Anti-DSP monoclonal antibody, clone EQ-3.26

Context: The formation of the multinucleated syncytiotrophoblast of the human placenta by the terminal differentiation and fusion of mononucleate cytotrophoblasts is a critical step in pregnancy. Previous studies have demonstrated that this cellular event is dependent on a progressive decrease in the levels of the cell-adhesion molecule, E-cadherin. Objective: The aim of the study was to examine the role of Twist, a transcription factor identified as a key repressor of E-cadherin expression, in the differentiation of human trophoblastic cells. Design: The expression of Twist or E-cadherin were first examined in first-trimester chorionic villi by immunohistochemistry. Gain-or loss-of-function studies on Twist were then performed in BeWo choriocarcinoma cells. The presence or absence of multinucleated syncytium was confirmed by indirect immunofluorescence using antibodies directed against Twist, E-cadherin, or desmoplakin, a cellular marker of mononucleate cytotrophoblasts. Results: The formation of multinucleated syncytium was associated with increased Twist and a decreased E-cadherin expression. Similarly, exogenous expression of Twist resulted in a continuous and progressive decrease in E-cadherin expression and the subsequent formation of syncytium in BeWo cells maintained under normal culture conditions. In contrast, small interfering RNA specific for Twist inhibited 8-Br-cAMP (8-bromoadenosine 3', 5'-cyclic monophosphate)-mediated differentiation and fusion of over time in culture. Conclusions: Twist is an upstream regulator of the E-cadherin-mediated terminal differentiation and fusion in a human trophoblastic cell line in vitro. (J Clin Endocrinol Metab 96: 3881-3890, 2011)

Desmoplakin (DP) is a cytoskeletal linker protein that connects the desmosomal cadherin/plakoglobin/plakophilin complex to intermediate filaments (IFs). The C-terminal region of DP (DPCT) mediates IF binding, and contains three plakin repeat domains (PRDs), termed PRD-A, PRD-B and PRD-C. Previous crystal structures of PRDs B and C revealed that each is formed by 4.5 copies of a plakin repeat (PR) and has a conserved positively charged groove on its surface. Although PRDs A and B are linked by just four amino acids, B and C are separated by a 154 residue flexible linker, which has hindered crystallographic analysis of the full DPCT. Here we present the crystal structure of a DPCT fragment spanning PRDs A and B, and elucidate the overall architecture of DPCT by small angle X-ray scattering (SAXS) analysis. The structure of PRD-A is similar to that of PRD-B, and the two domains are arranged in a quasi-linear arrangement, and separated by a 4 amino acid linker. Analysis of the B-C linker region using secondary structure prediction and the crystal structure of a homologous linker from the cytolinker periplakin suggests that the N-terminal similar to 100 amino acids of the linker form two PR-like motifs. SAXS analysis of DPCT indicates an elongated but non-linear shape with R-g = 51.5 angstrom and D-max = 178 angstrom. These data provide the first structural insights into an IF binding protein containing multiple PRDs and provide a foundation for studying the molecular basis of DP-IF interactions.