Mistletoe Lectins

ML are ribosome-inactivating proteins, composed of two polypetide chains (A and B) linked by disulfide bond. ML differ in specificity and molecular weight from between 50 kDa to 63 kDa. ML I binds to D-galactose, ML III to N-acetyl-D-galactosamine, and ML II to N-acetyl-D-galactosamine and D-galactose (Franz et al., 1981; Pfüller, this book). A number of plant proteins such as the toxic lectins from Ricinus communis, Abrus precatoris and Viscum album, which share a high grade of structural homology, have been identified that catalytically damage eukaryotic ribosomes, and the cells are consequently unable to perform the elongation step of protein synthesis (reviewed by Stirpe et al., 1992; Büssing, 1996). These "ribosome-inactivating proteins" (RIPs) possess carbohydrate-binding B chains linked by hydrophobic bonds and disulfide bridges to the catalytic A chain.

The lectin domains of RIPs can bind to any appropriate carbohydrate domain on cell surface receptors, enabling the protein to enter the cell by receptor-mediated endocytosis (Endo et al., 1988; Stirpe et al., 1982, 1992). Subsequently, the catalytic A chain of the ML inhibits protein synthesis (Stirpe et al., 1980; Olsnes et al., 1982; Endo et al., 1988; Stirpe et al., 1982, 1992), and the cells undergo apoptosis (reviewed by Büssing, 1996). In agreement with these observations, also ricin and abrin were recognised to induce apoptosis (Griffiths et al., 1987; Hughes et al., 1996; Oda et al., 1997). Results of Endo and co-workers (Endo et al., 1987, 1988; Endo and Tsurugi, 1988) indicated that the ML I A chain is similar to the A chain of ricin: a N-glycosidase that releases adenine from position 4324 of 28 S RNA of 60 S ribosomal subunits. This irreversible modification of the ribosomes impairs its ability to interact with elongation factor 2 (EF-2) during the translocation reaction and inhibits the elongation process of the polypeptide chains (Benson et al., 1975; Montanaro et al., 1975). In addition, it has been suggested that ricin inhibits protein synthesis through disturbance of EF-1-dependent aminoacyl-tRNA binding to ribosomes (Furutani et al., 1992). Beside the impairment of macromolecule synthesis, RIPs were suggested to possess polynucleotide: adenosine glycosidase activity and thus, resulting in depurination of DNA and RNA (Barbieri et al., 1997). This may explaine the observed inhibition of RNA and DNA synthesis in response to VA-E and ML (Hülsen et al., 1986; Metzner et al., 1987; Dietrich et al., 1992; Göckeritz et al., 1994; Urech et al., 1995). Protein synthesis was found to be inhibited by ML and other RIPs before that of DNA and RNA (Sargiacomo and Hughes, 1982), indicating that the effect on protein synthesis is of major importance for the fate of the cells. Similar findings were reported for whole plant VA-E (Hülsen et al., 1986). A 50% inhibition of [3H]-thymidine uptake was obtained with 0.06 ng/mL ML I, 0.015 ng/mL ML II and 0.010 ng/mL ML III (Dietrich et al., 1992). However, other researchers observed comparable cytotoxicity for ML I and ML III to mitogen-stimulated lymphocytes since their IC50 was about 0.4 ng/mL (Göckeritz et al., 1994). In mice trreated i.p. with the ML, the LD50 was was 28 ^g/kg BW for ML I, 1.5 pgj kg BW for ML II, and 55 ^g/kg BW (Franz, 1986).

Cytotoxicity of ML was inhibited by plasma proteins (Franz et al., 1977; Ribereau-Gayon et al., 1995, 1997), the specific carbohydrates (Ziska et al., 1978; Luther et al., 1980; Ribereau-Gayon et al., 1997), and CaCl2 (Büssing et al., 1999g). Although the underlying mechanisms of these Ca2+ effects are unclear, one may suggest a stimulation of apoptsis-preventing "calcium-sensing receptors".

