Interaction of volkensin with HeLa cells: binding, uptake, intracellular localization, degradation and exocytosis.
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.
Battelli
M G
MG
Dipartimento di Patologia Sperimentale, Alma Mater Studiorum Università di Bologna, Via S. Giacomo 14, 40126, Bologna, Italy. mariagiulia.battelli@unibo.it
Musiani
S
S
Buonamici
L
L
Santi
S
S
Riccio
M
M
Maraldi
N M
NM
Girbés
T
T
Stirpe
F
F
D005089:Exocytosis / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D054790:Ribosome Inactivating Proteins, Type 2; D012276:Ricin / Q000378:metabolism
D006023:Glycoproteins; D037121:Plant Lectins; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054790:Ribosome Inactivating Proteins, Type 2; D012276:Ricin; C049134:volkensin; D009699:N-Glycosyl Hydrolases