Marc De Wolf

Selective blocking of clathrin-mediated endocytosis by RNA interference: epsin as target protein

Epsin is an essential accessory protein exclusively implicated in clathrin-mediated endocytosis and therefore an ideal target to study involvement of this entry route in the uptake of bioligands. T...

Interaction of a cholera toxin derivative containing a reduced number of receptor binding sites with intact cells in culture

Hybrid CTB (hCTB), having only one or two functional binding sites, has been constructed from two chemically inactivated derivatives of CTB. One inactive derivative consisted of CTB formylated in the lone Trp-88 of each beta-chain (fCTB), whereas the other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site (sssCTB). hCTB, fCTB and sssCTB were able to reassociate with CTA and form the corresponding holotoxins hCT, fCT and sssCT as measured by gel filtration chromatography. In contrast to fCT and sssCT, hCT could increase the cAMP content of intact Vero cells in a time- and dose-dependent way: concentrations as low as a few nanograms of hCT per milliliter caused a significant increase in the intracellular cAMP level. The maximal cAMP level induced by hCT (1 microgram/ml) was, however, more than 2-fold lower than that elicited by its native counterpart. At saturating ligand concentrations and at 37 degrees C, the lag periods and rates of CT and hCT induced cAMP accumulation were essentially the same. Treatment of Vero and HeLa cells with GM1 did not affect their difference in response to CT and hCT. When Vero cells treated with hCT were incubated for longer periods of time, a further slow accumulation of cAMP occurred until after about 20 h cAMP levels of cells exposed to CT or hCT were essentially the same. In contrast to Vero and HeLa cells, human skin fibroblasts exhibited an almost identical response to CT as well as to hCT. Acidotropic agents such as chloroquine and monensin affected the CT and hCT induced increase in cAMP content of Vero cells, fibroblasts and GM1 treated Hela cells in a similar way. The results are consistent with the view that CT receptor recognition domains are shared between adjacent beta-chains, that pentavalent binding appears not to be essential for cytotoxicity and that in the cell types studied intracellular processing of CT, hCT is involved.

A Dipeptide Metalloendoprotease Substrate Completely Blocks the Response of Cells in Culture to Cholera Toxin

Prior exposure (15 min at 37 °C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mm N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH2), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation. The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH2, an inactive analogue of Cbz-Gly-Phe-NH2. The effect was reversible and dose- (IC50 as low as 0.2 mm depending on the cell type) and time-dependent. Adding Cbz-Gly-Phe-NH2 during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA). Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced. The A1 peptide of CT and NAD+ activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH2-treated cells or when Cbz-Gly-Phe-NH2 was added directly to the assay. The inhibitory effects of suboptimal amounts of Cbz-Gly-Phe-NH2and BFA were not additive pointing to a similar mode of action of the two substances. However, Madin-Darby canine kidney cells of which the Golgi structure is BFA-resistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH2 on CT cytotoxicity. Several lines of evidence indicate that a perturbation of intracellular Ca2+ homeostasis by Cbz-Gly-Phe-NH2 is not responsible for the inhibitory effect of the dipeptide. The dipeptide had also no effect on the binding of 125I-CT to cells and even increased its intracellular internalization. In contrast with BFA, Cbz-Gly-Phe-NH2 did not completely suppress the formation of the catalytically active A1 fragment from bound CT. The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH2cannot be excluded.

Regeneration of active receptor recognition domains on the B subunit of cholera toxin by formation of hybrids from chemically inactivated derivatives

In order to test the hypothesis that binding sites of cholera toxin for its receptor, the monosialoganglioside GM1, are shared between adjacent beta-polypeptide chains, two inactive chemical derivatives of the B subunit of cholera toxin (CTB) were prepared and were subsequently used for the construction of hybrid CTB pentamers. One inactive derivative consisted of CTB specifically modified in the single essential Trp-88 residue of each beta-chain. This residue was modified by formylation, a treatment preserving the structural integrity of CTB. The other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site. Using [1,4-14C]succinic anhydride for the site-specific succinylation and analysis of radiolabeled tryptic fragments of S-carboxymethylated [14C]sssCTB revealed that the amino groups specifically modified were the alpha-amino group of Thr-1 and the epsilon-amino groups of respectively Lys-34 and Lys-91. Upon submitting equal amounts of formylated CTB and site-specific succinylated CTB to a denaturation-renaturation cycle, hybrid pentamers were formed which in contrast to the parental compounds were able to bind GM1. The affinity of hybrid CTB for GM1, as estimated by a competitive solid-phase radiobinding assay was unexpectedly high and only 2.5-fold lower than that of its native counterpart. The number of active binding sites on hybrid CTB was determined from: (i) titration with the oligosaccharide moiety of GM1 (oligo-GM1) and monitoring the reversal of the Trp fluorescence quenching by iodide ions and (ii) rapid gel filtration over a superdex HR column of a mixture of hybrid CTB and an excess of 3H-labeled oligo-GM1. The data are in agreement with the formation of one active binding per four reconstituted binding sites in hybrid CTB, which is consistent with a random association of CTB monomers during the denaturation-renaturation cycle.

