate that, as their length increases, VE cadherin recombinant fragments generate increasingly complex self-associating structures; although single module fragments do not oligomerize, some two or three module-containing fragments self-assemble as dimers, and four module-containing fragments associate as hexamers. Our results also suggest that, before elaborating a hexameric structure, molecules of VE cadherin self-assemble as intermediate dimers. A synergy between the extracellular modules of VE cadherin is thus required to build homotypic interactions. Placed in a cellular context, this particular self-association mode may reflect the distinctive biological requirements imposed on VE cadherin at adherens junctions in the vascular endothelium. Vascular endothelial (VE) cadherin is an endothelial specific cadherin that plays a major role in remodeling and maturation of vascular vessels. Recently, we presented evidence that the extracellular part of VE cadherin, which consists of five homologous modules, associates as a Ca2+-dependent hexamer in solution (Legrand, P., Bibert, S., Jaquinod, M., Ebel, C., Hewat, E., Vincent, F., Vanbelle, C., Concord, E., Vernet, T., and Gulino, D. (2001)J. Biol. Chem. 276, 3581–3588). In an effort to identify which extracellular modules are involved in the elaboration and stability of this hexameric structure, we expressed various VE cadherin-derived fragments overlapping individual or multiple successive modules as soluble proteins, purified each to homogeneity, and tested their propensity to self-associate. Altogether, the results demonstrate that, as their length increases, VE cadherin recombinant fragments generate increasingly complex self-associating structures; although single module fragments do not oligomerize, some two or three module-containing fragments self-assemble as dimers, and four module-containing fragments associate as hexamers. Our results also suggest that, before elaborating a hexameric structure, molecules of VE cadherin self-assemble as intermediate dimers. A synergy between the extracellular modules of VE cadherin is thus required to build homotypic interactions. Placed in a cellular context, this particular self-association mode may reflect the distinctive biological requirements imposed on VE cadherin at adherens junctions in the vascular endothelium. vascular endothelial matrix-assisted laser desorption ionization hydrodynamic radius N-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride glutathione S-transferase 4-morpholineethanesulfonic acid Adhesion between cells of identical phenotypes is mediated by receptors belonging to the cadherin superfamily (1.Takeichi M. Annu. Rev. Biochem. 1990; 59: 237-252Crossref PubMed Scopus (1113) Google Scholar, 2.Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2948) Google Scholar). This protein family contains 50 different classic cadherins in both vertebrates and invertebrates and the recently described human protocadherins (3.Nollet F. Kools P. van Roy F. J. Mol. Biol. 2000; 299: 551-572Crossref PubMed Scopus (591) Google Scholar, 4.Wu Q. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3124-3129Crossref PubMed Scopus (97) Google Scholar). Based on their amino acid sequence, classic cadherins were first classified into two subgroups, type I and type II (5.Suzuki S. Sano K. Tanihara H. Cell Regul. 1991; 2: 261-270Crossref PubMed Scopus (317) Google Scholar, 6.Tanihara H. Sano K. Heimark R.L. St. John T. Suzuki S. Cell. Adhes. Commun. 