Preparation and characterization of the antibody recognizing AMACR inside its catalytic center

Popov,Boris  V.; Sutula,Gleb  I.; Petrov,Nikolay  S.; Yang,Ximing J.

doi:10.3892/ijo.2017.4220

Preparation and characterization of the antibody recognizing AMACR inside its catalytic center

Authors:
- Boris V. Popov
- Gleb I. Sutula
- Nikolay S. Petrov
- Ximing J. Yang
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Affiliations: Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia, Robert H. Lurie Comprehensive Cancer Center, Department of Pathology, Northwestern University, Chicago, IL 60611, USA
Published online on: December 11, 2017 https://doi.org/10.3892/ijo.2017.4220
Pages: 547-559

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Abstract

Alpha-methylacyl-CoA racemase (AMACR) catalyzes the β-oxidation of fatty acids and is overexpressed in carcinomas in various organs, while its inactivation results in the inhibition of cancer growth. In the present study, we prepared and characterized 20 different mouse monoclonal antibodies against human AMACR. In the course of biopanning of a phage peptide commercial library against in-house prepared 6H9 and 2A5, and commercial 13H4 antibodies, 10 phage mimotopes recognized by each type of the antibody were selected. Using the program Pepitope and the crystal structure of AMACR from Mycobacterium tuberculosis, we reveal for the first time, at least to the best of our knowledge, that the epitopes recognizing the antibody against AMACR are composed of conformation sequences localized inside the AMACR catalytic center. When delivered into live HeLa cells using cationic lipid-based PULSin reagent, the specific antibodies against AMACR were co-localized with peroxisomes. The in-house made 6H9 antibody exhibited a low level of this co-localization compared to the commercially available 63340 antibody, and did not inhibit the growth rate of HeLa and T98G cells. The results obtained suggest that antibody against AMACR may possess anti-AMACR catalytic activity and needs to be further investigated as a potential drug for use in anticancer therapy. On the whole, in this study, we generated several clones of AMACR antibodies and demonstrated that these antibodies can be colonized into live cells. Currently, we are testing the growth inhibitory properties of these antibodies against AMACR.

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