Protein Toxins

Protein Toxins

With the continuous deepening of research on the structure and mechanism of natural protein toxins, many toxins have entered the clinical application stage. The powerful killing effect of toxins has been used in the treatment of tumors, and therapies using antibodies to carry toxins to target and kill tumor cells have been realized. BOC Sciences is a leading provider of protein toxin development services, offering a broad range of services to support the development and production of protein toxins for antibody-drug conjugates (ADCs). Our comprehensive proteotoxin manufacturing services include the development of targeted toxins, conjugation of toxins to targeting ligands, and optimization of toxin delivery methods.

Structural and schematic models of Shiga toxin and ricinFig. 1. Structural and schematic models of Shiga toxin and ricin (FEBS Letters. 2010, 584: 2626-2634).

Mechanism of Action of Protein Toxins

Target Site of Protein Toxin

Although the sources of various plant poison proteins are different, their structures and mechanisms of action are very similar. The A-chain of phytotoxins or plant single-stranded ribosome inactivating protein (RIP) usually has the activity of N-glycosidase, which can catalyze the specific site of the ribosomal component 28S RNA to deactivate the ribosomal 60S subunit. Because A-chain or RIP has enzymatic properties, theoretically one A-chain molecule can catalyze and inactivate 1,500 ribosomes per minute, inhibiting the synthesis of all proteins in a short period of time, eventually leading to cell death. The main sites of action of bacterial toxins are elongation factor-2 (EF-2) and adenylate cyclase. In the process of cell synthesis of proteins, the elongation of the peptide chain requires a variety of elongation factors, but the most critical one is EF-2. For example, diphtheria toxin and Pseudomonas aeruginosa exotoxin have ADP-ribosyltransferase activity, which can cause EF-2 to undergo ADP-ribosylation, directly leading to its loss of efficacy. It is estimated that one toxin molecule can catalyze the ADP-ribosylation of 2000 EF-2 molecules per minute. Therefore, in theory, as long as one toxin molecule enters a cell, it can cause cell death. Cholera toxin, Escherichia coli heat-labile toxin, pertussis toxin, etc. mainly act on the cAMP signal transduction pathway. Their A subunit also has ADP ribosyltransferase activity, but its substrate is the G protein on the cytoplasmic membrane. For mycotoxins such as α-Sarcin, Res and Mit, they all have specific endonuclease activity and can catalyze the hydrolysis of a single phosphate bond on the 3' side of 28S RNA. The enzyme cleavage site is at the phosphate bond between guanine nucleotide G4325 and adenine nucleotide A4326, resulting in the inactivation of the ribosome 60S subunit, thereby inhibiting cellular protein synthesis.

Internalization of Protein Toxin

Protein toxin molecules are large and difficult to cross the membrane directly into the cytoplasm. The internalization process from outside the cell membrane into the cytoplasm is considered a key step in protein toxin poisoning and is also a hot topic in current toxin research. Because the target molecules of phytotoxins, bacterial toxins and mycotoxins are located in the cytoplasm, toxin molecules can only attack ribosomes and EF-2 when they enter the cytoplasm. It is generally believed that toxin molecules enter cells through receptor-mediated phagocytosis. Typically, the B chain of natural toxins is usually glycosylated. Therefore, the B chain of the toxin has the function of a lectin and can bind to glycoproteins or glycolipid receptors containing specific sugar groups. The binding of the B chain to the receptor can trigger the endocytosis of the entire toxin molecule. After entering the cell, the toxin molecule is mainly located in the phagosome or tubular structure formed by a unit membrane. Take the translocation mechanism of diphtheria toxin as an example. It enters endosomes through receptor-mediated endocytosis. In the acidic environment of the endosome (pH 4.5), the hydrophobic region of the B segment is exposed and inserted into the lipid bilayer of the endosome to form a channel, allowing the A segment to smoothly translocate from the channel into the cytoplasm.

Classes of bacterial protein toxinsFig. 2. Classes of bacterial protein toxins (Cold Spring Harb Perspect Med. 2013, 3: a013573).

Our Protein Toxin Development Capabilities

There are many ways to classify toxins, and they can be divided into animal, plant, bacterial and fungal toxins according to their sources. According to the chemical structure, they can be divided into small molecule organic compounds, peptides and protein toxins. Based on this, BOC Sciences can provide natural protein toxins extracted from microorganisms or plants for targeted cancer therapy, drug delivery and ADC research.