1994; 2: 15-26Crossref PubMed Scopus (138) Google Scholar). More recently, cadherin-5 and -15 that share little sequence similarity with other cadherins or among themselves were considered as two distinct non-type I or II cadherins (7.Shimoyama Y. Tsujimoto G. Kitajima M. Natori M. Biochem. J. 2000; 349: 159-167Crossref PubMed Scopus (134) Google Scholar). Classic cadherin molecules exhibit a similar organization, in particular in their extracellular domain, that consists of five homologous repeats designated EC1 to EC5 and numbered from the N to the C terminus. Type I members show a high degree of protein sequence similarity when compared with the E cadherin sequence and possess, in their N-terminal extracellular module EC1, the HAV cell adhesion recognition sequence (3.Nollet F. Kools P. van Roy F. J. Mol. Biol. 2000; 299: 551-572Crossref PubMed Scopus (591) Google Scholar). By contrast, the HAV sequence is absent on the extracellular domain of either type II members or non-type I or II cadherins (8.Shimoyama Y. Shibata T. Kitajima M. Hirohashi S. J. Biol. Chem. 1998; 273: 10011-10018Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 9.Lampugnani M.G. Resnati M. Raiteri M. Pigott R. Pisacane A. Houen G. Ruco L.P. Dejana E. J. Cell Biol. 1992; 118: 1511-1522Crossref PubMed Scopus (552) Google Scholar). The highly conserved cytoplasmic domain of cadherins interacts with β-catenin or γ-catenin (also called plakoglobin) in a mutually exclusive fashion (10.Gumbiner B.M. Neuron. 1993; 11: 551-564Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 11.Kemler R. Ozawa M. Ringwald M. Curr. Opin. Cell Biol. 1989; 1: 892-897Crossref PubMed Scopus (80) Google Scholar). Moreover, β- or γ-catenins also interact with α-catenin which links the cadherin-adhesion complex either directly or indirectly to the actin cytoskeleton (12.Jou T.S. Stewart D.B. Stappert J. Nelson W.J. Marrs J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5067-5071Crossref PubMed Scopus (305) Google Scholar, 13.Knudsen K.A. Soler A.P. Johnson K.R. Wheelock M.J. J. Cell Biol. 1995; 130: 67-77Crossref PubMed Scopus (564) Google Scholar). Cell-cell adhesion is constantly rearranged suggesting that the cadherin-catenin complex is dynamically remodeled. By mediating homotypic interactions, cadherins are responsible for segregation of different cell types and, consequently, are fundamental for the establishment and maintenance of multicellular structures. Several results demonstrate that the homotypic binding regions reside in the extracellular part of cadherins (14.Koch A.W. Pokutta S. Lustig A. Engel J. Biochemistry. 1997; 36: 7697-7705Crossref PubMed Scopus (130) Google Scholar). High resolution structure determination sheds light on the molecular determinants and organization of homotypic cadherin interactions at cell-cell junctions. Based on the first structure derived from the N-terminal domain of neural cadherin (N-EC1 fragment) (15.Shapiro L. Fannon A.M. Kwong P.D. Thompson A. Lehmann M.S. Grubel G. Legrand J.F. Als-Nielsen J. Colman D.R. Hendrickson W.A. Nature. 1995; 374: 327-337Crossref PubMed Scopus (978) Google Scholar), a model for cadherin-mediated homophilic interactions was proposed (the zipper model) (16.Patel D.J. Gumbiner B.M. Nature. 1995; 374: 306-307Crossref PubMed Scopus (14) Google Scholar). It suggests the formation of parallel dimer interfaces (cis dimers) and anti-parallel alignments (trans dimers). Both types of association may reflect interactions occurring between cadherins at the cell surface. Cis dimers involving the five extracellular modules of cadherins may mimic the alignment of two molecules emerging from the same cell, whereas trans dimers mediated by the N-terminal EC1 module may be elaborated by molecules protruding from adjacent cells. Structural data obtained for the two module fragments of E (E-EC1–2) (17.Nagar B. Overduin M. Ikura M. Rini J.M. Nature. 1996; 380: 360-364Crossref PubMed Scopus (565) Google Scholar, 18.Pertz O. Bozic D. Koch A.W. Fauser C. Brancaccio A. Engel J. EMBO J. 1999; 18: 1738-1747Crossref PubMed Scopus (344) Google Scholar) or N (N-EC1–2) (19.Tamura K. Shan W.S. Hendrickson W.A. Colman D.R. Shapiro L. Neuron. 