Progranulin Adsorbs to Polypropylene Tubes and Disrupts Functional Assays: Implications for Research, Biomarker Studies, and Therapeutics.

Sushmitha Gururaj,Paul J Sampognaro,Andrea R Argouarch,Aimee W Kao

doi:10.3389/fnins.2020.602235

Sushmitha Gururaj, Paul J Sampognaro + Show 2 more

Open Access

https://doi.org/10.3389/fnins.2020.602235

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Abstract

Progranulin (PGRN) is a tightly regulated, secreted glycoprotein involved in a wide range of biological processes that is of tremendous interest to the scientific community due to its involvement in neoplastic, neurodevelopmental, and neurodegenerative diseases. In particular, progranulin haploinsufficiency leads to frontotemporal dementia. While performing experiments with a HIS-tagged recombinant human (rh) PGRN protein, we observed a measurable depletion of protein from solution due to its adsorption onto polypropylene (PPE) microcentrifuge tubes. In this study, we have quantified the extent of rhPGRN adsorption to PPE tubes while varying experimental conditions, including incubation time and temperature. We found that ∼25–35% of rhPGRN becomes adsorbed to the surface of PPE tubes even after a short incubation period. We then directly showed the deleterious impact of PGRN adsorption in functional assays and have recommended alternative labware to minimize these effects. Although the risk of adsorption of some purified proteins and peptides to polymer plastics has been characterized previously, this is the first report of rhPGRN adsorption. Moreover, since PGRN is currently being studied and utilized in both basic science laboratories to perform in vitro studies and translational laboratories to survey PGRN as a quantitative dementia biomarker and potential replacement therapy, the reported observations here are broadly impactful and will likely significantly affect the design and interpretation of future experiments centered on progranulin biology.

Highlights

Progranulin (PGRN) is an evolutionarily conserved, cysteine-rich, secreted glycoprotein with a diverse set of functions
It is well established that most proteins and peptides readily bind to experimental surfaces owing to their amphipathic nature (Nakanishi et al, 2001). This very property is exploited beneficially in several bio-techniques, such as the enzyme-linked immunosorbent assays (ELISA) immuno-assay where the coating of 96-well plastic plates relies on non-specific adsorption, and ion exchange chromatography whose central principle is the binding between charged proteins and resins
We observed in our experiments that the incubation of recombinant HIS-tagged human Progranulin (rhPGRN) protein in PPE microcentrifuge tubes depleted the protein amounts in solution over time

Summary

INTRODUCTION

Progranulin (PGRN) is an evolutionarily conserved, cysteine-rich, secreted glycoprotein with a diverse set of functions. PGRN gene (Pgrn) delivery has demonstrated ameliorative effects in animal models of both Alzheimer’s (Van Kampen and Kay, 2017) and Parkinson’s disease (Van Kampen et al, 2014) Based on these observations, circulating PGRN has garnered recent attention as a potential quantitative biomarker for neurodegeneration, for FTD. In several of our biochemical assays involving human plasma and CSF, we routinely use an ∼80kDa recombinant HIS-tagged human Progranulin (rhPGRN) protein as a control for the full-length human PGRN protein While performing these experiments, we observed inconsistencies in our rhPGRN control measurements in enzyme-linked immunosorbent assays (ELISA) and mass spectrometry assays. Given the significant evidence for PGRN’s tight in vivo regulatory control and in light of recent diagnostic and therapeutic efforts aimed at modulating PGRN levels in patients (those with neurodegenerative disease), we present these data in an effort to optimize future work in these areas

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DATA AVAILABILITY STATEMENT