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Brefeldin A and Exo1 completely releave the block of cholera toxin action by a dipeptide metalloendoprotease substrate

Vanden Broeck, D;De Wolf, MJS;

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
The subunits are arranged in such a manner that CT-A occupies the central channel of the
CT-B pentamer extending well above the plane of the pentameric ring (Sixma et al., 1991;
Zhang et al., 1995). The CT-A2peptide goes through the pore in the doughnut-like structure
of the CT-B pentamer, and protrudes on the side, which binds cell surface receptors with its
COOH-terminal KDEL sequence exposed. CT elicits a secretory response from intestinal
epithelia by binding to the apical cell membrane through interaction between CT-B and the
monosialoganglioside GM1, followed by entry of polypeptide A1 into the cell, where it is
able to stimulate the basolateral adenylatecyclase by catalyzing the ADP-ribosylation of
Arg201 of the Gs subunit of the stimulatory GTP-binding regulatory protein (de Haan and
Hirst, 2004; Spangler, 1992; Vanden Broeck et al., 2007a; Sixma et al., 1991).
There is a distinct lag period between toxin binding and the activation of adenylatecyclase,
during which the toxin must be internalized and processed.At the end of this lag period small
amounts of CT-A1 appear in the cells parallel to activation of the cyclase(Kassis et al., 1982).
Early morphologic studies showed that CT is preferentially clustered into non-coated
membrane invaginations characteristic of caveolae and enters several cell types via smooth,
non clathrin coated vesicles (Lencer et al., 1999).
Studies using cholesterol perturbing agents and chimeric toxins have shown that GM1-
mediated association with detergent-resistant membrane fractions (DRMS) or lipid rafts is
required for toxic entry of CT (Orlandi and Fishman, 1998; Wolf et al., 1998, 2002)

Although CT is currently used as a marker for endocytosis without utilising clathrin-coated
pits,it appears to be endocytosed simultaneously through both clathrin-dependent
and independent routes (Orlandi and Fishman, 1998; Nichols et al., 2001; Shogomori and
Futerman, 2001; Torgersen et al., 2001; Vanden Broeck et al., 2007b).
In a recent study using fluorescence microscopy (Massol et al., 2004)it has been shown that
apart from clathrin-, caveolin-endocytic pathways, CT also enters cells via a pathway that is
regulated by the small GTPase Arf6 and possibly a fourth pathway that is dynamin and
Arf6-independent. However, after blocking all three known endocytic payhways
simultaneously by over expression of negative dominant mutants of dynamin and Arf6,
fluorescent CT in the Golgi and ER became undetectable, although CT induced toxicity was
hardly affected (Massol et al., 2004).These findings illustrate the difficulty in correlating
morphologic data with the functional entry of a potent toxin such as CT.
Consistent with the multiple ports of entry into the cell, CT can be found in early and recycling
endosomes (Tran et al., 1987; Nichols, 2002)and in caveolin-1 containing endocytic
intermediates (Nichols, 2002),which have been proposed to be responsible for the functional
transport of CT. For CT to be toxic it must be transported through the Golgi to the ER.
Brefeldin A (BFA), a fungal metabolite that disrupts the structural and functional integrity of
the Golgi apparatus (Klausner et al., 1992),renders cells resistant to CT cytotoxicity and blocks
intracellular formation of CT-A1(Orlandi et al., 1993; Nambiar et al., 1993; Lencer et al., 1993).
Movement into the Golgi can also be inhibited by blockage of COPI- and COPII- mediated
vesicular transport, and this affects toxin function further implicating trafficking through the
Golgi apparatus as a necessary step in toxin action (Richards et al., 2002; Majoul et al., 1998).
On reaching the ER, the reduced form of the lumenal chaperone protein disulfideisomerase
binds to the A1 chain, dissociates it from the B subunit and unfolds it(Tsai et al., 2001; Tsai
and Rapoport, 2002; Fujinaga et al., 2003). Subsequent oxidation of PDI by the ER lumenal
ERO1, the A1 chain is released (Tsai and Rapoport, 2002)and is translocated to the cytosol
probably via the Sec61 channel, identifying the rough ER as the compartment from which
translocation occurs (Schmitz et al., 2000). It has been suggested that the rapid refolding
(Tsai and Rapoport, 2002)may render the A1 chain resistant to poly ubiquitination and
provide the driving force for retro translocation to the cytosol (Rodighiero et al., 2002).
We previously reported that the metalloendoprotease substrate
N-benzoyloxycarbonyl-GlyPhe-NH2(Cbz-Gly-Phe-NH2) completely blocked the response of different celltypes in
culture to CT. The effect was reversible, dose-and time-dependent. The dipeptide had no
effect on the binding of CT to the cell surface and did not decrease its internalization but
appeared to affect a later step in toxin action (De Wolf, 2000).
In this study we further investigated the mechanism by which Cbz-Gly-Phe-NH2 blocks CT