1998; 20: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) cadherin revealed that the length of a single cadherin extracellular module is ∼45 Å. It can be deduced that the cell to cell distance in the zipper model is about 405 Å, a value incompatible with the dimension of cell-cell adherens junctions that ranges from 200 to 250 Å based on electron microscopy analysis. By using direct-force measurements, Leckband and co-workers (20.Leckband D. Sivasankar S. Curr. Opin. Cell. Biol. 2000; 12: 587-592Crossref PubMed Scopus (73) Google Scholar, 21.Sivasankar S. Brieher W. Lavrik N. Gumbiner B. Leckband D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11820-11824Crossref PubMed Scopus (145) Google Scholar) proposed a new model for homotypic interactions in which cadherin molecules emerging from adjacent cells elaborate antiparallel, completely interdigitated contacts. These structures, which exhibit multiple adhesive contacts involving successive domains along the extracellular region of the protein, possess a length in the range of 250 Å. Here we investigate the mechanism of homotypic interactions mediated by the non-type I or II cadherin 5 (or VE1 cadherin). The distinct phylogenetic attribution of this cadherin is likely to be at the basis of its specialized function. Indeed, VE cadherin is expressed at the surface of a peculiar tissue, the vascular endothelium (9.Lampugnani M.G. Resnati M. Raiteri M. Pigott R. Pisacane A. Houen G. Ruco L.P. Dejana E. J. Cell Biol. 1992; 118: 1511-1522Crossref PubMed Scopus (552) Google Scholar). This tissue is formed of a continuous monolayer of endothelial cells that constitute a physical barrier between blood and underlying tissues. The highly dynamic modulation of endothelial adherens junctions allows the passage of leukocytes from blood toward inflamed tissues (22.Johnson-Leger C. Aurrand-Lions M. Imhof B.A. J. Cell Sci. 2000; 113: 921-933PubMed Google Scholar). In fact, recently, we and others (23.Gulino D. Delachanal E. Concord E. Genoux Y. Morand B. Valiron M.O. Sulpice E. Scaife R. Alemany M. Vernet T. J. Biol. Chem. 1998; 273: 29786-29793Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24.Hordijk P.L. Anthony E. Mul F.P. Rientsma R. Oomen L.C. Roos D. J. Cell Sci. 1999; 112: 1915-1923Crossref PubMed Google Scholar) established a critical role for VE cadherin in the modulation of endothelial monolayer permeability and consequently in the control of leukocyte trafficking. Moreover, targeted inactivation of the VE cadherin gene in mice affects remodeling and maturation of endothelial vessels leading to the death of the embryos from severe vascular defects at mid-gestation (25.Gory-Faure S. Prandini M.H. Pointu H. Roullot V. Pignot-Paintrand I. Vernet M. Huber P. Development. 1999; 126: 2093-2102PubMed Google Scholar, 26.Carmeliet P. Lampugnani M.G. Moons L. Breviario F. Compernolle V. Bono F. Balconi G. Spagnuolo R. Oostuyse B. Dewerchin M. Zanetti A. Angellilo A. Mattot V. Nuyens D. Lutgens E. Clotman F. de Ruiter M.C. Gittenberger-de Groot A. Poelmann R. Lupu F. Herbert J.M. Collen D. Dejana E. Cell. 1999; 98: 147-157Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar). This demonstrates that VE cadherin is also required for vascular morphology. Furthermore, in contrast to other cadherin family members, VE cadherin is both connected to the actin cytoskeleton and to intermediate filaments (27.Valiron O. Chevrier V. Usson Y. Breviario F. Job D. Dejana E. J. Cell Sci. 1996; 109: 2141-2149Crossref PubMed Google Scholar, 28.Kowalczyk A.P. Navarro P. Dejana E. Bornslaeger E.A. Green K.J. Kopp D.S. Borgwardt J.E. J. Cell Sci. 1998; 111: 3045-3057Crossref PubMed Google Scholar). Recently, we have presented evidence (29.Legrand P. Bibert S. Jaquinod M. Ebel C. Hewat E. Vincent F. Vanbelle C. Concord E. Vernet T. Gulino D. J. Biol. Chem. 2001; 276: 3581-3588Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) for the Ca2+-dependent hexameric association of the extracellular region of VE cadherin. This type of oligomerization is difficult to reconcile with structural data obtained for shorter fragments of other cadherins. We have now extended our previous study by analyzing a series of VE cadherin-derived arrays of modules. We show that the degree of oligomerization is related to the number of modules within the array indicating that each module acts in synergy with the neighboring ones during the homotypic assembly of VE cadherin. Altogether, our results suggest that VE cadherin molecules first self-assemble as intermediate dimers involving