A Review of Literature on Toxin Cholera and its Receptor Cell Binding with a Focus on B Subunit Hua-an Tai TA:

Gary Shen Section: Wednesday 2:30pm to 5:30pm March 31, 2010

A Review of Literature on Cholera Toxin and its Receptor Cell Binding with a Focus on B Subunit Introduction: To begin we must briefly observe the mechanical structure and composition of cholera toxin. Essentially cholera toxin is a fairly small oligomeric protein complex composed of six protein subunits excreted by Vibrio cholera, a bacterium. These subunits are labeled subunit A with a single copy and subunit B with five copies. The two parts are interconnected by a disulfide bond. It should be noted that subunit A has two significant pieces, CTA1 and CTA2. CTA1 can be described as a spheroprotein that proceeds to ADP-ribosylation on guanine nucleotide-binding proteins while the CTA2 is an extended alpha helix which is centered in the middle of the ring formed by the five B subunits. B subunits specific receptor is ganglioside monosialoganglioside GM1, which can bind up to five locations on the ring formed by the B subunit. Mechanism of cholera toxin can be described as the function of CTA1, in which the enzyme precedes to ADP-ribosylation a g-protien which normally would be the inhibitor of adenylate cyclase thus increasing cAMP levels within an organism. Significantly increased cAMP levels can also lead to increase release of chloride ions and water molecules from intestinal absorptive cells in the gut lumen leading to watery diarrhea. Cholera toxins are very similar to heat-labile enterotoxin which can be found in Escherichia coli. Cholera toxin is the key agent that causes Cholera disease. Cholera disease is a devastating disease that still affects many third world countries. Cholera is usually passed through contaminated water supplies that causes massive watery diarrhea leading to rapid dehydration and death. Throughout history cholera disease has become a worldwide epidemic causing much death and

suffering to humanity, it is important that we devote time and research into understanding the mechanism in order to prevent more suffering and loss of life. Knowledge of how the cholera toxin binds and its mechanism of actions will be very helpful to researchers as they search for possible cures against it. Throughout this review of literature many new scientific research will be presented in order to better understand the Cholera Toxin and its relationship with it receptor binding.

Basic Receptor Binding Mechanism of the Cholera Toxin Now that the basic structure and the basic mechanism of cholera toxins are actualized we can begin to undeveloped into the details. Firstly we can look at the basic receptor bind mechanism in which the important receptor binding sites attaches on their respective receptors. In an experiment conducted by Stanford University School of Medicine, cholera toxin B subunit was extensively examined under a panel of monoclonal antibodies 40B10 to check the conformational integrity and to help clarify the direct versus the indirect effects of chemical derivatizations. Researchers Ludwig, Holmes, and Schoolnik carried an analysis of results and determined that the monoclonal antibody 40B10 is a competitive inhibitor against the ganglioside monosialoganglioside GM1. The ganglioside monosialoganglioside GM1 was then used to identify important residue locations. Farther studies show that tryptophan played an important role in the attachment of ganglioside monosialoganglioside GM1 to the cholera toxin B subunit. In the experiment, they found that the concentration of ganglioside monosialoganglioside GM1 stayed roughly the same when they disrupted the tryptophan reside so that the cholera toxin B subunit could not effectively bind to the monoclonal antibody (Ludwig,

Holmes, & Schoolnik, 1985). The researchers also observed the disulfide bond between the two subunits and it was proposed that the amino acid lysine served two critical functions; they help conserve an electrostatic attraction essential for recognition of receptors and one or more lysine amino acids nearest the receptor binding domain may cooperate with the ganglioside monosialoganglioside GM1 (Ludwig, Holmes, & Schoolnik, 1985). This result also reinforces the fact that the disulfide bond is needed in order to preserve structural integrity and function of the cholera toxin B subunit and also concludes that the tyrosyl residue is of no significant use. The experiment laments on how previous experiments failed to demonstrate reversibility by positively identifying chemical specificity of the modification, failure to preserve higher protein structure and minimize conformational distortion, and the general insensitivity of assays used to detect binding affinity (Ludwig, Holmes, & Schoolnik, 1985). The article puts value in the intramolecular disulfide bond whereas other previous experiments before it do not. Using these information future researchers may look into using the disulfide bond against cholera toxins, and try to inhibit cholera toxins by denaturing the disulfide bond so that they cannot bind to their receptor bind domain. Researchers from the National Institution of Health used Fourier-transform infrared spectroscopy was used to study the effects of receptor binding of ganglioside GM1 to its cholera toxin receptor in this study, as well as the overall structure of the cholera toxin itself. Cholera toxin B subunit is densely folded including many beta sheets in its secondary structures. Temperature dependence of the infrared spectrum indicates that the B subunit undergoes thermal unfolding in the temperature between sixty six and seventy eight degree Celsius (Surewicz, Leddy, & Mantsch, 1990). When ganglioside

