Camp

ADP'fiboEO +

MAD'

Figure 4 Mode of action of cholera toxin, LT toxins and pertussigen. There are five main features in this diagram:

1. cAMP is an important second messenger involved in the intracellular amplification mechanism of many cellular responses to external signals including hormones. The nature of the physiological response reflects the biochemical differentiation of the cell responding to the stimulus. For example, in gut cells the response would be altered ion transport and hence fluid secretion; in muscle cells it would be glycogen breakdown in response to the call for more energy. The production of cAMP is controlled both positively and negatively at two different levels. The central cycle represents a normal membrane-bound hormone receptor and the heterotrimeric (a07) G protein regulator complex which is activated upon binding of hormone to the receptor. There are two receptors, each with regulatory G proteins: one system responds to stimulatory and the other responds to inhibitory stimuli; only one system is shown. In gut cells these receptors would be on the basolateral (nonluminal) side of enterocytes enabling responses to stimuli from the circulation or neuroactive hormone-like substances of which there are quite a few.

2. The endogenous GTPase properties of both stimulatory (ocj and inhibitory («¡) subunits of the G protein regulator may be outlined as follows. Stimulation: Stimulation of receptor results in the dissociation of the as0-y G protein from the receptor and of the subunits from each other, and the binding of GTP to as. us-GTP will productively interact with the catalytic unit of adenylyl cyclase (not shown) and stimulate the production of cAMP from ATP. Endogenous GTPase activity in as results in a delayed conversion of as-GTP to inactive as-GDP which can no longer stimulate the cyclase but allows the reassociation of the trimeric G protein and loss of GDP. Inhibition: Stimulation of receptor results in an analogous situation for inhibiting cyclase activity via <*,: inhibitory «¡-GTP inactivates the cyclase; a,-GDP does not inactivate the cyclase.

3. The level of cAMP may be affected by physiological stimuli (which stimulates removal and modification of the G protein complex) or by intoxication which results in the perturbation of the normal regulatory cycle as illustrated for cholera toxin (CT) or pertussis toxin (PTx, pertussigen).

4. CT acts first by interacting (via CTB) with its receptor resulting in the internalization of the active Al subunit. A1 ADP-ribosylates as-GTP which promotes continued dissociation of the heterodimer; also the endogenous GTPase is no longer functional hence the stimulation of the cyclase continues. LT toxins act in a similar manner. In the case of the enterotoxins the receptors are on the brush border membranes, i.e. on the luminal apex, of enterocytes.

5. PTx acts first by interacting (via PTxB) with its receptor resulting in the internalization of the active S1 subunit. S1 ADP-ribosylates the (».-GDP,,, heterodimer, which can no longer associate with the receptor (or lose GDP) to undergo another cycle of GTP activation; activated cyclase can no longer be turned off. (Reproduced with permission from Mims et al, 1995.)

This description (Figure 4) is correct but too simplistic. Studies by Lundgren's group in Sweden using gut preparations in situ have shown that up to 60% of CT-induced secretion is dependent on a functional enteric nervous system (ENS). Earlier studies by American workers (which have never been refuted) had shown that up to 60% of CT-induced secretion in ligated rabbit ileal loops was suppressed by cycloheximide, which inhibited protein synthesis and mitotic activity (presumably in crypt cells), without affecting the elevation of levels of adenylate cyclase and cAMP. More recently, the colon has been implicated in CT-induced secretion via the ENS. The effect of ZOT has been demonstrated by electron microscopy in human biopsies but the relative importance of ZOT and ACE in human disease has not been quantified.

E. coli produces LT-I, and LT-II whose structure and mode of action is similar to CT. LTh (human).

LTp (porcine) are minor variants of LT-I differing only by one amino acid in the A subunit and four amino acids in the B subunit. LT-I is plasmid encoded. LT-II (isolated from water buffalo) is chromo-somally encoded, exists as Ha or lib (which show partial serological identity), is a major variant of LT-I and is not neutralized by antisera to LT-I or CT. LT-II shows biological activity similar to LT-I, except that it does not cause secretion in ligated rabbit ileal loops, and (unlike LT-I) does not bind to ganglioside GM1. LT-IIb requires trypsin treatment for Y-l adrenal cell assay.

CT and LT toxins are among the most potent antigens known and are also powerful adjuvants.

Bordetella pertussis produces several toxins including a heat-labile toxin, tracheal cytotoxin, endotoxin, adenylate cyclase-hemolysin (AC-Hly), and pertussis toxin (pertussigen). The latter has many biological activities (histamine sensitization, leukocytosis promotion, islets of Langerhans activations and adjuvanticity), is believed to be the most important but by no means the only virulence determinant of B. pertussis, and forms the basis of the vaccine against whooping cough. It is an AB5 toxin in which the B, pentamer consists of four peptides comprising two heterodimers (S2, S4; S3, S4) held together by a fourth peptide (S5); A subunit is designated SI. The mode of action of A subunit is to ADP-ribosylate the arGTP complex of adenylate cyclase; this can no longer inhibit cyclase activity, thereby elevating cAMP. It is not yet clear how such biochemical activity relates to the clinical syndrome.

Clostridium tetani and C. botulinum both produce neurotoxins. Recently huge strides have been made in our knowledge of the structure and mode of action of these toxins (see Figures 5 and 6). There is very little overall sequence homology between BoNTs and TeTx; this could account for their immunological characteristics and the animal types normally affected. However, there is one structural feature common to all these neurotoxins located in the middle of the light chain and that is a zinc-binding motif. In fact it has now been formally proven that these neurotoxins are zinc endopeptidases. Their targets are protein constituents which comprise the synaptic fusion machine responsible for the exocytotic release of neurotransmitters. The intracellular target cleaved by BoNTs B, D, and F and TeTx is synaptobrevin (VAMP, vesicle-associated membrane protein) present in small synaptic vesicles. BoNTs A and E cleave SNAP-25 (synaptosomal-associated protein of 25 kDa) a highly conserved protein known to be involved in exocytosis. BoNT C acts on syntaxin, a synaptic membrane protein also involved in exocytosis.

s-s

sc-TeTx I

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