Study of the interaction between the antitumour protein α-sarcin and phospholipid vesicles

Abstract

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.

Fungal extracellular ribotoxins as insecticidal agents

“…Consequently, although any ribosome could be potentially inactivated by these proteins, due to the universal conservativeness of the SRL, ribotoxins have been described as especially active on transformed or virus-infected cells (Fernández-Puentes and Carrasco, 1980;Olmo et al, 2001;Olson et al, 1965). This observation has been explained in terms of an altered permeability of these cells combined with the ability of ribotoxins to interact with acid phospholipid-containing membranes (Gasset et al, 1989(Gasset et al, , 1990Herrero-Galán et al, 2008, 2012bMartínez-Ruiz et al, 2001;Olmo et al, 2001). Insect cells have a different plasma membrane composition from mammalian cells due to their higher content of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol and a significant lower cholesterol/phospholipid ratio (Marheineke et al, 1998).…”

Role of the basic character of α-sarcin’s NH2-terminal β-hairpin in ribosome recognition and phospholipid interaction

“…In fact, mutation of some basic residues that lie outside the restrictocin-SRL interface has been shown to decrease markedly the ribosomes cleaving rates without having any effect on the kinetics of assays employing an isolated SRL-like oligoribonucleotide [37]. It is also well proven how electrostatic interactions are needed for the establishment of the associations needed to allow the passage of ribotoxins across acid phospholipid membranes [24,25,27] and the α-sarcin NH 2 -terminal-β-hairpin is one of the protein regions that has been involved in this membrane recognition mechanism [23,48].…”

“…It is well known how α-sarcin interacts with lipid vesicles through electrostatic and hydrophobic interactions promoting events of vesicle aggregation that are followed by lipid mixing occurring between the bilayers of the aggregated vesicles, as would be expected for fusing liposomes [1,24,25,28]. Stopped-flow measurement of the initial rates of this aggregation induced by α-sarcin showed a second-order dependence on phospholipid concentration, suggesting the formation of vesicle dimers as the initial steps of the process [33].…”

“…Aggregation was monitored as previously described [24,28] by measuring the increase in absorbance at 400 nm of a suspension of vesicles (30 mM final lipid concentration) after addition of a small aliquot of a freshly prepared solution of protein.…”

“…In addition to their specific and lethal ribonucleolytic activity, ribotoxins can also cross phospholipid membranes due to their ability to interact with acid phospholipid-containing bilayers [6,[24][25][26][27][28]. This is the basis to explain why they are especially active on transformed or virus-infected cells [6,29,30], although any ribosome could be potentially inactivated by them, given the universal conservativeness of the SRL.…”