monosialoganglioside GM1 receptor binds, this action only causes a small change in secondary structure; then when compared to the B subunit, the A subunit is much less stable and ordered which contributes to its effective membrane translocation (Surewicz, Leddy, & Mantsch, 1990). The purpose of this experiment was to verify the conformational change in the B protomer caused by the entry binding of A subunit via Fourier-transform infrared spectroscopy. Even though the mechanism is very detailed it is poorly understood, this experiment seeks to explain various reasons to address proper understanding. This paper concludes that no significant conformational change in the ganglioside monosialoganglioside GM1 receptor during receptor binding, which leads the paper to conclude that B subunit is only used as a polar carrier for the A subunit so that the cholera toxin may be transported closer to the membrane surface (Surewicz, Leddy, & Mantsch, 1990). It also concluded in accordance to other studies the general structure of cholera toxins A subunit and B subunits making note that it is the A subunit that is toxic.

Specifics of the Cholera Toxin Receptor Binding for Toxin Assembly After knowing a bit about the receptor mechanics, it is important to spend time on specifics of the cholera toxin as a tertiary structure protein and how it interacts with itself thus it can be properly understood in the environment. An experiment conducted by the University of Colorado Health Science Center theorizes that cholera toxins have a specific hydrophobic region located at the AB5 interface which is very important for toxin assembly. In order to exam this farther, aspartic acid substituted the hydrophobic residues of CTA1 F223, and hydrophobic residues of CTA2 174, L77 and T78. Then the

resulting mutants were observed for any changes in their ability to create holotoxins. The holotoxin CTA-F223D increased susceptibility to proteolysis by trypsin and decreased stability and toxicity (Tinker, Erbe, Hol, & Holmes, 2003). Function pentamers were not able to be formed by CTB-L77D while CTB174Dand CTB-T78D could form pentamers but failed to connect with their cholera toxin A subunits to form the holotoxin (Tinker, Erbe, Hol, & Holmes, 2003). In respect to holotoxin assembly, this research concludes the importance of hydrophobic interactions between the two subunits, which leads us into how they bind to their target cells. Another research from the National Institution of Health focused on an experiment designed to discover the way cholera toxin binds to their targets with direct observation and biochemical tests used to determine the percentage bound. Effects of molecules are heavily depended on their stereochemistry and this experiment was used to determine the facing of cholera toxins in their interactions with their target cells. The results showed that cholera toxin binds with CTA1 facing opposite of the membrane, which is supported by evidence of active assembly of cholera toxins on cell surfaces of cultured cells (Orlandi & Fishman, 1993). Also observed was that at thirty seven degrees Celsius they both become inaccessible to antibodies (Orlandi & Fishman, 1993). It is proposed by the article that the holotoxin must be bound with A subunit facing opposite from the membrane so that it can enter the cell to release CTA1, access to ganglioside monosialoganglioside GM1 can be achieved, and adenylycyclase can thus be activated (Orlandi & Fishman, 1993). Results are similar to other conclusions showing similar conformations. This begs the question about how cholera toxin A undergoes translocation

through the pore of the B subunit unless it unfolds itself or dissociates from the B pentamer.

