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

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.

Blood-based biomarkers of inflammation in amyotrophic lateral sclerosis

“…With many of the subpopulations of blood cells producing different expression levels of genes or soluble factors, these analyses in blood provide an averaged data point, losing the cell-type specific resolution of the obtained data. This may partly explain why there is no established diagnostic biomarker for ALS (yet) by using single factor analyses, in addition to the other possible explanations including technology used [ 63 ], sample sizes, co-morbidities, (anti-inflammatory) medications, and disease state at biosample donation [ 64 ].…”

Regulation of extracellular progranulin in medial prefrontal cortex

“…NaCl from Fisher Scientific, all other components from MilliporeSigma) supplemented with 4% bovine serum albumin (BSA, MilliporeSigma). Dilutions were made in low protein-binding tubes (ThermoFisher) to prevent progranulin from binding to the tubes ( Gururaj et al, 2020 ), and post-experimental analysis revealed no detectable loss of progranulin due to non-specific binding to tubes. Progranulin concentrations for in vitro microdialysis were 1, 2, and 5 ng/mL, which approximates the range of concentrations reported in human cerebrospinal fluid ( Batzu et al, 2020 ; Berghoff et al, 2016 ; De Riz et al, 2010 ; Meeter et al, 2016 ; Morenas-Rodriguez et al, 2016 ; Nicholson et al, 2014 ; Van Damme et al, 2008 ; Wilke et al, 2016 ).…”

“…Microdialysis sampling was conducting using an Atmos (Amuza) protein microdialysis system, consisting of a syringe pump (Amuza #ESP-180LD) to push dialysate through the probe, a peristaltic pump (Amuza #ERP-10) to pull dialysate from the probe, and a refrigerated fraction collector (Amuza #FC-90) for sample collection. Probes were perfused with aCSF supplemented with 4% BSA (MilliporeSigma) to maintain osmotic pressure ( Trickler and Miller, 2003 ) and prevent progranulin from binding to the tubing ( Gururaj et al, 2020 ). Each concentration of progranulin was measured with two one-hour samples at a flow rate of 1 μL/min, with a two-hour equilibration phase allowed when switching to each new concentration.…”