Saporin

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Function

Saporin is a ribosome-inactivating protein (RIP); alone, saporin does not selectively inactive ribosomes but rather conjugate with other molecules like peptides ^[3]. Saponaria officinalis is the plant from which saporin is extracted ^[4]. Type I and type II RIPS exist. Of these types, saporin is a type I. Ribosome inactivating proteins catalyze a cleavages N-glycosidic bond that is formed between the ribosome and adenine ^[5]. This adenine has the role of binding EF-1 and EF-2 to a ribosome ^[5]. EF stands for elongation factor. Since adenine no longer has a bond to the ribosome, the elongation step in translation cannot occur because the elongation factors cannot bind to just the ribosome. The specific elongation factor that is inhibited is elongation factor 2, which causes irreversible damage and disallows protein synthesis ^[4].

Structural highlights

Type 1 RIPS are monomeric, meaning they have one part ^[6]. Saporin-S6 at maturity is 256 amino acids long ^[4]. While saporin consists of different residues and molecules, there is only one Chain A in it, and thus is monomeric. Chain A is a polypeptide weighing 30 KDa ^[7]. This chain consists of beta-sheets and alpha-helixes. The β-sheets make up the N-terminal domain, while the 𝛼-helix portion is the C-terminal domain ^[5]. In the figure of Chain A, the 𝛼-helices are spiral-shaped strands, while the β-sheets are more of a flat strand.

There is an active site within this chain that consists of five residues. These residues are , Tyr¹²⁰, Glu¹⁷⁶, Arg¹⁷⁹, and Trp²⁰⁸ ^[4]. Other RIPs also have these same residues in their active sites. The saporin active has Glu¹⁷⁶, Arg¹⁷⁹, and Trp²⁰⁸ in the exact same position as the other ribosome-inactivating proteins. There is a difference in Tyr⁷², which has different side-chain conformations in RIPs and thus is not the same in saporin and other RIPs. This Tyr⁷² is the residue that interacts with the adenine in the cleavage of adenine and the ribosome ^[5].

Saporin can also be complexed with other inhibitors. One of these is cyclic tetranucleotide inhibitor in . This can be used because the cyclic tetranucleotide can take the place of the recognition loop for saporin of 28S rRNA ^[8]. It is also interesting to note that Ricin can also be complex with other inhibitors like saporin. Saporin is a homologue of Ricin A-Chain which means they are similar in structure ^[8]. This ability also allows for saporin-S6 to be conjugated with specific targeting proteins, and thus the saporin-S6 is able to be delivered to the cell. This happens due to the antibodies and is referred to as an immunotoxin ^[4]. The antibodies are recognized by the cell, and the cell binds them. Since the saporin-S6 is in complex with the antibody, it is also taken to the cell. Some other carries can also be used, such as growth factors, antigens, and growth hormones ^[4].

Medical Uses

Medical uses for saporin have been studied as well. Most notably, the use of saporin as an anti-tumor agent or anti-cancer agent. Research has been done to determine if and how saporin could be used to treat cancer. Cancer treatments include many things, some of which are tumor debulking surgeries and chemotherapy. Although toxins like saporin can cause cell death, if saporin can be used to target cancer, it would kill off cancer cells. The delivery of drugs to a specific tissue can be used to avoid healthy tissue but harm the diseased tissue or cancer tissue ^[9]. Saporin is toxic to cancer cells due to its level of enzymatic activity and saponin’s resistance to conjugation. This toxicity causes apoptosis, cell death to the targeted cancer cells ^[7]. Assays of saporin-S6’s ability to induce apoptosis and necrosis are also being studied ^[4]. Saporin-S6 has been used in mice to study its effects on tumors and cancers. Mice were injected with saporin-S6 at a dose that was non-lethal to them. They were then left and later examined for tumors throughout their bodies. The best results were in the liver, but then no tumors were reported 14 days after the initial saporin-S6 injection ^[4].

One way that has been researched to treat cancer with saporin is by using an amino-terminal fragment (ATF) and fusing it with saporin (SAP) ^[9]. The specific ATF used was the human urokinase-type plasminogen activator (uPA) which is the ligand to the urokinase plasminogen activator receptor (uPAR). This means that uPAR will bind uPA, and if saporin is fused with uPA, it will also be bound to the uPAR. This is important because uPAR is upregulated, increasing its levels in cancer cells ^[9]. With increased levels, it means that more uPA will be in the cancer cell and again more saporin since the saporin is bounded to the uPA. This particular study found that the ATF-SAP was able to get into the cancer cells and kill the cells in cancer cells. Although, with lower and undetectable levels of uPAR, there was no effect on cancer with ATF-SAP ^[9]. Saporin can be bound to other molecules to make it effective for specific things in the body. Although, there need to be appropriate levels of what is being targeted, or the saporin won't be able to get to where it needs to be to achieve the goal.

The study explained above has saporin being used as a chimeric toxin, which is different from that of an immunotoxin ^[4]. This just means that the saporin is conjugated with another carrier rather than one, such as antibodies ^[4]. One way that this is helpful is so that the saporin does not activate an immune response. In general, plant toxin-based chimerae have shown less immune response when compared to bacterial origin toxins ^[6]. Since there is less immune response, the body will not attack the saporin conjugates, at least not as much, and the saporin can get to the target area, most likely cancer.

It is apparent through different studies that the delivery method of saporin is essential. Overall, saporin does not penetrate cancer cells that well. As seen previously, saporin can be bound to another molecule to achieve this. Some mechanisms have been thought of for how saporin enters the cell. Some of which are passive mechanisms, although due to cells in the body showing resistance to saporin-S6, the idea of a receptor for saporin ^[4].

Another study used a lipid-based nanoparticle and loaded saporin into it ^[7]. These nanoparticles can serve as a sort of delivery system to the target cells. The problem trying to be solved in this particular study was getting around multidrug resistance (MDR), which happens when cancer is resistant to many unrelated drugs. One reason that cancer cells become resistant is due to the ATP-binding cassette (ABC) transporters that are overexpressed in the cancer cells ^[7]. The use of saporin in the nanoparticle was to inhibit the ABC transporters. Not only did the study find that saporin successfully accomplished this, but it inhibited the growth of tumors in mice ^[7].

There are other uses than just cancer treatment for saporin being studied as well. One of which is the ability for saporin to have some antimicrobial activity. This activity is the same that occurs from pokeweed antiviral protein (PAP) ^[6]. Substance P-saporin (SP-SAP) is being studied to target a specific neuron that expresses the substance P pain-related receptor ^[6]. This could help with the pain that terminal cancer patients may experience ^[6]. While this still connects with cancer, it is for pain management rather than treatment of cancer itself. Other RIPs like saporin are also being studied for these same effects, and since other RIPs are similar in structure to saporin, it is easier to study them when comparing them to saporin.

The use of saporin-S6 and other RIPs are still being studied, and their structures are as well. Including what saporin-S6 can be complexed with and what the end result would be. As of right now, there is a promise that saporin-S6 could be used to treat cancers in humans at some point in the future. Hurdles needed to be overcome, like the delivery system, but there could come a day. Also, while saporin-S6 is toxic to cells and could cause damage when targeting cancer cells, it might be more limited than the damage produced by chemotherapy or other cancer treatments. Through research, there could also be more uses for saporin than just cancer treatment as well.