extracellular modules EC3 and EC4 before elaborating mature hexameric structures. The single module fragments were named VE-ECi, with i corresponding to the position of the respective extracellular module within the extracellular domain of VE cadherin. The multidomain fragments were designated VE-ECi-j, where i and j correspond to the positions of the most N- and the most C-terminal modules, respectively. For instance, VE-EC1–3 starts at the N terminus of module EC1, ends at the C terminus of module EC3, and consequently overlaps the three modules EC1, EC2, and EC3 (Fig. 1A). The oligonucleotide pairs used to produce the cDNA fragments encoding the VE cadherin-derived proteins are summarized in Table I. PCR-amplified products were cloned into different expression vectors. The single module fragments, cloned into the vector pGEX-4T1 (Amersham Biosciences), were expressed as glutathione S-transferase fusion proteins, whereas multimodule fragments, cloned into the vector pET30b+ (Novagen), were expressed as native proteins. The resulting pET-VE cadherin plasmids allowed the expression of VE cadherin multiple module fragments fused to an N-terminal methionine. The coding sequence of all VE cadherin-derived constructs was verified by sequence analysis.Table IOligonucleotides used to produce the different VE cadherin-derived recombinant fragmentsFragmentsOligonucleotidesVE-EC15′5′GGA TCC GAT TGG ATT TGG AAC CAG ATG CAC 3′ BamHI Asp13′5′ GAA TTC TTA CCG ATG CGT GAA CAC AGG CCA GTT GTC 3′ EcoRI stop Arg107VE-EC1–25′5′ TATA CAT ATG GAT TGG ATT TGG AAC CAG ATG CAC 3′ NdeI Asp13′5′ CTC GAA TTC TCA CCG GTT CTG GGG CTC ATC TGG GTC 3′ EcoRI stop Arg244VE-EC1–35′5′ TATA CAT ATG GAT TGG ATT TGG AAC CAG ATG CAC 3′ NdeI Asp13′5′ CTC GAA TTC TCA CTC GTC CAC ATC TGT GAT GTT GAT 3′ EcoRI stop Glu321VE-EC1–45′5′ TATA CAT ATG GAT TGG ATT TGG AAC CAG ATG CAC 3′ NdeI Asp13′5′ CTC GAA TTC TCA CTC CGG GGC ATT GTC ATT CTC ATC 3′ EcoRI stop Glu431VE-EC45′5′ CGT GGA TCC CGA TAC ATG AGC CCT CCC GCG GGA AAC 3′ BamHI Arg3003′5′ GAA TTC TCA CTC CGG GGC ATT GTC ATT CTC ATC 3′ EcoRI stop Glu431VE-EC3–45′5′ TATA CAT ATG ACC CAG ACC AAG TAC ACA TTT GTC 3′ NdeI Thr2123′5′ CTC GAA TTC TCA CTC CGG GGC ATT GTC ATT CTC ATC 3′ EcoRI stop Glu431VE-EC2–45′5′ TATA CAT ATG ACG CAT CGG TTG TTC AAT GCG TCC 3′ NdeI Thr1053′5′ CTC GAA TTC TCA CTC CGG GGC ATT GTC ATT CTC ATC 3′ EcoRI stop Glu431VE-EC1–4m5′5′ GTT CAT GAG GTG GCC GCC AAC TGG CCT GTG 3′ Ala98Ala993′5′ CAC AGG CCA GTT GGC GGC CAG GTC ATG AAC 3′ Ala99 Ala98Restriction sites (underlined) and stop codons are shown in bold. The first and the last encoded amino acids as well as the mutated positions are also indicated. Open table in a new tab Restriction sites (underlined) and stop codons are shown in bold. The first and the last encoded amino acids as well as the mutated positions are also indicated. The mutated fragment VE-EC1–4m was elaborated using the QuickChange site-directed mutagenesis kit (Stratagene Europe, Amsterdam, The Netherlands). The mutations N98A and D99A were introduced using the synthetic oligonucleotide primers shown in Table I. The single module fragments VE-EC1 and VE-EC4 were purified as GST fusion proteins directly from bacterial lysates using the affinity matrix glutathione-Sepharose 4B (Amersham Biosciences). Removal of the GST tail was achieved by cleavage with thrombin (2× 1 unit/mg fusion protein, 2× for 2 h at room temperature). Following complete digestion, GST was retained by an affinity chromatography step on glutathione-Sepharose 4B (Amersham Biosciences), whereas the VE cadherin fragments eluted in the flow-through. After concentration to 1 mg/ml, VE cadherin-derived proteins were run on a gel filtration Superdex S200 column (Amersham Biosciences) to eliminate degradation products. The multimodule fragments VE-EC1–2, VE-EC1–3, VE-EC1–4, VE-EC3–4, and VE-EC2–4 (Fig. 1A) were produced and purified as described (29.Legrand P. Bibert S. Jaquinod M. Ebel C. Hewat E. Vincent F. Vanbelle C. Concord E. Vernet T. Gulino D. J. Biol. Chem. 2001; 276: 3581-3588Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Molar