Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids

LOMBARD, Sophie; Helmy, Mohamed; Piéroni, G

doi:10.1042/bj3580773

Cited by 21 publications

(16 citation statements)

References 29 publications

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“…This demonstrates the catalytic role of RTA-Ser-221 in lipid hydrolysis and is consistent with the 89% inhibition obtained using a serine hydrolase inhibitor, i.e. E 600 (18). The lipase site predicted by structural analysis is therefore functional in ricin and unique within the molecule.…”

Section: Resultssupporting

confidence: 65%

“…Ricin lipase activity is more active (ϳ5-fold) on neutral lipids, such as triglycerides (18), than on glycerophospholipids (17). We therefore investigated the crystal structure of ricin to find common structural features with ␣/␤ hydrolases, using the typical neutral lipase of Pseudomonas as reference (26).…”

Section: Resultsmentioning

confidence: 99%

“…After 30 min at 37°C, 2 ml of acetone were added before measuring the A 412 as described previously (18). Specific lipolytic activities were expressed as millikatal/mol.…”

Section: Methodsmentioning

confidence: 99%

“…Following our initial observation that ricin displays significant homology to a lipase from another Euphorbiaceae (16), we recently demonstrated that ricin shows lipolytic activity (17) and further characterized its specificity toward neutral lipids (18). Such lipase activity could facilitate RTA access to the cytosol by providing local destabilization of the membrane (18) and might therefore be implicated in the potentiation of RTA toxicity by RTB.…”

mentioning

confidence: 99%

“…Such lipase activity could facilitate RTA access to the cytosol by providing local destabilization of the membrane (18) and might therefore be implicated in the potentiation of RTA toxicity by RTB. The B-chain would thus directly assist the A-chain during its translocation step as is the case for another heterodimeric toxin, diphtheria toxin (19).…”

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confidence: 99%

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Identification of the Ricin Lipase Site and Implication in Cytotoxicity

Morlon-Guyot

Helmy

Lombard-Frasca

et al. 2003

Journal of Biological Chemistry

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Ricin is a heterodimeric plant toxin and the prototype of type II ribosome-inactivating proteins. Its B-chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the membrane of intracellular compartments to reach the cytosol where its Nglycosidase activity inactivates ribosomes, thereby arresting protein synthesis. We here show that ricin possesses a functional lipase active site at the interface between the two subunits. It involves residues from both chains. Mutation to alanine of catalytic serine 221 on the A-chain abolished ricin lipase activity. Moreover, this mutation slowed down the A-chain translocation rate and inhibited toxicity by 35%. Lipase activity is therefore required for efficient ricin A-chain translocation and cytotoxicity. This conclusion was further supported by structural examination of type II ribosome-inactivating proteins that showed that this lipase site is present in toxic (ricin and abrin) but is altered in nontoxic (ebulin 1 and mistletoe lectin I) members of this family.Ricin, isolated from seeds of the plant Ricinus communis, is the prototype of type II ribosome-inactivating proteins (RIPs) 1 and one of the most powerful toxins capable of killing animal cells. This 66-kDa glycoprotein is composed of two chains (RTA and RTB) linked via a disulfide bond. RTA is responsible for cytotoxicity. This N-glycosidase catalyzes the depurination of a specific adenine on the 28 S ribosomal RNA, thereby inactivating protein synthesis and leading to cell death. RTB is a lectin that recognizes terminal galactose residues and is responsible for toxin binding to cells (1).X-ray structures of the heterodimer and the recombinant RTA subunit (rRTA) have been solved at 2.5 and 2.3 Å, respectively (2, 3). These studies described both the RTA N-glycosidase active site and the RTB galactose binding pockets. rRTA lacks the glycans present on native RTA but shows complete biological activity (1, 4).Ricin has been used to study molecular mechanisms involved in intracellular trafficking (5). After cell binding, ricin is endocytosed and can be visualized in endosomes and the trans-Golgi network (6). Biochemical evidence indicates that it could be transported back to the endoplasmic reticulum (7). Ricin escape to the cytosol has been reported to occur from endosomes (8), the Golgi apparatus (9), and the endoplasmic reticulum (7). It is not known whether the affinity of RTA for model membranes (10, 11) facilitates its trans-membrane transport. This translocation step is rate-limiting for cytotoxicity (12).Ricin has been extensively studied for its potential use in cancer treatments. Toward this objective, immunotoxins (ITs) were prepared by attaching ricin to a monoclonal antibody (13). For clinical use and to avoid nonspecific binding of the IT, B-chain lectin sites were chemically inactivated. ITs containing such blocked ricin are usually much more efficient in vivo than those prepared with RTA alone (13). Similar data were obtained in vitro when lactose was used to preve...

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Section: Resultssupporting

confidence: 65%

Section: Resultsmentioning

confidence: 99%

“…After 30 min at 37°C, 2 ml of acetone were added before measuring the A 412 as described previously (18). Specific lipolytic activities were expressed as millikatal/mol.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Identification of the Ricin Lipase Site and Implication in Cytotoxicity

Morlon-Guyot

Helmy

Lombard-Frasca

et al. 2003

Journal of Biological Chemistry

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Membrane destabilization by ricin

et al. 2004

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Ricin is a promising candidate for the treatment of cancer because it can be selectively targeted to tumor cells via linkage to monoclonal antibodies. Biochemical evidence suggests that escape of ricin or its ribosome-inactivating subunit from an intracellular compartment is mediated by retrograde transport to the endoplasmic reticulum and subsequent direction into the ER-associated degradation pathway. Alternatively, lipase activity of ricin may facilitate leakage from endocytic vesicles. We have observed ricin-mediated release of macromolecular dyes from lipid vesicles that mimic the composition of endosomal membranes. Release of small molecules occurs to the same extent, suggesting an all-or-none mechanism due to bilayer destabilization. The level of accompanying membrane fusion depends on vesicle composition. Since it takes 24 h of incubation before the first traces of lysolipids are detectable by matrix-assisted laser desorption/ionization mass spectrometry, membrane destabilization is not due to the lipase activity of ricin.

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