Mutational Analysis on Cholera Toxin and Their Effects Knowing both the mechanics and the specifics of cholera toxin, researchers from the University of Colorado Health Science Center experimented on cholera toxin B by modifying it at several positions in order to understand the functions of those general regions. The purpose of this research was to actually put theory into practice so they can confirm many speculations. They used radial passive immune hemolysis assay RPIHA to obtain variants of cholera toxin B so they can observe for loss of binding ability to certain cells. They also tested other characterization; such as the formation of immunoreactive pentamers, in vitro binding ability of ganglioside GM1 and levels of reactivity against monoclonal antibodies. Results showed that substitutions at eight locations intensively reduced the immunoreactive numbers of cholera toxin B, these locations all had lossed their halo, in contrast those who had altered halo phenotypes did not have problems binding to ganglioside monosialoganglioside GM1 (Jobling & Holmes, 2001). Modification of asparate substituted for tyrosine at position 12 caused significant failure to bind to ganglioside GM1, which is contrasted in substitution of glycine which greatly increased their binding abilities (Jobling & Holmes, 2001). The paper hypothesizes that this is due to steric hinderance and charge effects of the carbonyl oxygen and nitrogens. Results also indicate that if substitution of valine for alanine at position 10 and 46, this will dramatically effect negatively on the immunoreactivty of cholera toxin B (Jobling & Holmes, 2001). The paper explains many small modifications to the cholera holotoxin

that can be used to nullify its effect but does not go in to detail how this could be put into wide spread commercial practice. This research shows the many possibilities a research team can contribute to the process of gathering knowledge as many experiments were to be viewed as stepping stones for farther in-depth studies. Conclusion In this review of literature it is impossible to cover all aspects even if it is such a narrow topic, there is just too much material. Firstly topics about cholera toxins structure and receptor binding abilities were presented, and then its mechanism was explained. After that specifics of cholera toxins were pointed out and questions rose about this topic. Lastly innovative research field options were presented to show how this can be expanded and fitted into the modern w orld of biochemistry. For structural binding of cholera toxins the most important find was that tryptophan is essential to the mechanism as was the disulfide bond connecting both subunits- if any of these components were missing or inhibited the binding rate drastically reduces GM1 (Ludwig, Holmes, & Schoolnik, 1985). FTIS researchers confirmed the b subunit as a densely folded complex composed of beta sheets whose main function is to act as a polar carrier for the A subunit so that they may be transported closer to the target cell membrane surface (Surewicz, Leddy, & Mantsch, 1990).. In the specifics of cholera toxin receptor binding, holotoxins were shown that they needed the hydrophobic interactions between the two subunits in order to be effect (Tinker, Erbe, Hol, & Holmes, 2003), as suggested by their conformational change when attaching to the surface of the target cell membrane when they face the opposite direction (Orlandi & Fishman, 1993). Unanswered questions include how the A subunit is entangled into the B subunit, specifics of particular

mechanisms, and the exact purpose of the disulfide bond. This was followed by mutational analysis for farther research of possibilities of how to deal with cholera toxins, showing many important locations that may be removed to inhibit the effectiveness of the cholera toxins. In conclusion, much is still needed to be learned from this field. Other researchers could perform experiments to confirm findings of the previous experiments. Another experiment could be used to hinder the development of the B subunit in order to find out how the A subunit interacts with its environment, and through observations perhaps a mechanism could be derived from the situation. Also, what could be done is mix proportional amounts of A and B subunits together and see if they naturally combine, then find the concentration of cholera toxins in that solution. These are just some of the possible experiments that could be conducted in the future for improvement upon this field.

Literature Citation
References Ludwig, D., Holmes, R., & Schoolnik, G. (1985). Chemical and immunochemical studies on the receptor binding domain of cholera toxin B subunit. The Journal Of Biological Chemistry, 260, 12528-12534. Surewicz, W., Leddy, J., & Mantsch, H. (1990). Structure, stability, and receptor interaction of cholera toxin as studied by Fourier-transform infrared spectroscopy. Biochemistry, 29, 8106-8111. Tinker, J., Erbe, J., Hol, W., & Holmes, R. (2003). Cholera holotoxin assembly requires a hydrophobic domain at the A-B5 interface: Mutational analysis and development of an in vitro assembly system. Infection and Immunity, 71, 4093-4100. Jobling, M., & Holmes, R. (2001). Mutational analysis of ganglioside GM1-biding ability, pentamer formation, and epitopes of cholera toxin B (CTB) subunits and CTB/heat-labile enterotoxin B subunit chimeras. Infection and Immunity, 70, 12601271. Orlandi, P., & Fishman, P. (1993). Orientation of cholera toxin bound to target cells. The Journal of Biological Chemistry, 268, 17038-17044.