extinction coefficients for VE cadherin fragments were calculated based on their respective amino acid composition using the computer program Protparam Tools of the Expasy server (www.expasy.ch/tools/protparam.html). Multimers of VE cadherin fragments were fractionated at 4 °C by analytical gel filtration on a Superdex S200 column (fractionation range 10,000–500,000 Da, Amersham Biosciences). The hydrodynamic radii (Rh) corresponding to the oligomeric forms of the different VE cadherin fragments were deduced from chromatograms as described previously (29.Legrand P. Bibert S. Jaquinod M. Ebel C. Hewat E. Vincent F. Vanbelle C. Concord E. Vernet T. Gulino D. J. Biol. Chem. 2001; 276: 3581-3588Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Two calibration curves connecting Rh to the molecular weights of the corresponding proteins were established using either standard globular proteins (30.Le Maire M. Aggerbeck L.P. Monteilhet C. Andersen J.P. Moller J.V. Anal. Biochem. 1986; 154: 525-535Crossref PubMed Scopus (105) Google Scholar) or VE cadherin fragments. Molecular weight values, used to establish the VE cadherin curve, were calculated from sedimentation coefficients (see the experimental section under "Analytical Ultracentrifugation") or deduced from the degree of oligomerization determined using cross-linking experiments. The equilibrium between the monomeric (M) and dimeric (D) forms of both VE-EC3–4 and VE-EC2–4 fragments can be described as 2M ↔ D with apparent dissociation constants KD = [M]2/[D], where [M] and [D] correspond to the concentrations of monomer and dimer, respectively. To estimate KD, serial dilutions of the fragments VE-EC3–4 and VE-EC2–4 (10–210 μm) were prepared from 250 μm stock solutions and equilibrated at 4 °C for periods exceeding 48 h. 100 μl of each dilution were injected on the Superdex S200 column using 50 mm Tris, pH 8, containing 150 mm NaCl, and 5 mm CaCl2 as running buffer (0.5 ml/min). No significant dissociation was noticed in a protein concentration range of 10–210 μm despite dilution occurring during the chromatographic run. This very slow dissociation rate allowed estimation of the percentages of monomeric (%M) and dimeric (%D) forms from chromatographic profiles. They were calculated, from their respective chromatographic peaks, after fitting and integration with two Gaussian curves. From these percentages, [M] and [D] were deduced using the following equation: [D] = (%D)Ci/200 and [M] = (%M) Ci/100, where Ci corresponds to the total concentration of the fragments.KD can be finally deduced from the equation ln[D] = 2ln[M] − KD. Prior to digestion, the fragments were equilibrated in 5 mm Ca2+, and their concentrations were adjusted so that they remained oligomeric. Limited proteolysis of the VE cadherin-derived fragments was performed with trypsin at room temperature for 20 min and then blocked using 2 mm phenylmethylsulfonyl fluoride (ICN, Biomedical Inc, Aurora, OH). To establish a comparison between trypsin sensitivity, the various VE cadherin-derived fragments were simultaneously digested using identical standard conditions. The mixtures of peptides were separated by SDS-PAGE, and the gels were stained by Coomassie Blue. The different protein fragments generated by trypsin digestion were individually excised from polyacrylamide gels (lanes 5, Fig. 2A) and subjected to in-gel total proteolysis with trypsin. Practically, excised bands were washed with 50 mm ammonium bicarbonate, destained with acetonitrile (diluted 50/50 in 50 mm ammonium bicarbonate), and dried in a Speedvac evaporator. The gel pieces were re-swollen in 20 μl of 50 mm acetate buffer, pH 7.2, containing 1 μg of trypsin. Following a 2-h digestion at 37 °C, the peptides were extracted with 20 μl of 60% acetonitrile containing 1% trifluoroacetic acid. The combined extracts were desalted on Poros R2 resin. One microliter of the eluate was applied on the dried matrix spot and analyzed as described (31.Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Shevchenko A. Boucherie H. Mann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Crossref PubMed Scopus (1303) Google Scholar). The molecular masses of the resulting peptides were determined using MALDI mass spectrometry that allowed assignment of the cleavage sites. Mass spectra of the recombinant fragments, cross-linked or not, were determined as described previously (29.Legrand P. Bibert S. Jaquinod M. Ebel C. Hewat E. Vincent F. Vanbelle C. Concord E. Vernet T. Gulino D. J. Biol. Chem. 2001; 276: 3581-3588Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The recombinant fragments were cross-linked using N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC from Pierce). EDC was chosen because the coupling reaction is restricted to carboxyl and amino groups separated by only 6 Å, thus preventing unwanted cross-linking between two oligomers. To maintain the oligomeric states, concentrations of the fragments and calcium were adjusted to 50 μm and 5 mm, respectively. Various cross-linking assays were systematically performed with molar ratios between the recombinant fragments and the cross-linker reagent of 37.5, 75, 150, 300, and 600. These experiments were carried out for 2 h at 20 °C in MES buffer, pH 7.0. The cross-linking reactions were terminated by adding 1 m Tris, pH 8.0. Analysis of cross-linked products was then performed on 4–15% gradient Phast gels (Amersham Biosciences). Sedimentation velocity experiments were performed using a Beckman model XL-A analytical centrifuge as described previously (29.Legrand P. Bibert S. Jaquinod M. Ebel C. Hewat E. Vincent F. Vanbelle C. Concord E. Vernet T. Gulino D. J. Biol. Chem. 2001; 276: 3581-3588Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). By combining the sedimentation coefficients (s) and the Rh of VE-EC1–3, determined by ultracentrifugation and gel filtration chromatography experiments, the molar mass (M) of this fragment could be calculated using the following equation: s = M(1 − ρv̄)/6πNηRh, where N is the Avogadro number. The partial specific volumes (v̄) for VE-EC1–3 (0.718 ml/g), the density (ρ) (1.0059 g/ml), and the viscosity (η) (1.022 centipoise) were calculated according to the program Sedinterp supplied by D. B. Hayes, T. Laue, and J. Philo (software available at www.bbri.harward.edu/rasmb/rasmb.html). Sedimentation velocity experiments were performed at 42,000 and 60,000 rpm. To evaluate the relative contribution of each extracellular module of VE cadherin to homotypic binding, several recombinant fragments, encompassing variable parts of the extracellular region of human VE cadherin, were expressed in Escherichia coli. The boundary of these fragments was defined according to the cadherin domain organization proposed by Tanihara et al. (6.Tanihara H. Sano K. Heimark R.L. St. John T. Suzuki S. Cell. Adhes. Commun. 1994; 2: 15-26Crossref PubMed Scopus (138) Google Scholar). To improve the stability of some of them, the boundaries of the fragments were also delineated by means of limited proteolysis experiments. Individual modules or arrays of modules are depicted in Fig. 1A. The single module fragments VE-EC1 and VE-EC4 were expressed as N-terminal glutathione S-transferase (GST) fusion proteins and purified by affinity chromatography using glutathione-Sepharose prior to proteolytic cleavage of the GST tag. The multimodule fragments were purified from inclusion bodies and refolded by dilution as described previously (29.Legrand P. Bibert S. Jaquinod M. Ebel C. Hewat E. Vincent F. Vanbelle C. Concord E. Vernet T. Gulino D. J. Biol. Chem. 2001; 276: 3581-3588Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The purity of the VE cadherin fragments was verified by SDS-PAGE analysis (Fig. 1B). N-terminal sequencing revealed that cleavage by thrombin left the exogenous sequence GS at the N termini of fragment VE-EC1 (Table II). Concerning the multimodule fragments, N-terminal sequences corresponded to the expected ones, except for those of the inner fragments VE-EC3–4 and VE-EC2–4 for which the N-terminal methionine was cleaved off (Table II). MALDI mass spectrometry analysis