C-Mannosylated tryptophan-containing WSPW peptide binds to actinin-4 and alters E-cadherin subcellular localization in lung epithelial-like A549 cells.

The Trp-x-x-Trp (W-x-x-W) peptide motif, a consensus site for C-mannosylation, is the functional motif in cytokine type I receptors or thrombospondin type I repeat (TSR) superfamily proteins. W-x-x-W motifs are important for physiological and pathological functions of their parental proteins, but effects of C-mannosylation on protein functions remain to be elucidated. By using chemically synthesized WSPW peptides and C-mannosylated WSPW peptides (C-Man-WSPW), we herein investigated whether C-mannosylation of WSPW peptides confer additional biological functions to WSPW peptides. C-Man-WSPW peptide, but not non-mannosylated WSPW, reduced E-cadherin levels in A549 cells. Via peptide mass fingerprinting analysis, we identified actinin-4 as a C-Man-WSPW-binding protein in A549 cells. Actinin-4 partly co-localized with E-cadherin or ß-catenin, despite no direct interaction between actinin-4 and E-cadherin. C-Man-WSPW reduced co-localization of E-cadherin and actinin-4; non-mannosylated WSPW had no effect on localization. In actinin-4-knockdown cells, E-cadherin was upregulated and demonstrated a punctate staining pattern in the cytoplasm, which suggests that actinin-4 regulated cell-surface E-cadherin localization. Thus, C-mannosylation of WSPW peptides is required for interaction with actinin-4 that subsequently alters expression and subcellular localization of E-cadherin and morphology of epithelial-like cells. Our results therefore suggest a regulatory role of C-mannosylation of the W-x-x-W motif in interactions between the motif and its binding partner and will thereby enhance understanding of protein C-mannosylation.

Hsc70 facilitates TGF-ß-induced activation of Smad2/3 in fibroblastic NRK-49F cells.

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-ß-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-ß signaling, was also suppressed in the early phase of TGF-ß stimulation. Upon stimulation with TGF-ß, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-ß-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-ß-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-ß-induced Smad signaling in NRK-49F cells.

High frequency and large deposition of acid fog on high elevation forest.

We have collected and analyzed fogwater on the mountainside of Mt. Oyama (1252 m) in the Tanzawa Mountains of Japan and observed the fog event frequency from the base of the mountain with a video camera. The fog event frequency increased with elevation and was observed to be present 46% of the year at the summit. The water deposition via throughfall increased with elevation because of the increase in fogwater interception and was about twice that via rain at the summit, where the air pollutant deposition via throughfall was several times that via rainwater. The dry deposition and the deposition via fogwater were dominant factors in the total ion deposition at high elevation sites. In a fog event, nitric acid, the major acid component on the mountain, is formed during the transport of the air mass from the base of the mountain along the mountainside, where gases including nitric acid deposit and are scavenged by fogwater. Therefore, high acidity caused by nitric acid and relatively low ion strength are observed in the fogwater at high elevation sites.