Pancreatic Lipase Deficiency: Characterization of Novel PNLIP Mutations

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Abstract Title: Pancreatic Lipase Deficiency: Characterization of Novel PNLIP Mutations Background: Pancreatic triglyceride lipase (PNLIP) is the primary lipase responsible for dietary fat digestion in humans. Studies have reported that mutations in PNLIP lead to pancreatic lipase deficiency, steatorrhea, and fat-soluble vitamin deficiencies. Previously, we illustrated that a rare homozygous missense mutation (p.T221M) in PNLIP causes steatorrhea through its secretion defect. This led to a loss of digestive lipase function, insoluble aggregation via protein misfolding, and a gain of proteotoxic function increasing the risk for pancreatic diseases like pancreatitis. Recently, two more novel PNLIP mutations (p.R188C and p.W419X) have been identified and associated with congenital pancreatic lipase deficiency in symptomatic children. The aim of this study was to characterize the lipase function and proteotoxicity of these two PNLIP mutants in vitro. Methods: We generated a mammalian protein expression construct pcDNA3/PNLIP WT. Next, we made plasmid DNA constructs pcDNA3/PNLIP R188C or W419X by site-directed mutagenesis. We then transfected HEK293T cells to express PNLIP WT, R188C, or W419X. Three days post-transfection, we harvested conditioned media and cells. We assessed secretion of lipase in medium through lipase activity assay and immunoblot against PNLIP. We fractionated the cell lysates into detergent soluble and insoluble fractions to assess PNLIP misfolding and endoplasm reticulum (ER) stress by immunoblot against PNLIP and BiP (an ER stress marker), respectively. Results: The conditioned media from cells expressing PNLIP WT had steady lipase activity whereas the R188C or W419X media lacked detectable lipase activity. Immunoblot revealed a strong PNLIP band in the WT media, but no visible bands in R188C or W419X media. These results suggest the defective secretion of both mutant PNLIP proteins. Moreover, a much higher proportion of PNLIP R188C and W419X was present in the detergent insoluble fraction compared with WT indicating increased misfolding of the PNLIP mutant proteins. Further immunoblot analysis of cell lysates indicated markedly up-regulated BiP in the mutant samples relative to WT controls. Conclusion: Our data illustrate that both R188C and W419X mutations in PNLIP result in loss of lipase function due to abolished secretion. Additionally, both mutations may increase the risk to develop chronic pancreatitis through its gain of proteotoxic function.