Recent advances in trichosanthin, a ribosome-inactivating protein with multiple pharmacological properties

Abstract

Trichosanthin (TCS), a ribosome-inactivating protein extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii Maximowicz, has multiple pharmacological properties including abortifacient, anti-tumor and anti-HIV. It is traditionally used to induce abortion but its antigenicity and short plasma half-life have limited the repeated clinical administration. In this review, work to locating antigenic sites and prolonging plasma half-life are discussed. Studies on structure–function relationship and mechanism of cell entry are also covered. Recently, TCS has been found to induce apoptosis, enhance the action of chemokines and inhibit HIV-1 integrase. These findings give new insights on the pharmacological properties of TCS and other members of ribosome-inactivating proteins.

Introduction

Trichosanthin (TCS) is a 27 kDa protein extracted from the root tuber of the Chinese medicinal herb Trichosanthes kirilowii Maximowicz (Tian Hua Fen). TCS is synthesized as a preproprotein consisting of 289 amino acids, with a 23-residue signal peptide at the N-terminus and a 19-residue propeptide at the C-terminus. TCS is a type I ribosome-inactivating protein (RIP). It inactivates eukaryotic ribosomes by cleaving the N-glycosidic bond at adenine-4324 of 28S rRNA (Zhang and Liu, 1992). This alters the conformation of the rRNA and inhibits the binding of elongation factors. TCS has long been used to induce mid-term abortion, treat ectopic pregnancies and hydatidiform moles, reset menstruation and expel retained placenta. TCS has also been found to have anti-tumor and anti-HIV activities. Ten years ago, we have reviewed the various pharmacological properties of TCS (Shaw et al., 1994). Recently, ricin and Shiga toxin, members of type II ribosome-inactivating protein with a catalytic subunit similar to TCS, have received increased attention due to their potential use in terrorism and biological warfare. This report provides a timely updated review on our knowledge in the mechanism of action and applications of TCS, which may be extrapolated to related ribosome-inactivating proteins.

TCS has many therapeutic uses including abortifacient (Jin, 1985) anti-tumor (Tsao et al., 1986) and anti-HIV (Mayer et al., 1992). TCS elicits the production of IgG and IgE once injected to the body (He et al., 1999b), and the antigenicity of TCS restricts its repeated clinical use. Several studies have been conducted to elucidate the antigenic sites of TCS, and to reduce the antigenicity without affecting the potency of TCS.

A 2.7-fold decrease in antigenicity and a 10-fold decrease in ribosome-inactivating activity were observed after deleting the C-terminal seven amino acids from TCS (Chan et al., 2000a). Another study found that the smallest antigenic fragment mapped correspond to the N-terminal 20 amino acids of TCS (Mulot et al., 1997). Mutation of K173 and R174 of TCS to glycine reduced the IgE production in immunized mice (Cai et al., 2002).

Attempts have been made to couple PEG or dextran to TCS to block its antigenic sites. IgE response was 4–16-fold lower when PEG was coupled to potential antigenic sites S7, K173 and Q219 of TCS (He et al., 1999a, He et al., 1999b) (Table 1). Number of death cases caused by acute hypersensitivity reaction in guinea pigs was reduced from five to nil after coupling of [K173C]TCS to dextran T40 (Chan et al., 1999) (Table 2).

As a relatively small protein, TCS is rapidly cleared by glomerular filtration and lost in urine. Wild-type TCS had a plasma half life of 9 min (Ko et al., 1991). Coupling of PEG and dextran can prolong plasma circulating half-life of TCS. Coupling of dextran to R29 and K173 on TCS increased the plasma half-life in rat by 27 folds (Chan et al., 1999). Coupling of PEG 5000 to Q219, K173 and S7 of TCS increased the plasma half-life in rat by 6 folds (He et al., 1999a, He et al., 1999b) (Table 1). PEG 20000 is a better coupling agent than PEG 5000 for reducing antigenicity and prolonging plasma half-life due to the increase in effective size of the coupled TCS. The increase in resistance of the coupled proteins towards proteolysis may also contribute to the longer plasma half-life (Abuchowski et al., 1977, Tsutsumi et al., 1995).

Coupling PEG or dextran to TCS often reduces its in vitro cytotoxicity and ribosome-inactivating activities. This is due to the fact that the coupled PEG or dextran blocks the active site of TCS and may prevent entry of modified TCS into the cell (Wang et al., 2004). However, the in vivo abortifacient activity was only slightly decreased, unchanged or even increased in some cases (He et al., 1999a), suggesting that the decrease in activity is compensated by the increase in circulating half-life.

TCS preferentially inhibited the replication of human immunodeficiency virus type 1 (HIV-1) in both acutely infected T-lymphoblastoid cells and chronically infected macrophages in vitro (McGrath et al., 1989). TCS was found to decrease the serum HIV-1 p24 antigen level and to increase the percentage of CD4⁺ cells in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (Kahn et al., 1994).

