Abstract
In newborn mice, PLRP2 is essential for fat digestion. In human infants, the role of PLRP2 in fat digestion is unclear, as it has poor activity against long-chain triglycerides in vitro. Also, many infants carry a genetic polymorphism resulting in a truncated protein, PLRP2 W340X, which may impact function significantly. We re-examined the properties of recombinant human PLRP2 and studied the impact of W340X mutation on its function. In the presence of bile salt micelles and colipase, human PLRP2 hydrolyzed long-chain tri-, di-, and monoglycerides. It hydrolyzed triolein at a level much lower than that of pancreatic triglyceride lipase, but close to that of carboxyl ester lipase, after a long lag phase, which could be eliminated by the addition of oleic acids. Human PLRP2 W340X was poorly secreted and largely retained inside the cell. The retention of the mutant protein triggered endoplasmic reticulum stress and unfolded protein responses. Our results show that earlier studies underestimated human PLRP2 activity against triolein by employing suboptimal assay conditions. In vivo, dietary fat emulsions contain fatty acids as a result of the action of gastric lipase. Consequently, PLRP2 can contribute to fat digestion during early infancy. Furthermore, infants with homozygous W340X alleles will not secrete functional PLRP2 and may have inefficient dietary fat digestion, particularly when breastfeeding is unavailable. Additionally, the aberrant folding of W340X mutant may cause chronic cellular stress and increase susceptibility of pancreatic exocrine cells to other metabolic stressors.
Highlights
Pancreatic lipases are critical for efficient dietary fat digestion
PLRP2 should have an important role in dietary fat digestion in humans, but in vitro studies suggest that human PLRP2 may not function as a triglyceride lipase
The addition of oleic acids did not significantly improve the absorption of human PLRP2 to triolein. These results suggest that human PLRP2 requires colipase to absorb to trioctanoin and triolein, the ability to absorb to triolein emulsions is poor, even in the presence of oleic acids
Summary
DNA Manipulations—All DNA manipulations were done by standard methods unless stated otherwise. Lipase Activity Assay—Lipase activity of human PLRP2 was determined at 25 °C by using the standard pH-stat method with the emulsion of tributyrin (114 mM), trioctanoin (27 mM), triolein (6.9 mM), diolein (5.1 mM), or monoolein (10.7 mM) in a total volume of 15 ml as described previously [17, 18]. Human CEL lipase activity was assayed by the pH-stat method using 10 g of purified CEL, with the assay buffer containing 150 mM NaCl, 1 mM Tris-HCl, 2 mM CaCl2, pH 8.0, 3.75% defatted BSA, and varying concentrations of sodium cholate or sodium taurocholate [20]. Preparation of Cell Lysates—To study the total intracellular PLRP2 and W340X mutant protein levels, harvested cell pellets were resuspended in ice-cold PBS with EDTA-free Complete protease inhibitor mixture (Roche Diagnostics), with 200 l for HEK 293T and 50 l for COS7 cells, mixed with equal volume of 2ϫ Laemmli buffer, followed by 10-min boiling. PCR products were run on 2% agarose gels, and the bands were visualized by ethidium bromide staining
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