CYANOBACTERIAL TOXINS - Cyanobacteria - page 517

E) Mode of action of Hepatotoxins: Signal transduction pathways are mediated by enzymes that
catalyze phosphorylation and dephosphorylation of proteins at specifi c serine, threonine and
tyrosine sites by protein kinases and protein phosphatases, respectively (Cohen, 1989). Substrate
specifi city, dependence on metal ions and sensitivity to inhibitory agents are taken into consideration
for classifying protein phosphatases (PPs). Ser/Thr protein phosphatases are mainly divided into
two categories. Type-1 phosphatases (PP-1) that are inhibited by two heat-stable proteins termed
as inhibitor 1 and inhibitor 2 and preferentially dephosphorylate the β-subunit of phosphorylase
kinase. Type-2 phosphatases (PP-2) are not inhibited by the heat-stable inhibitors and show preference
to the α-subunit of phosphorylase kinase (Cohen, 1989). PP-2 are subdivided into spontaneously
active (PP-2A), Ca 2+ -dependent (PP-2B) and Mg 2+ -dependent (PP-2C) classes. Thus PP-1 and PP2-A
enzymes show metal-independent activities. Wera and Hemmings (1995) described the structure of
each Ser/Thr protein phosphatase, its regulation and probable physiological role. By now as many
as 40 tyrosine phosphatases have been characterized.
Okadaic acid was the fi rst specifi c inhibitor of PPs and subsequently other inhibitors such as
tautomycin, dinophysistoxin, calyculin, MCs and nodularins were discovered. The activities of
PP-1 and PP-2A were inhibited by MC-LR at 1.7 nM and 0.04 nM concentrations (IC 50 ), respectively.
Thus inhibition of PP-2A was greater than PP-1 and the inhibition of the activities of both PP-1
and PP-2A was 10-folds more than that of okadaic acid (Honakanen et al ., 1990). The Ki value for
inhibition of PP-1 and PP-2A by MC-LR was found to be below 0.1 nM. However, the inhibition of
PP-2B was 1000-folds less potent. Further, okadaic acid prevented the binding of MC-LR to PP-2A
while protein inhibitors 1 and 2 prevented the binding of MC-LR to PP-1 and PP-2A. The potential
value of MC-LR in the detection and analysis of protein kinases and phosphatases was highlighted
(MacKintosh et al ., 1990).
MCs and nodularins in solution assume a chemical shape that is similar especially in the Adda-
glutamate part of the cyanotoxin molecule (Rudolph-Boehner et al ., 1994; Annila et al ., 1996). Thus
this region is important for the interaction with PPs and constitutes the essential part for exerting
the toxicity (Barford and Keller, 1994; Goldberg et al ., 1995). The molecular basis of interactions
of MCs with PPs has been explained (Bagu et al ., 1997; Dawson and Holmes, 1999). MCs interact
covalently with PP-1c and PP-2Ac but nodularins do not bind covalently to PP-1c and PP-2Ac.
MCs form this covalent link between the Mdha residue of MCs and Cys-273 of PP-1c bringing them
closer to catalytic center of PP-1c. MCs exert little effect upon PP-2B. The difference in position of
N-methyldehydrobutyrine residue in nodularin relative to N-methyldehydroalanine residue of MC-
LR is responsible for the nature of binding. Further, Bagu et al . (1997) suggested that both okadaic
acid and calyculinA are similar to MCs and nodularins in their tertiary structure and binding domain
to PP1c. Besides these, X-ray crystal structures (Gauss et al ., 1997) and mutational analysis (Zhang
et al ., 1994) provide additional evidences for the nature of interaction of MCs and nodularins with
PP1. In this regard, Maynes et al . (2006) elucidated the crystal structures of nodularin-V (motuporin)
and MC-LA bound to human protein phosphatase-1c (gamma isoform). This is another additional
evidence for the covalent binding of MCs to PP1c but not nodularins to an active site cysteine residue
(Cys-273). MC-HtyR, a dehydrobutyrine containing MC variant [Asp 3 -ADM Adda 5 -Dhb 7 ], isolated
from Nostoc sp., showed a non-covalent interaction with PPs. It could potently inhibit PP1, PP2A,
PP4 and PP5 with IC 50 values similar to MC-LR. Besides, a new cyclic peptide containing 3-amino-
6-hydroxy-2-piperidone named as nostocyclin exhibited 500-folds less potency for inhibition of PP1,
PP2A, PP4 and PP5 (Hastie et al ., 2005).
CYN caused nearly 50% decrease in glutathione levels of cultured rat hepatocytes and this
makes the hepatocytes more vulnerable for CYN toxicity (Runnegar et al ., 1994). The lowering of