Initially, it is believed that anti-HIV activity is related to its ribosome inactivating activity. Wang and colleagues showed that two TCS mutants, [KIRE120-123SAGG]TCS and [E160A-E189A]TCS, having 4000- and 1800-fold reduction in ribosome-inactivating activity, respectively, lost almost all the anti-HIV activity. Another TCS variant R122G which showed milder decrease in ribosome-inactivating activity retained some anti-HIV activity (Wang et al., 2002). However, the same group later found that two TCS variants, TCS_C19aa and TCS_KDEL, having a 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, respectively, lost most of the anti-HIV activity without losing ribosome-inactivating activity (Wang et al., 2003).

Some recent findings also suggest TCS does not just inactivate ribosomes. First, not all RIPs have antiviral activity (Wang et al., 2002). Second, Momordica charantia anti-HIV protein inhibits HIV-1 at a concentration that shows little effects on ribosome function (Lee-Huang et al., 1990, Wang et al., 1999). Third, TCS was able to inhibit HIV-1 integrase (Au et al., 2000). Fourth, TCS was found to associate with chemokine receptors that are also co-receptors for HIV-1 fusion and TCS co-stimulated the activation of chemokine receptors by their chemokines (Zhao et al., 1999). Therefore, other action of TCS may contribute to the anti-HIV property of TCS.

TCS is a ribosome-inactivating protein and targets at the ribosome in the cytosol. Before TCS carrying out its functions, it first needs to enter the cell (Zhang et al., 2003). TCS is a type-I RIP, and therefore it lacks the lectin-like B chain of type II RIPs for binding to glycosylated membrane receptors (Stirpe, 2004). Many studies have been carried out to elucidate the mechanism of how TCS enters the cell.

Effect of phospholipids on TCS adsorption at the air–water interface has been studied. It was shown that the presence of phospholipids increased the concentration of TCS at the air–water interface by stabilizing TCS at the interface, suggesting that interaction occurs between TCS and phospholipid membrane (Xia et al., 2001).

Electrostatic interactions were found to be the major interaction between TCS and negatively charged phospholipid containing membranes under acidic condition. Other interaction might be present between TCS and negatively charged membrane as increasing the Na⁺ concentration to 1.5 M did not detach all the membrane-bound TCS (Lu et al., 2001) and TCS after incorporating to the phospholipid monolayer could not be completely squeezed out until the monolayer collapsed (Xia et al., 2004). It was also found that the tertiary conformation of TCS would be changed when TCS was placed in a low pH microenvironment, which exposed more hydrophobic sites (Xia and Sui, 2000). Therefore, TCS may interact with the negatively charged phospholipid-containing monolayer under acidic conditions through hydrophobic interaction (Lu et al., 2001). The low pH microenvironment of membrane may change the charges on some residues, resulting in breakage of salt bridges and charge–charge repulsion, which partially denature TCS to become ‘molten globular state’, and finally lead to the membrane insertion of TCS (Xia and Sui, 2000). The deletion of C-terminal seven amino acids from TCS reduced its membrane insertion ability, suggesting that the C-terminal of TCS is essential for its translocation into the cytosol (Zhang et al., 2003).

It has long been known that TCS is highly toxic to choriocarcinoma JAR cells but is relatively less toxic to hepatoma H35 cells (Tsao et al., 1986, He et al., 1999a, He et al., 1999b). It is believed that the differential toxicity of TCS towards JAR cells and hepatoma H35 cells is due to the different rate of entry of TCS into the cells and different efficacy of the intracellular transport of TCS to reach its targets (Chan et al., 2003). Study on the rate of entry of TCS showed that about 3-fold more TCS had entered the choriocarcinoma JAR cells than the hepatoma H35 cells in a fixed period of time (Chan et al., 2003). This clearly showed that there is a special mechanism for the entry of TCS into choriocarcinoma JAR cells (Chan et al., 2000b). TCS was found to interact with LDL-receptor related protein (LRP) and megalin and the binding can be inhibited by receptor-associated protein (RAP) (Chan et al., 2000b). JAR cells are rich in LRP (Jensen et al., 1990). It was shown that the uptake of TCS to these cells was inhibited by RAP, suggesting that the internalization of TCS into the JAR cells is mediated by the LDL receptor family (Chan et al., 2000b). Megalin is abundant in proximal tubule epithelial cells in kidney and is important for the uptake of filtered proteins (Christensen et al., 1998). High concentration of TCS damaged the proximal tubules in kidney (Nelson et al., 1997) and it was suggested that the cytotoxicity is due to megalin mediated endocytosis of TCS (Chan et al., 2000b). After endocytosis, TCS may induce vesicle leakage and escape from its package vesicle at low pH because TCS at low pH may destabilize the membrane of vesicle (Xia et al., 2003).