Fluidity of Thyroid Plasma Membranes

The main function of the thyroid cell is the secretion of thyroid hormones (T4, thyroxine; T3, triiodothyronine). Thyroid hormones are first synthesized as part of thyroglobulin. Newly synthesized thyroglobulin is iodinated and vectorially transported to the apical region of the cell and released for storage in the lumen by exocytosis. When hormones are needed, thyroglobulin is removed from the luminal content by endocytosis and transferred to the lysosomes, where it is hydrolysed by cathepsin D liberating the hormones. Nonhormonal active iodotyrosines (3-monoiodotyrosine; 3,5-diiodotyrosine) are also formed. The hormones are released in the bloodstream and the iodotyrosine deiodinated. The iodide formed is partly reutilized by the cell (Taurog, 1978). This bidirectional transport of thyroglobulin is compartmented and implies concomitant transfer of membrane (Herzog, 1981). Every step in thyroid metabolism is activated by TSH (thyroid stimulating hormone, thyrotropin). It evokes intracellular metabolic responses by (1) recognition and binding to the cell surface, (2) transmission of information across the cell membrane, and (3) activation of internal metabolic pathways.

Characterization of dolichol kinase from bovine thyroid microsomes

Bovine thyroid microsomes are able to phosphorylate exogenous [1-3H]dolichol as well as endogenous dolichol. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [1-3H]dolichol to [1-3H]DMP as well as the formation of [32P]DMP from endogenous dolichol and [gamma-32P]CTP. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca2+- and Mn2+-ions. In accordance, EDTA was strongly inhibitory. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. The apparent Km-value for exogenous dolichol amounted to 4 microM. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [1-3H]dolichol depending on the source of CTP. With endogenous dolichol Km-values for CTP of 27.8 and 6.1 microM were calculated in respectively the absence and presence of 5 mM VO4(3-). Triton X-100 (0.15%) was necessary in the [1-3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [gamma-32P]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). The levels of the enzymatic activity could be doubled by the inclusion of 18-21 mM NaF [( 1-3H]dolichol kinase) as phosphatase inhibitor: VO4(3-) had practically no effect. In contrast with [gamma-32P]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO4(3-) while F- resulted into no appreciable effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Brefeldin A and Exo1 Completely Releave the Block of Cholera Toxin Action by a Dipeptide Metalloendoprotease Substrate

Cholera toxin (CT), the enterotoxin secreted by Vibrio cholerae classical as well as El Tor biotypes, is the major causative agent of the acute diarrheal disease of humans. CT and the Escherichia coli heat labile enterotoxin (LT),are structurally and immunologically highly homologous,seeing that they belong to the same enterotoxin family (de Haan and Hirst, 2004; Spangler, 1992; Vanden Broeck et al., 2007). Both are oligomeric proteins of the A-B type. CT is composed of one A or activating subunit (CT-A Mr 27,400), which consists of two distinct polypeptide chains CT-A1 (Mr 22,000) and CT-A2 (Mr 5,400), linked by a single disulfide bridge, and 5 identical B subunits (Mr 11,600) arranged in a ring like configuration (CT-B).

Interaction of Cholera Toxin with its Receptor the Monosialoganglioside GM1: A Fluorescence Study

Cholera toxin (CT) is an enterotoxin secreted by Vibrio cholerae producing its pathological effects by increasing the c-AMP level in intestinal epithelial cells1,2. It is an oligomeric protein (Mr ~ 84,000) composed of two structural and functional distinct subunits CT A and CT B (Mr ~ 29,000 and 55,000 respectively). CT B contains five identical polypeptide chains (Mr = 11,600), most likely arranged in a ring-like pentameric configuration and CT A consists of two non-identical polypeptide chains A or a-chain (Mr = 23,000) and A2 or γ-chain (Mr = 5,500) linked by a single disulfide bridge (for reviews see refs. 1, 3–5). CT A is synthesized as a single polypeptide chain which is “nicked” between the two cysteine residues by an extracellular bacterial protease. During this proteolysis two serine residues are removed at the COOH terminus of A1 6. The first event in the action of CT on cells is the rapid, irreversible binding to receptors on the cell surface. It is generally accepted that the receptor for the toxin is the mono-sialoganglioside GM1 (for reviews see refs. 3,4,7).

Identification and characterization of a specific dolichylmonophosphate phosphatase activity in bovine thyroid microsomes

Bovine thyroid membranes are able to dephosphorylate exogenous [1-3H]DMP as well as endogenous prelabeled [32P]DMP. The kinetics, properties and specificity of the dolichylmonophosphatase activity have been studied by monitoring respectively the release of [1-3H]dolichol from [1-3H]DMP and the residual amount of [32P]DMP. The DMP-phosphatase activity is not linear with respect to time and exhibits a neutral pH optimum. There is only linearity in a narrow range of protein concentration when 0.25% Triton X-100 is included in the incubation mixture. Studying the enzymatic activity in function of protein concentration, the detergent requirement shows to be very critical. Triton X-100 is necessary for enzymatic activity with [1-3H]DMP (only 10% of enzymatic activity in the absence of detergent) although the detergent inhibits the hydrolysis of endogenous prelabeled [32P]DMP. Divalent cations are not essential for enzymatic activity, Ca2+-ions being even inhibitory. In accordance, EDTA (EGTA) is slightly stimulatory. The DMP-Pase activity is not influenced by the ionic strength of the incubation system and sulphydryl groups are not involved. NaF, VOS and VO4(3-) are strongly inhibitory. The inhibition by dolichol and PO3-4 can be explained as the result of product inhibition. An apparent Km-value of 2.5 X 10(-5) M is computed for [1-3H]DMP. Bacitracin inhibits DMP-phosphatase in contrast with other reports. Propylthiouracyl, cAMP, TSH and several other bio-effectors are without effect on the in vitro system. The specificity of the DMP-Pase activity is discussed, showing that the phosphatase is distinctly different from other phosphatases especially phosphatidic acid phosphohydrolase.