Cell death mechanisms

The first effect observed in lymphocytes treated with the ML or its lectin B chain is a rapid receptor-mediated "signal" that increases the content of intracellular Ca2+ (Göckeritz et al., 1994; Büssing et al., 1996; Wenzel-Seifert et al., 1997), which is involved in certain activation pathways leading to endonuclease activation and subsequently DNA fragmentation. After the interaction of lectin B chain with an appropriate surface receptor, and endocytosis of the protein, the A and B chains dissociate in the cytosol (Figure 3). While the A chain enzymatically inhibits the ribosomes, the cytosolic actions of the B chains are unclear. Recent experiments with intensively purified A and B chains from ML I confirmed that neither the B chain nor the A chain alone was able to induce apoptosis (Vervecken et al., 2000). However, the enzymic A chain was reported to induced a slight blastogenic transformation of lymphocytes (Metzner et al., 1987). In contrast, Hostanska et al. (1997) reported an induction of apoptosis by the enzymic A chain of ML I but no effect with the lectin B chain. One cannot exclude the possibility that cross-contaminations of isolated ML chains with the hololectin or the opposite chains may be a reason for the conflicting results.

Subsequently, the ML induce expression of mitochondrial membrane molecules Apo2.7, generation of ROI, cytochrome C release, induction of caspase-3 and caspase-9, Bcl-2 protein degradation, binding of Annexin-V to phosphatidyl serine exposed on the outer leaflet of the cell membran, and loss/fragmentation of DNA (Janssen et al., 1993; Büssing et al., 1996c, 1997, 1998a, b, 1999b, f; Hostanska et al., 19961997; Möckel et al., 1997; Bantel et al., 1999). These apoptosis-associated changes were also observed in lymphocytes treated with protein and/or RNA synthesis inhibitors cycloheximide and actinomycin D, and protein transport inhibitor brefeldin A (Büssing et al., 1999b).

The caspases belong to a family of cystein proteases contributing to apoptosis through direct disassemmbly of cell structures (reviewed by Green and Reed, 1998). In human lymphocytes, we observed both, casapase-3+ cells with and without simultaneous expression of mitochondrial Apo2.7 molecules, indicating that the caspase cascade may be activated in response to mitochondrial triggers (Büssing et al., 1999f). However, leukemic Molt-4 cells were more sensitive to ML I as compared to differentiated lymphocytes; here, almost all cells were Caspase-3+ Apo2.7+. An activation of "death receptors" with Fas-associated death domains (FADD) such as APO-1/Fas, TNF-RI (tumour necrosis factor receptor type 1) and TRAIL (TNF receptor related apoptosis inducing ligand), which play a major role in apoptotic signalling, is unlikely as ML-induced apoptosis was observed in both, Fas-sensitive and -resistant Jurkat T cells, and also in BJAB cells stably transfected with a dominant-negative FADD mutant (Bantel et al., 1999). Moreover, blocking of Fas-molecules on human lymphocytes did not prevent ML-induced cell death (Büssing et al., in preparation).

However, several aspects of ML-induced cell death are unclear, such as the impact of protein synthesis inhibition, DNA damages or altered chromosomal stability, and consequently, the involvement of nuclear p53, Bcl-2 proteins, and IL-4. Since cycloheximide, actinomycin D, brefeldin A, ricin and ML I induced apoptosis (Kochi and Collier, 1993;

Figure 3 Schematic presentation of apoptosis-associated changes induced by the interaction of ML with appropriate surface receptors, internalisation of the protein, and dissociation of A and B chains in the cytosol. Inhibition of protein synthesis may result in decreased level of suggested so-called "apoptosis-preventing factors" (APF) which may inhibit the delivery of "death signals" to the mitochondria. Mitochondrial release of cytochrome c (Cyto c) activates caspases by binding of Apaf-1 (apoptosis-activating factor 1), inducing it to associate with procaspase-9, and thereby initiating the proteolytic caspase cascade that culminates in protein degradation and DNA fragmentation. Bcl-2 is suggested to regulate apoptosis by binding of Apaf-1 and by regulating the induction of mitochondrial permeability transition (reviewed by Kroemer et al., 1997; Green and Reed, 1998).

