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bioRxiv · 10.1101/2020.10.02.311787

Development and validation of nanobodies specific to the oncogenic phosphatase Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3)

Smith, C. N.·Wei, M.·Williamson, Z. A.·Chernyavskyay, Y.·Chow, K. M.·Hersh, L. B.·Korotkov, K. V.·Blackburn, J. S.
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Abstract

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis in various solid tumors and leukemias. The mechanisms that drive PRL-3s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. In particular, small molecules do not exhibit binding specificity for PRL-3 over highly homologous family members PRL-1 and PRL-2, and antibodies directed against PRL-3 are limited by assay type. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards PRL-1 and PRL-2. Hydrogen deuterium exchange mass spectrometry (HDX-MS) and co-immunoprecipitation with a known PRL-3 substrate showed the nanobodies bind PRL-3 outside of the active site, meaning they can be used to study PRL-3 interaction with binding partners. The nanobodies were also specific to PRL-3 over other PRLs in immunoprecipitation and immunofluorescence experiments in human cancer cells that overexpressed the PRL family. We found that N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression.

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Smith, C. N., Wei, M., Williamson, Z. A., Chernyavskyay, Y., Chow, K. M., Hersh, L. B., Korotkov, K. V., Blackburn, J. S.. 2020-10-03. Development and validation of nanobodies specific to the oncogenic phosphatase Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3). https://doi.org/10.1101/2020.10.02.311787

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