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Development of a human epidermal growth factor derivative with EGFR-blocking and depleted biological activities: A
Masomeh Mehrabi1, Kamran Mansouri1, Bijan Soleymani1
1Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Abstract:
Epidermal growth factor (EGF) is a local growth factor that stimulates cell growth, proliferation, and differentiation by binding to its receptor EGFR. EGF and EGFR are involved in many aspects of the development of carcinomas. Because EGFR has been found to be over-expressed in many tumors of epithelial origin, it is a potential target for antitumor therapy. In this study we designed a mutated form of hEGF (mEGF) with a deletion of four amino acids residues (Gln43, Tyr44, Arg45 and Asp46) in order to show importance of Leu spatial location for EGFR binding/activation. Expression vector pET32a+ and E. Coli, strain Rosetta-gami B (DE3) were used to enhance solubility of the recombinant protein with yielding approximately 10mg/l of cell culture. The purified cleaved hEGF as well as non-cleaved fusion protein were biologically active, which was confirmed by their equal ability to stimulate proliferation of MCF7 cells. The mEGF showed specificity and high affinity for EGFR binding, however binding affinity of mEGF for EGFR was reduced about 11.5 fold compared with that of hEGF. The mEGF effect on the MCF7 cell proliferation had a relatively different outcome; mEGF simulated differential cell growth in a dose dependent manner. On the other hand, in MDA-MB468 cells, hEGF and mEGF induced growth inhibition, which was much more severe for hEGF than that of mEGF. Also, hEGF strongly induced the phosphorylation of EGF receptor in MDA-MB468 cells while mEGF induced poor EGFR phosphorylation. The same observations were also made for migration of cancer cells, especially induction of MDA-MB468 migration by mEGF was significantly lower than that of hEGF, suggesting a connection between tyrosine phosphorylation of EGFR and cell migration. Docking analysis revealed that the binding affinity and the buried surface area of mEGF to EGFR complex are lower than those of hEGF/EGFR. Although theoretical studies confirmed reduction in mEGF-EGFR binding affinity, the data of the present study indicate that mEGF is a potential EGFR blocker but may highlight it as excellent delivery agent of protein/non-protein toxins as well as for α-, β-, γ-emitting radio-immunotherapy.
Insights
A mutated epidermal growth factor (EGF) shows reduced binding affinity to its receptor EGFR but retains biological activity. This mutated EGF may serve as an EGFR blocker or a delivery agent for cancer therapies.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Epidermal growth factor (EGF) and its receptor EGFR are crucial in cell growth and implicated in carcinoma development.
- EGFR overexpression in epithelial tumors makes it a promising target for antitumor therapies.
Purpose of the Study:
- To investigate the importance of specific amino acid spatial locations in human EGF (hEGF) for EGFR binding and activation.
- To design and characterize a mutated form of hEGF (mEGF) with altered binding properties for potential therapeutic applications.
Main Methods:
- Engineered a mutated hEGF (mEGF) by deleting four amino acid residues.
- Expressed and purified recombinant mEGF and hEGF using an E. coli expression system.
- Assessed biological activity through cell proliferation assays (MCF7 cells) and EGFR phosphorylation studies (MDA-MB468 cells).
- Analyzed binding affinity and complex formation using docking analysis.
Main Results:
- Purified hEGF and mEGF were biologically active, stimulating MCF7 cell proliferation.
- mEGF exhibited specificity and high affinity for EGFR binding, but with an ~11.5-fold reduced binding affinity compared to hEGF.
- mEGF induced differential cell growth in MCF7 cells and growth inhibition in MDA-MB468 cells, with less severe inhibition than hEGF.
- hEGF strongly induced EGFR phosphorylation and MDA-MB468 cell migration, while mEGF showed significantly weaker induction.
- Docking analysis confirmed lower binding affinity and buried surface area for the mEGF-EGFR complex compared to hEGF-EGFR.
Conclusions:
- Despite reduced binding affinity, mEGF demonstrates potential as an EGFR blocker.
- mEGF's altered interaction with EGFR suggests its utility as a delivery agent for toxins or radio-immunotherapy agents.
- The study highlights the critical role of specific amino acid residues in EGF-EGFR interactions and their therapeutic implications.