Plant Protein Toxins

Generally, such toxins include plant toxins and plant single-chain ribosome inactivating proteins (RIP). Typical plant toxins include ricin, abrin, volkensin, viscumin and modeccin. These toxins are glycoproteins with molecular weights between 60 and 65 kD. Their molecules are composed of two chains, A and B, connected by disulfide bonds. Compared with plant toxins containing two subunits, A and B, the structure of RIP is only equivalent to the A chain, so it is also called a hemitoxin. Typical RIP family members include luffin, trichosanthin, phytolacca antiviral protein (PAP), saporin, dianthin and trichosanthin (gelonin) etc.

BOC Sciences has expertise in the isolation, purification and characterization of plant protein toxins and the synthesis of toxin analogs and derivatives for research and pharmaceutical purposes. Our capabilities also include the development of toxin detection methods and the design and synthesis of potential toxin inhibitors and neutralizers. Our toxin development services can be tailored to meet the specific needs of our clients in the pharmaceutical, biotechnology and academic research sectors.

Bacterial & Fungal Protein Toxins

Typical bacterial toxins include diphtheria toxin (DT), Pseudomonas exotoxin (PE), cholera toxin (CT), and E.coli heat-labile toxin, shigatoxin, pertussis toxin, etc. There are certain differences in the structures of bacterial toxins. Both DT and PE are single peptide chains (molecular weight 60~70 kD), and their molecules contain cell receptor binding regions (equivalent to the B chain of phytotoxins) and enzyme active regions (equivalent to the A chain of phytotoxins). The PE molecule also contains a translocation region that helps it translocate into the cytoplasm. The molecular structures of cholera toxin and E.coli heat-labile toxin are the same, consisting of 1 A chain and 5 B chains. The molecular weight of chain A is large, about 30 kD, and that of chain B is about 11.5 kD. They are connected into pentamers through weak non-covalent interactions. Shiga toxin consists of 1 A chain (molecular weight 30 kD) and 6 or 7 similar B chains (molecular weight 5 kD). Pertussis toxin is one of the most complex molecules among known toxins, consisting of one A chain (molecular weight 28 kD) and five B chains. The five B chains have different sizes, two of which have molecular weights of 11.7 kD, and the remaining three are 9.3 kD, 22 kD, and 23 kD respectively. There are only a few fungal protein toxins. The protein toxins α-Sarcin, restrictocin (Res) and mitogillin (Mit) isolated from different molds only consist of a polybrain chain, with a molecular weight of 16~17 kD, belonging to single-stranded ribosome inactivating proteins.

In addition to phytotoxin extraction, BOC Sciences also has extensive capabilities in the development of bacterial and mycotoxins, including toxin isolation, purification and characterization, and the production of recombinant toxins using advanced biotechnological methods. Our team of experienced scientists is well versed in the study of bacterial and mycoprotein toxins and has the ability to develop custom solutions for the production and purification of these toxins. They can also provide comprehensive support in the development of assays to assess toxin activity and screen potential inhibitors.

Protein Toxins We Support
RicinLuffinCholera Toxin (CT)Restrictocin (Res)
AbrinTrichosanthinE.coli Heat-Labile ToxinMitogillin (Mit)
VolkensinSaporinShigatoxinTetrodotoxin
ViscuminDiphtheria Toxin (DT)Pertussis ToxinConotoxin
ModeccinPseudomonas Exotoxin (PE)α-SarcinEtc.

Highlights of Our ADC Services

  • Comprehensive custom ADC payload solutions: Ability to provide custom development services for ADC payloads, including biological payloads, chemical payloads and protein toxins.
  • Comprehensive analytical characterization and quality verification services: Advanced analytical techniques such as HPLC, LC-MS, NMR and other spectroscopic methods to accurately characterize and verify product purity, identity and potency.
  • Advanced antibody conjugation technology support: Providing guidance and support for the design and optimization of antibody conjugation processes to ensure the successful development of ADCs with desired properties.
  • One-stop ADC cGMP manufacturing and purification services: Covering the entire process from antibody production, cytotoxin development, linker synthesis to conjugation, purification and formulation of payload.

References

  1. Sandvig, K. et al. Protein toxins from plants and bacteria: Probes for intracellular transport and tools in medicine. FEBS Letters. 2010, 584: 2626-2634.
  2. Barbieri, J.T. et al. General Aspects and Recent Advances on Bacterial Protein Toxins. Cold Spring Harb Perspect Med. 2013, 3: a013573.
* Only for research. Not suitable for any diagnostic or therapeutic use.
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