KeywordsProtein Phosphatase Okadaic Acid Microcystis Aeruginosa Round Form Primary Human Fibroblast
Microcystin-LR is cyclo(-D-Ala-L-Leu-D-erythro-beta methylisoaspartic acid-(Masp)- L-Arg-(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-(4E,6E)-dienoic acid (Adda)-D-isoGlu-N-methyldehydroalanine (Mdha)-).
Microcystin-YR (microcystin containing tyrosine instead of the leucine in microcystin-LR).
- Microcystin-RR (microcystin containing arginine instead of the leucine in microcystin-LR) (Fig. 1a).
Microcystin-LR is a hepatotoxin, one of the microcystins isolated from cyanobacteria (blue-green algae), which include Microcystis aeruginosa, Microcystis viridis, Microcystis wesenbergii, Microcystis flos-aquae, and Oscillatoria agardhii. Microcystin-LR is found in waterblooms of toxic cyanobacteria and in eutrophic freshwater municipal and residential water supplies; it is associated with increasing environmental hazards in various areas of the world.
Toxins of cyanobacteria are grouped into cytotoxin, neurotoxin, and hepatotoxin. The hepatotoxins are microcystin-LR, microcystin-YR, and microcystin-RR, and there are 50 microcystin derivatives. Microcystin-LR is a tumor promoter in rat liver, a potent inhibitor of serine/threonine protein phosphatase 1 and 2A (PP1 and PP2A) (IC50 values 0.1 nM and 0.1 nM, respectively). Protein dephosphorylation by PP1 and PP2A is an opposite biochemical reaction of protein phosphorylation by protein kinases. Thus, inhibition of protein phosphatase causes accumulation of phosphoproteins in the cells. The World Health Organization (WHO) International Agency for Research on Cancer (IARC), Lyon, in 2006 assessed the carcinogenicity of microcystin-LR for humans, based on three perspectives: carcinogenicity in rodents, epidemiological evidence, and unique mechanisms of action of the compound; WHO concluded that microcystin-LR is “possibly carcinogenic to humans” (group 2B).
Tumor-Promoting Activity in Rat Liver
Liver organotropy of microcystins is unique because intraperitoneal administration of [3H]dihydromicrocystin-LR results in the highest uptake into the liver of mice – 17.0 ± 4.1 % of the total administered radioactivity after 5 min and 71.5 ± 6.9 % after 1 h – whereas oral administration results in 0.68 % of the total administered amount in the liver from 6 to 19 h after treatment. After a single intraperitoneal injection of 1.5 μg microcystin-YR, i.e., 75 μg/kg body weight, the liver became dark red and the mouse died within 2 h. The histological examination revealed massive intrahepatic hemorrhages resulting in acute death of liver cells by necrosis.
Microcystins are not mutagenic in the Ames test with Salmonella typhimurium. Thus, microcystin-LR has a tumor-promoting activity in rat liver and does not have any initiating activity. Microcystin-YR and microcystin-RR also are assumed to be liver tumor promoters, because they have the same specific activity in various biochemical assays as does microcystin-LR.
Inhibition of Specific [3H]okadaic Acid Binding
The tertiary structure of microcystin-LR is similar to that of okadaic acid, since okadaic acid is thought to have a flexible cavity formed by an intramolecular hydrogen bond between C-1 carbonyl and C-24 hydroxyl groups. Microcystin-LR, microcystin-YR, and microcystin-RR inhibited the specific [3H]okadaic acid binding to cytosolic fraction with the same potencies. Thus, microcystins bind to the okadaic acid receptors PP1 and PP2A as strongly as okadaic acid does.
Inhibition of Protein Phosphatases 1 and 2A
Microcystin-LR inhibits the catalytic subunit of PP1 purified from rabbit skeletal muscle (IC50 value 0.1 nM) and that of PP2A purified from human erythrocytes (IC50 value 0.1 nM). Microcystin-LR inhibited both PP1 and PP2A with the same potencies, but it did not inhibit protein tyrosine phosphatase. Microcystin-LR, microcystin-YR, and microcystin-RR inhibited protein phosphatase 2A, with the similar potencies, and the results correlate well with inhibition of specific [3H]okadaic acid binding.
Cellular Biochemical Response
Microcystin-LR and okadaic acid have different effects on primary human fibroblasts. Microcystin-LR at concentrations up to 9.6 μM did not induce any biochemical or biological effects on primary human fibroblasts, but 250 nM okadaic acid was enough to induce morphological changes in cells, from spindle-like to a round form, within 2 h of incubation. A solution of microcystin-YR at a very high concentration of 670 μM was injected into primary human fibroblasts resulted in morphological changes from spindle-like to a round form 45 min after microinjection. Obviously, there are differences in tissue specificity between microcystins and okadaic acid.
Structure-Function Relationship of Microcystins
The long hydrophobic portion of microcystins (Adda) is significant in their activity. Two geometrical isomers at C-7 in the Adda portion of microcystins, (6Z)-Adda microcystin-LR and microcystin-RR, were isolated from cyanobacteria (Fig. 1b). The specific [3H]dihydromicrocystin-LR binding to the cytosolic fraction was inhibited by microcystin-LR and microcystin-RR (IC50 values 0.38 and 0.42 nM, respectively) and by (6Z)-Adda microcystin-LR and microcystin-RR (IC50 values 32 and 52 nM, respectively). Thus, (6Z)-Adda microcystin-LR and microcystin-RR are 100 times weaker than their maternal (6E)-Adda microcystins, and the conjugated diene with (4E,6E) geometry in the Adda portion is essential in the interaction with protein phosphatases.
Furthermore, microcystin-LR and microcystin-LA have the same specific activity on the inhibitions of PP1 and PP2A and specific [3H]okadaic acid binding, suggesting that the arginine residue in microcystin-LR does not significantly contribute to its biochemical activity.
Crystal Structure of PP1-Microcystin-LR Complex
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