Figure 3 Schematic presentation of apoptosis-associated changes induced by the interaction of ML with appropriate surface receptors, internalisation of the protein, and dissociation of A and B chains in the cytosol. Inhibition of protein synthesis may result in decreased level of suggested so-called "apoptosis-preventing factors" (APF) which may inhibit the delivery of "death signals" to the mitochondria. Mitochondrial release of cytochrome c (Cyto c) activates caspases by binding of Apaf-1 (apoptosis-activating factor 1), inducing it to associate with procaspase-9, and thereby initiating the proteolytic caspase cascade that culminates in protein degradation and DNA fragmentation. Bcl-2 is suggested to regulate apoptosis by binding of Apaf-1 and by regulating the induction of mitochondrial permeability transition (reviewed by Kroemer et al., 1997; Green and Reed, 1998).

Martin, 1993; Büssing et al., 1999b), one may suggest that each obstacle resulting in an inhibition of protein synthesis or transport will result in an apoptotic cell death, and thus, survival of cells may depend on the constant production of "survival promotors" and/or "death suppressing factors". ML-affected protein synthesis may inhibit homolougs of the Bcl-2 family or protein kinases which may have an anti-apoptotic effect by the phoshorylation of apoptosis-regulating proteins, as suggested by Bantel et al. (1999). In fact, Apo2.7+ caspase-3+ cells were induced also by the protein kinase C inhibitor staurosporine. Also, ML III decreased the intracellular level of Bcl-2 proteins and p53 proteins (Büssing et al., 1998b). Moreover, in response to ML I, Apo2.7 molecules and caspase-3 were detected only in cells with low level of Bcl-2 proteins (Bcl-2'°), while these death markers were not observed in Bcl-2hi cells (Büssing et al., in preparation), indicating degradation of Bcl-2 proteins by the caspases during apoptosis (Büssing et al., 1999f).

Induction of chromosomal instability

In lymphocytes treated with ML III, an increase of telomeric associations (TAs) and C-anaphases was observed (Büssing et al., 1998b). Cancer cells, which lack normal p53 and retinoblastoma protein functions, show chromosomal instability leading to TAs, ring chromosomes and dicentric chromosomes (Healy, 1995; Sharma et al., 1996). TAs have been considered as a cellular manifestation of telomeric loss. The telomeres on both ends of a chromosome serve as a cap and protect the chromosomes from fragmentation, help in the attachment to the nuclear membrane, and in the pairing of homologues during meiotic division (Pathak et al., 1994a). Loss of even a single telomer could render a chromosome instable. Thus, one may assume that the cells with higher p53 level will be killed by the ML via apoptosis, while cells with low level of p53 proteins survive but may harbour chromosomal affections, which are incompatible with normal life. The exact contribution of these obstacles to the onset of apoptosis are yet unclear.

Selectivity of killing

The lectin chains of the various RIPs differ in their cellular interactions. This is suggested by the different lesions each toxin causes in animals (Stirpe et al., 1992), with ricin at high concentrations damaging primarily Kupffer and other macrophagic cells, whereas modeccin and volkensin affect both parenchymal and non-parenchymal liver cells. Since the type 1 RIPs are lacking a lectin subunit, these single chain RIPs are less toxic than type 2 RIPs; however, they are highly toxic to some cells, for instance macrophages and trophoblasts, possibly due to their high pinocytic activity (Stirpe et al., 1992). Obviously, the sensitivity of cells to the lectin-mediated cytotoxicity differs.

A selective killing of CD8+ lymphocytes with a "memory" phenotype (CD62L'°) was observed by low concentrations of the galNAc-binding ML III (Büssing et al., 1998a, b). The reason why ML III at 10 ng/ml selectively killed this subset as cML-1 FITC [3) vs CD19 PE (4)

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