A humán NADPH-oxidáz 5 (NOX5) enzim vizsgálata új monoklonális antitesttel
Szeles Zsolt
Molecular Medicine
Dr. Enyedi Péter
Semmelweis Egyetem Elméleti Orvostudományi Központ Hári Pál Előadóterem
2024-07-03 11:00:00
Dr. Geiszt Miklós, Dr. Petheő Gábor
Dr. Wunderlich Lívius
Dr. Szöllősi András
Dr. Fekete Andrea
Dr. Gál Anikó
Dr. Szeri Flóra
In the family of NADPH oxidases, our knowledge about NOX5 is the least comprehensive. We know the distribution of enzyme in the human body, primarily expressed in the spleen and testes. However, there was previously no widely available, high-quality antibody for the detection of NOX5 at the protein level. Therefore, we developed a new monoclonal antibody that recognizes human NOX5, in collaboration with Immunogenes Ltd., and conducted its detailed characterization. The antibody is now commercially available. We hope that our developed antibody will give new momentum into research exploring the localization and function of NOX5.
We established a HEK293T cell line stably expressing NOX5. Predominantly, the overexpressed protein was observed intracellularly in the endoplasmic reticulum and the nuclear membrane. Through superoxide measurements, we confirmed that the cells express a functional enzyme that can be activated by calcium. To study endogenous NOX5, we used the UACC-257 melanoma cell line. Using Western blot and immunostaining procedures, along with NOX5 siRNA-controlled experiments, we demonstrated that the developed antibody recognizes the endogenous NOX5 protein. In these cells, the enzyme is mainly distributed in the compartments mentioned above. By activating the TRPV4 channel (with GSK1016790A) and inhibiting SERCA (with thapsigargin), we induced a calcium signal in the cells, followed by superoxide production. The origin of the measured ROS as NOX5-derived was confirmed with the use of NOX inhibitor DPI and NOX5 siRNA.
With the novel monoclonal antibody, we detected the NOX5 protein in normal human spleen, testes, and ovaries with the help of Western blot and immunohistochemistry techniques. By analyzing RNA databases, we identified the cellular sources of the oxidase in these tissues, supporting our immunostaining results. In the testes, early and late spermatids; in the ovaries, oocytes and granulosa cells; and in the spleen, endothelial cells were the primary sites of enzyme expression. Contrary to literature but in agreement with mRNA database findings, we found no significant expression of NOX5 in primary vascular cells, except in spleen endothelium.
We also conducted „off-target” analysis for other NADPH oxidases of NOX5-deficient rabbits created in our laboratory. We determined that the NOX1, NOX2, and NOX4 genes were not affected by the modifications in the rabbits generated using the CRISPR/Cas9 technology.
