Dataset Information

Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin.

ABSTRACT: Human P-cadherin is a cell adhesion protein of the family of classical cadherins, the overexpression of which is correlated with poor prognosis in various types of cancer. Antibodies inhibiting cell-cell adhesion mediated by P-cadherin show clear therapeutic effect, although the mechanistic basis explaining their effectiveness is still unclear. Based on structural, physicochemical, and functional analyses, we have elucidated the molecular mechanism of disruption of cell adhesion by antibodies targeting human P-cadherin. Herein we have studied three different antibodies, TSP5, TSP7, and TSP11, each recognizing a different epitope on the surface of the cell-adhesive domain (EC1). Although all these three antibodies recognized human P-cadherin with high affinity, only TSP7 disrupted cell adhesion. Notably, we demonstrated that TSP7 abolishes cell adhesion by disabling the so-called X-dimer (a kinetic adhesive intermediate), in addition to disrupting the strand-swap dimer (the final thermodynamic state). The inhibition of the X-dimer was crucial for the overall inhibitory effect, raising the therapeutic value of a kinetic intermediary not only for preventing, but also for reversing, cell adhesion mediated by a member of the classical cadherin family. These findings should help to design more innovative and effective therapeutic solutions targeting human P-cadherin.

SUBMITTER: Kudo S

PROVIDER: S-EPMC5206748 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin.

Kudo Shota S Caaveiro Jose M M JM Nagatoishi Satoru S Miyafusa Takamitsu T Matsuura Tadashi T Sudou Yukio Y Tsumoto Kouhei K

Scientific reports 20170103

Human P-cadherin is a cell adhesion protein of the family of classical cadherins, the overexpression of which is correlated with poor prognosis in various types of cancer. Antibodies inhibiting cell-cell adhesion mediated by P-cadherin show clear therapeutic effect, although the mechanistic basis explaining their effectiveness is still unclear. Based on structural, physicochemical, and functional analyses, we have elucidated the molecular mechanism of disruption of cell adhesion by antibodies ta ...[more]

PMID: 28045038

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Project description:The maturation of dendritic cells (DCs) after exposure to microbial products or inflammatory mediators plays a critical role in initiating the immune response. We found that maturation can also occur under steady state conditions, triggered by alterations in E-cadherin-mediated DC-DC adhesion. Selective disruption of these interactions induced the typical features of DC maturation including the upregulation of costimulatory molecules, MHC class II, and chemokine receptors. These events were triggered at least in part by activation of the b-catenin pathway. However, unlike maturation induced by microbial products, E-cadherin-stimulated DCs failed to release immunostimulatory cytokines, exhibiting an entirely different transcriptional profile. As a result, E-cadherin-stimulated DCs elicited an entirely different T cell response in vivo, generating T cells with a regulatory as opposed to an effector phenotype. These DCs induced tolerance in vivo and may thus contribute to the elusive steady state âtolerogenic DCsâ. Experiment Overall Design: We performed a genome-wide microarray analysis to study the expression profiles of DCs matured by CD as opposed to a conventional TLR agonist (E. coli, which stimulates multiple TLRs). RNA was isolated from human CD34+ DCs at various times after stimulation and used to probe Affymetrix U95Av2 chips. A time course was followed after various stimuli, with single chips used per time point. >700 genes were found differentially regulated upon maturation by either CD or bacterial stimulation. Cluster analysis revealed that after an early phase (1-3 hr) of similarity, expression profiles exhibited by the two sets of DCs diverged dramatically at later time points (>6 hr). A number of transcripts were markedly upregulated in the bacteria-stimulated set that remained relatively unchanged or actually decreased in the cluster-disrupted set. There were some transcripts upregulated in cluster-disrupted cells, however, with at least some of these increases prevented by adding anti-E-cadherin mAb under conditions that blocked maturation. Clearly, the transcriptional events induced by alteration of E-cadherin-mediated adhesion were quite distinct from those induced by TLR activation.

Project description:CD148 is a transmembrane tyrosine phosphatase that is expressed at cell junctions. Recent studies have shown that CD148 associates with the cadherin/catenin complex and p120 catenin (p120) may serve as a substrate. However, the role of CD148 in cadherin cell-cell adhesion remains unknown. Therefore, here we addressed this issue using a series of stable cells and cell-based assays. Wild-type (WT) and catalytically inactive (CS) CD148 were introduced to A431D (lacking classical cadherins), A431D/E-cadherin WT (expressing wild-type E-cadherin), and A431D/E-cadherin 764AAA (expressing p120-uncoupled E-cadherin mutant) cells. The effects of CD148 in cadherin adhesion were assessed by Ca2+ switch and cell aggregation assays. Phosphorylation of E-cadherin/catenin complex and Rho family GTPase activities were also examined. Although CD148 introduction did not alter the expression levels and complex formation of E-cadherin, p120, and ?-catenin, CD148 WT, but not CS, promoted cadherin contacts and strengthened cell-cell adhesion in A431D/E-cadherin WT cells. This effect was accompanied by an increase in Rac1, but not RhoA and Cdc42, activity and largely diminished by Rac1 inhibition. Further, we demonstrate that CD148 reduces the tyrosine phosphorylation of p120 and ?-catenin; causes the dephosphorylation of Y529 suppressive tyrosine residue in Src, a well-known CD148 site, increasing Src activity and enhancing the phosphorylation of Y228 (a Src kinase site) in p120, in E-cadherin contacts. Consistent with these findings, CD148 dephosphorylated both p120 and ?-catenin in vitro. The shRNA-mediated CD148 knockdown in A431 cells showed opposite effects. CD148 showed no effects in A431D and A431D/E-cadherin 764AAA cells. In aggregate, these findings provide the first evidence that CD148 promotes E-cadherin adhesion by regulating Rac1 activity concomitant with modulation of p120, ?-catenin, and Src tyrosine phosphorylation. This effect requires E-cadherin and p120 association.

Dataset Information

Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin.

Publications

Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin.

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