Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.

The membrane targeting and spatial activation of Src, Yes and Fyn is influenced by palmitoylation and distinct RhoB/RhoD endosome requirements.

Src activation is a tightly regulated process which requires RhoB endosome-associated actin assembly and transit to the cell periphery. We show here that although two other ubiquitous Src family kinases (SFKs) Yes and Fyn also require intact actin filaments for peripheral membrane targeting, they display distinct spatial activation and endosomal requirements. Unlike Src, both Yes and Fyn are constitutively membrane-localized to some extent, and Fyn is present in RhoD-positive endosomes whereas Yes does not visibly colocalize with either of the endosomal markers RhoB or RhoD. By modulating amino acid acceptor sites for palmitoylation in Src, Yes and Fyn, we show that Src S3C/S6C, which is palmitoylated (unlike wild-type Src) behaves in a manner more similar to Fyn, by predominantly colocalizing with RhoD endosomes, and the targeting of both Fyn and Src S3C/S6C is inhibited by siRNA-mediated knockdown of RhoD. Moreover, Fyn C3S/C6S, which is no longer palmitoylated, behaves much more like Src by colocalizing with RhoB endosomes and by requiring RhoB for activation and membrane translocation. These data imply that distinct modes of spatial activation and membrane delivery, at least partly under the control of specific acylation attachment sequences and endosome sub-type requirements, define distinct properties of the three ubiquitously expressed SFKs.

Accumulation of RhoA, RhoB, RhoG, and Rac1 in fibroblasts from Tangier disease subjects suggests a regulatory role of Rho family proteins in cholesterol efflux.

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. However, the exact relationship of many of the biochemical and morphological abnormalities in TD to ABCA1 is unknown. Since small GTP-binding proteins are important regulators of many cellular functions, we characterized these proteins in normal and TD fibroblasts using the [alpha-32P]GTP overlay technique and Western blotting of SDS and isoelectric focusing gels. Our results indicate that GTP-binding proteins of the Rho family (RhoA, RhoB, RhoG, Rac-1) are enriched in fibroblasts from TD patients. The accumulation of small G proteins may have potential implications for the TD phenotype and the regulation of cholesterol excretion in TD cells.