Abstract

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis. The mechanisms that drive PRL-3’s oncogenic functions are not well under- stood, partly due to a lack of research tools available to study this protein. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobo- dies, targeting PRL-3 with a KD of 30–300 nM and no activity towards highly homologous family members PRL-1 and PRL-2. We found that longer and charged 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 nanobodies perform equally, if not better, than commercially available antibodies in immunofluorescence and immunoprecipitation. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed that the nanobodies bind partially within the PRL-3 active site and can interfere with PRL-3 phosphatase activity. Co-immunoprecipitation with a known PRL-3 active site binding partner, the CBS domain of metal transporter CNNM3, showed that the nanobodies reduced the amount of PRL-3:CBS inter-action. The potential of blocking this interaction is highly relevant in cancer, as multiple research groups have shown that PRL-3 binding to CNNM proteins is sufficient to promote metastatic growth in mouse models. 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.

Document Type

Article

Publication Date

5-2023

Notes/Citation Information

© 2023 Smith et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Digital Object Identifier (DOI)

https://doi.org/10.1371/journal.pone.0285964

Funding Information

NCI R37CA227656 and NIH DP2CA228043 to J.S.B., GMaP Region 1 North Stimulus Award to C.N.S. (Research reported in this manuscript was supported by the National Cancer Institute of the National Institutes of Health under Award Number P30CA177558. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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