confirmed the identity of the VE cadherin fragments (Table II).Table IICharacterization of VE cadherin recombinant fragmentsFragmentsN-terminal sequencingMassTheoreticalExperimentalTheoretical2-aDetermined using the computer program Protparam Tools of the Expasy server (www.expasy.ch/tools/protparam.html).Experimental2-bMeasured by MALDI mass spectrometry.DaVE-EC1GSDWIWNQMGSDWIWNQ12,55212,554VE-EC1–2MDWIWNQMMDWIWN27,45527,473VE-EC1–3MDWIWNQMMDWI–NQM36,27836,269VE-EC1–4MDWIWNQMMDWIWNQM48,94148,948VE-EC4GSPEFQQPGSPEFQQP12,89012,892VE-EC3–4MTQTKYTFVTQTKYTFV25,19825,198VE-EC2–4MTHRLFNASTHRLFNAS36,81436,816Exogenous methionines at the N termini are shown in bold. Exogenous sequences generated following cleavage with thrombin are given in italics.2-a Determined using the computer program Protparam Tools of the Expasy server (www.expasy.ch/tools/protparam.html).2-b Measured by MALDI mass spectrometry. Open table in a new tab Exogenous methionines at the N termini are shown in bold. Exogenous sequences generated following cleavage with thrombin are given in italics. The capacity of the fragments to self-associate was analyzed by gel filtration chromatography, chemical cross-linking, and analytical ultracentrifugation experiments. As illustrated in Fig. 2, the chromatographic profiles differ according to the nature of the fragments. Thus, fragments VE-EC1, VE-EC4, VE-EC1–2 (not shown), and VE-EC1–3 gave single elution peaks for fragment concentrations tested up to 200 μm. In contrast, double distributions (peaks I and II) were observed for the fragments VE-EC3–4, VE-EC2–4, and VE-EC1–4, even at the relatively low concentration of 10 μm, indicating that they possess two different oligomeric states. From the elution volumes of the peaks observed on the chromatograms, the hydrodynamic radii of the different species of each fragment were deduced (Table III and "Materials and Methods").Table IIIOligomeric states of different VE cadherin fragmentsVE cadherin fragmentsRhS (Svedberg)Molar massesMultimerC50%ÅkDamVE-EC1–438; 672.9; 9.849; 3013-aDetermined by analytical centrifugation.Monomer, hexamer0.5VE-EC1–3332.2363-aDetermined by analytical centrifugation.MonomerVE-EC1–226MonomerVE-EC118.5123-bDetermined by cross-linking experiments.MonomerVE-EC420.5153-bDetermined by cross-linking experiments.MonomerVE-EC3–430; 4030; 603-bDetermined by cross-linking experiments.Monomer, dimer80VE-EC2–436; 5036; 673-bDetermined by cross-linking experiments.Monomer, dimer25The oligomeric states of the different fragments were determined either by analytical centrifugation or by cross-linking experiments. For each fragment, the first number corresponds to the monomeric form, and the second one corresponds to the multimeric form. Hydrodynamic radii (Rh) and sedimentation coefficients (s) were determined from gel filtration chromatography and ultracentrifugation experiments, respectively. C50% values indicate the concentration at which 50% of the respective fragments is oligomeric.3-a Determined by analytical centrifugation.3-b Determined by cross-linking experiments. Open table in a new tab The oligomeric states of the different fragments were determined either by analytical centrifugation or by cross-linking experiments. For each fragment, the first number corresponds to the monomeric form, and the second one corresponds to the multimeric form. Hydrodynamic radii (Rh) and sedimentation coefficients (s) were determined from gel filtration chromatography and ultracentrifugation experiments, respectively. C50% values indicate the concentration at which 50% of the respective fragments is oligomeric. To determine the oligomeric states of the fragments, they were first cross-linked using the heterobifunctional reagent EDC that covalently couples primary amino to carboxyl groups located in close proximity. Fig. 3 shows the electrophoretic separation of the cross-linked products. Cross-linked fragments VE-EC3–4 and VE-EC2–4 exhibited a two band pattern. The upper bands have molecular masses of ∼50 and 72 kDa for the cross-linked fragments VE-EC3–4 and VE-EC2–4, respecti