TCS was found to stimulate the production of reactive oxygen species (ROS) and apoptosis in JAR cells (Zhang et al., 2000, Zhang et al., 2001). It was shown that the ROS production may be a consequence of Ca²⁺ signaling and the production of ROS occurs before the activation of caspase-3 (Zhang et al., 2001). It is possible that TCS induces the apoptosis of JAR cells via ROS production, internal Ca²⁺ elevation and caspase-3 activation (Zhang et al., 2001) (Fig. 1). It was also demonstrated that the cytotoxicity and the TCS-induced ROS production in JAR cells were reduced in [Y55G] and [FYY140-142GSA]TCS variants in which there were changes in secondary and surface structure of TCS (Zhang et al., 2002). It was also found that mutation on mistletoe lectin A chain (E166N, R169N) caused concomitant decrease in ribosome-inactivating activity and apoptosis, suggesting that apoptosis may be a result of ribosome inactivation (Langer et al., 1999).

Based on the known three-dimensional structure of TCS (Gao et al., 1994, Zhou et al., 1994) and the limited proteolysis study, the putative active site of TCS was proposed to be located at amino acids 110–174 (Ke et al., 1997). It is interesting to find that deletion of the first 100 amino acids from TCS did not affect much of its RNA N-glycosidase activity (Ke et al., 1997). On the other hand, amino acids 120–123 of TCS were found to be important for biological activities and were postulated to be the potential active site as deletion and hydrophobic replacement caused 4000-fold decrease in ribosome-inactivating activity (Nie et al., 1998). E160 of TCS was found to be involved in the catalytic reaction as [E160A]TCS variant had about 15-fold increase in ID₅₀ (median inhibitory concentration of in vitro protein synthesis) (Wong et al., 1994). [E160A]TCS variant was 50-fold more potent than [E160D]TCS variant as E189 provides a backup carboxylate group when E160 was mutated to alanine. On the other hand, mutation of E160 to aspartate moved the carboxylate group from its optimal position (Wong et al., 1994). [E160A-E189A]TCS had a 1800-fold decrease of ribosome-inactivating activity (Wong et al., 1994). E160 and E189 in TCS do not play an important role in maintaining the active site conformation and binding adenine. However, removal of two glutamate residues changes a large patch of negatively charged surface to a positive charge, which may account for the destabilization of the oxocarbenium-like transition-state and the significant decrease in ribosome-inactivating activity in this variant (Shaw et al., 2003). Similar trend of potency was obtained in mouse embryonic development and tumor cell growth (Chan et al., 1995).

Y14 and R22 were suggested to take part in the folding process (Shaw et al., 1997) and W192 was found to define the active site and to maintain protein stability (Ding et al., 2003). It is found that Y70 is crucial to neo-trichosanthin, an isoform of TCS sharing 95% sequence identity, for its substrate recognition, binding and perhaps N-glycosidase activity (Yan et al., 1999). Y70 and Y111 form an aromatic stack of π electron system in which the adenine ring of substrate stacks between their phenol rings (Yan et al., 1999). R163 is crucial for N-glycosidase activity as neo-TCS mutant R163K, R163H and R163Q were found to have lower or even abolished N-glycosidase activity (Li et al., 1999).

N-glycosidase activity in TCS can be divided into two steps, recognition and hydrolysis. It was suggested that there are two subsites in the active pocket of TCS, one for initial substrate recognition as revealed by the AMP site in [E85A]TCS–AMP and [E85Q] TCS–AMP complexes. Another subsite as represented by the NADPH site in [E85R]TCS–adenine and TCS–NADPH complexes is for catalysis (Guo et al., 2003). As discussed, Y70, Y111, E160 and R163 are the most important active site residues. For hydrolysis of substrate, protonation of adenine substrate is needed. Two different hypotheses for protonation of the adenine substrate have been reported. First, N7 of adenine may be protonated by E85 of TCS (Huang et al., 1995). However, this view was disputed as E85 is not conserved, the side chain orientation of E85 in β-momorcharin, an homologous RIP, is different from TCS and the distances from N7 to OE1 and OE2 of E85 in TCS–NADPH and TCS–ADE are quite far apart (Gu and Xia, 2000). Second, it is suggested that atom N3 of adenine is most likely the protonation site based on the hydrogen bond between N3 and R163. (Ren et al., 1994). Recently, a stable water molecule was found to form hydrogen bonds with N3 of AMP and guanidinium of R163 in the [E85A or E85Q]TCS/AMP complex (Fig. 2) and this AMP resides in a new position in the active pocket (Fig. 3) (Guo et al., 2003). It is hypothesized that the substrate first binds to the enzyme in a similar position to AMP in the [E85A or E85Q]TCS/AMP complex where the stable water molecule acts as a proton donor and R163 stabilizes the partial negative charge on the water molecule. Then, the adenine is oriented to the second subsite, as observed in NADP–TCS (Fig. 3).