Production of the CD44 human receptor in Escherichia coli and study of its interactions with potential inhibitors.

Abstract

The cell transmembrane receptor CD44 is a member of type I transmembrane receptor family that is found on the outer surface of different types of both immune and cancer cells. It is used by the cells to detect as well as to respond on different changes in the near environment. CD44 receptors are involved in a variety of processes such as hematopoiesis, lymphocyte homing, or limb development. The receptors communicate with the extracellular matrix. Overexpression of various CD44 isoforms has been found in many types of human tumors. In colorectal cancer, specific CD44 isoforms are expressed according to the progression of the disease. An important substrate for the membrane receptor CD44 is Hyaluronan or Hyaluronic Acid (HA). It has been found to be important as well with different cancer progressions, especially in the case of breast cancer where it has been well documented. After a structural analysis of HA, it is possible to find suitable substitutes that might bind into the binding domain of CD44 and regulate its function. Some of these possible substitute molecules are chitooligosaccharides (CHOS) which are oligomers prepared from chitosan. Due to the relative abundance of this compound and the strong structural similarity with HA we suggest the use of this compound as a possible substrate analog with potentially interesting outcomes. Therefore different in silico simulations and wet laboratory experiments, between CD44, HA and six- and eight-sugar CHOS, were made to show the different thermodynamically conditions and probe, at least hypothetically, if these derivates of chitin are efficient regulators of the function of this membrane receptor. The research showed the next interesting results. The lack of expression of the CD44 protein obtain from the synthetic gene with optimize codons due to the possible existence of hairpins interfering with the translation of the RNA by the bacterial ribosome. The refolding of the membrane receptor was not satisfactory due to the contamination of the protein with nucleic acids (small fragments of genomic DNA and RNA) that prevented the bond formation between the amino acids, preventing the formation of an stable 3D protein structure. After performing several in silico simulations using the Yasara software, it was possible to see a possible interaction between the CD44 and a short chain chitooligosaccharides of six sugars (Chi6). Also it was possible to deduce a new binding domain and its possible interaction with small chain chitooligosaccharides of 2 or 3 sugars.

Popular Abstract

Haven’t we all heard about cancer at least once in our life? What is more incredible is that after so many years and millions of dollars poured into researching this disease, not a concrete cure has been found. It is true, the hurdles to find a cure are many and, as it is now, what works to stop the spreading one type of cancer may not work with another one. Therefore it might be a good idea to find out what is common among, at least, many different types of tumors and try to find a universal solution. Here the cell receptor CD44 enters the scene. CD44 is a multi-structural and multi-functional cell surface molecule involved in cell proliferation, differentiation, migration and signaling for cell survival. All these biological functions are essential for the physiological activities of healthy cells, but they are also associated with pathologic conditions such as cancer. The functions of CD44 are regulated by its interactions with a molecule called hyaluronic acid (HA). If we can control the interaction between the cell receptor and the HA; we might control its function and even prevent it from promoting tumors in all kinds of cells. But to make this possible, it is needed to somehow interfere with how CD44 and HA interact.
It is here where chitooligosaccharides (CHOS) come into play. We hypothesized that these saccharides would disturb the natural interaction between CD44 and HA, thereby affecting the biological functions of CD44. In addition, one of the advantages of using these particular oligosaccharides is their low cost, since they can be obtained from chitin, which is an abundant waste material from different industries, such as fisheries. Due to the relative abundance of this compound and as well as a strong structural similarity with hyaluronic acid we suggest the use of this compound as a possible analog substrate with potential interesting outcomes in the interaction with CD44. Therefore, different computational simulations and normal laboratory experiments, with CD44, HA, six (Chi6) and eight sugar (Chi8) CHOS, were made to explore different thermodynamically conditions and prove, at least hypothetically, that derivates of chitin might be good regulators of the function of this receptor.
The production of functional human proteins, such as CD44, in genetically modified bacteria is a very complex task. The main trouble was the production of a proper folded CD44. Protein folding was analyzed by an instrumental technique called circular dichroism, but did not show clear results. In a subsequent analysis of the CD44 it was found that contamination with DNA or RNA could be the main cause for not allowing the proper 3D structure to form. Another outcome of the laboratory work was the discovery of stable secondary structures in the mRNA that might prevent the production of CD44. The cellular machinery of the bacterium used to produce proteins might encounter problems when interacting with specific secondary structures of mRNA especially if this last one is really stable, thermodynamically speaking.
Using computational models to study the interaction between CD44 with HA and the possible antagonists Chi6 and Chi8, it was possible to see a trend in the interactions between CD44 and the chitooligosaccharides. After running simulations measuring the stress cause in the protein by the interaction with the sugars and the binding energy among this complex, it was possible to see that small oligosaccharides, of two to four sugars might be good antagonists. They manage to interact in the same place where HA binds and they seemed to be stable, hence it might be good to peruse this line of research and run more simulations with small chitooligosaccharides and monitor the possible interactions with CD44.
To find a cure against a complex disease like cancer will take more time and a lot of effort from different branches of science. It is possible to try to modulate hypothetical solutions in computer programs, as well as testing these initial assumptions with traditional experimental wet lab methods. In this particular case of finding a way to control CD44, one can encounter a lot of technical problems, from proteins that can’t be produced to misfolded ones that might limit the success of the experiment. However, when a door closes another one opens, and with the help of more precise computational models and simulation software, one might find new ways to test a hypothesis and obtain new results.

• author: Diaz Martinez, Juan Antonio
• supervisor: Javier Linares-Pastén
• organization: Department of Chemistry
• course: KEMT30 20171
• year: 2017
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: CD44, YASARA, RNA, hairpins, Chitooligosaccharides, Protein Production: Refolding, protein science, proteinvetenskap
• language: English
• id: 8932051
• date added to LUP: 2018-05-15 13:13:37
• date last changed: 2018-05-15 13:13:37