Interaction of volkensin with HeLa cells: binding, uptake, intracellular localization, degradation and exocytosis

Abstract

Among two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10(-10) M) and had a similar number of binding sites (2 x 10(5)/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.

Plant ribosome-inactivating proteins type II induce the unfolded protein response in human cancer cells

“…Type II RIP exposure should be able to overload the UPR machinery of cancer cells at concentrations that are still well tolerated by normal cells [32]. Apart from the differences in receptor binding, internalization pathways and degradation, the induction of the UPR is probably an important factor contributing to type II RIP specificity against cancer cells [17].…”

“…Since toxic type II RIPs are known to accumulate in the ER upon cell entry [17], it is conceivable that ER stress might occur in cells exposed to the toxic members of this class of agents. To answer this question, the two prominent, highly toxic type II RIPs ricin and volkensin were included in this study, in addition to riproximin.…”

Ribosome-inactivating proteins: progress and problems

“…The hypothesis was formulated [98] that, although both lectins can bind to serum glycoproteins and red blood cells, only the agglutinin, being divalent, can form precipitates or agglutinate red cells, thus being prevented from reaching cells in vital organs. Among other non-toxic type 2 RIPs, the uptake by cells and the intracellular routing and processing of nigrin b, from S. nigra, have been studied and this lectin was found to enter cells as well as ricin, but is more rapidly and extensively degraded in, and excreted by, cells [83,99].…”

“…Vol. 63,2006 Review Article 1855 Volkensin too is rapidly excreted by cells, but mostly in a non-degraded form, thus being available for further entering into cells, which may account at least in part for the higher toxicity of this RIP, compared with ricin [83]. The resistance to degradation of volkensin may be due to its low content of lysine [100], as ricin and abrin, which also have a low lysine content, were reported to be more resistant to degradation when some lysine residues were removed, whereas they were more easily degraded when the lysine content was increased [101].…”

“…The reasons for these differences are not completely known and could be many; they most likely involve the binding to, and entry into, cells and/or the intracellular destination, degradation and exocytosis of the proteins. Differences in the number of receptors do not account for the different cytotoxicities: thus on the surface of HeLa cells, there are more receptors for ricin (1-3 × 10 7 ) [81] than for the more toxic RIPs modeccin and volkensin (2 × 10 5 ) [82,83]. After the binding of type 2 RIPs to glycoproteins and glycolipids on the cell surface, different endocytosis processes, either clathrin dependent or independent, direct the molecules to early endosomal vesicles where they can be sorted for further routing, depending on the nature of the receptor, and led to various fates [reviewed in ref.…”