Routing of internalized ricin and ricin conjugates to the Golgi complex.

Abstract

Abstract. Receptor-mediated endocytosis and intracellular routing of native ricin, and of ricin conjugated to colloidal gold (Ri-Au) and to horseradish peroxidase (Ri-HRP), have been studied in cultured MCF-7 and Veto cells by electron microscopical techniques including serial section analysis. Both native ricin, as demonstrated by immunoperoxidase cylochemistry, and the ricin conjugates were internalized via a common coated pit-coated vesicle pathway to reach vacuolar and tubulo-vesicular portions of the endosomal system. In addition, native ricin and a purified monovalent fraction of Ri-HRP reached distinct Golgi cisterns, whereas Ri-Au and polyvalent Ri-HRP did not. The results delineate intracellular routing of native ricin and compare it with the routing of different ricin conjugates. Moreover, our study shows that conjugates of a particular ligand (ricin) and various probes (e.g., gold and peroxidase), may be handled differently by cells. Sorting apparently takes place in the endosomal system, allowing some but not other molecules to reach Golgi elements. This sorting seems to depend on the valency of the ricin conjugate.ICIN, a representative of a group of highly toxic plant and bacterial proteins (31, 32, 40), consists of two polypeptide chains (each of about 30 kD) linked by a disulfide bond. The B-chain is a lectin that binds to cell surface receptors (galactosc-terminating glycoproteins and giycolipids). The A-chain is an enzyme which, following translocation into the cytosol, interferes with protein synthesis (3 l, 32). Ricin has recently been paid considerable attention because of its application in the preparation of immunotoxins, where monoclonal antibodies are used to direct the toxin or its enzymatically active A-chain towards specific target cells (4,47,52).In order to act, ricin A-chain must gain access to its intracellular target, the ribosomal 60S subunit. As deduced from biochemical studies, the first step in this entry process is receptor-mediated endocytosis (RME) ~ of ricin (3 l, 32, 36, 38-40). Internalization of ricin has also been elucidated ultrastructurally by using ricin conjugated to markers such as ferritin (Ri-Fe) (15,28,29), horseradish peroxidase (Ri-HRP) (13), and colloidal gold (Ri-Au) (51), and in the form of a ricin-immunotoxin adsorbed to colloidal gold (6). However, these studies are contradictory as regards the intracellular routes followed by the conjugates. Moreover, it is important to stress that the route followed by a ligand conjugate does J Abbreviations used in this paper. CF, eationized ferritin; Ri-Au, Ri-Fe, and Ri-HRP, ricin conjugated to colloidal gold, ferritin, and horseradish peroxidase, respectively; Ri-HRPmv and Ri-HRPpv, purified monovalent and polyvalent fractions of Ri-HRP; RME, receptor-mediated endocytosis; SPDP, N-suceinimidyl-3-[2-pyridylthio]propionate: WGA, wheat germ agglutinin. not necessarily reflect the route followed by the native ligand (5 l). Using Ri-HRP, Gonatas et al. (13) observed labeling of Golgi elements, an observation which cou...

The effect of ricin B chain on the intracellular trafficking of an A chain immunotoxin

“…The Fib75-SS-ricin A with associated ricin B chain was found localised in coated pits as has been observed previously for ricin holotoxin (Van Deurs et al, 1986;McIntosh et al, 1990) as well as in discrete areas of the plasma membrane (Figure 2a). Fib75 antibody was seen as fairly evenly distributed label on the plasma membrane but was absent from coated areas.…”

Lipid-Mediated Endocytosis

“…Interestingly, when quantum dots are functionalized either with GD1a antibodies or cholera toxin, they are not recycled like ganglioside antibodies or transported to the Golgi apparatus like cholera toxin, but sorted to the ER, suggesting that the three-dimensional organization of binding site on colloids may play a role in intracellular sorting (Tekle et al 2008;Qian et al 2009). A similar clustering dependent defect of trafficking to the Golgi has been observed for ricin (van Deurs et al 1986). …”

Delivery into cells: lessons learned from plant and bacterial toxins

“…Furthermore, ricin was used to quantify sorting to the various compartments such as the Golgi apparatus versus lysosomes, 50 and it was discovered that the valency of this ligand was important for its destiny. 51 Multivalent complexes of ricin and HRP as well as ricin coupled to gold particles were sent directly to lysosomes, whereas monovalent ricin had the ability to be transported also to the Golgi apparatus. Similarly, evidence for the importance of valency for sorting has been obtained from other systems.…”