Abstract
Type 2 diabetes mellitus is a multifactorial and polygenic disorder with increasing prevalence. Recently, a polymorphism in the gene encoding procolipase, a cysteine for arginine substitution at position 92, was associated with type 2 diabetes in two human populations. Because procolipase plays a critical role in dietary fat metabolism, polymorphisms that affect the function of procolipase could influence the development of type 2 diabetes. We hypothesized that the Arg92Cys polymorphism has functional consequences. To test our hypothesis, we expressed recombinant cysteine 92 (Cys92) procolipase in a yeast expression system and compared the function and stability of purified Cys92 with that of the more common arginine 92 (Arg92) procolipase. Cys92 fully restored the activity of bile-salt inhibited lipase with short- and medium-chain triglycerides but only had 50% of Arg92 function with long-chain triglycerides. After storage at 4 degrees C, Cys92 lost the ability to restore pancreatic triglyceride lipase activity with medium- and long-chain triglycerides. The loss of function correlated with the inability of Cys92 to anchor lipase on an emulsion surface and oxidation of the cysteine. No detectable degradation or intramolecular disulfide formation occurred in Cys92 after storage. Our findings demonstrate that the Arg92Cys polymorphism decreases the function of Cys92 colipase. This change may contribute to the development of type 2 diabetes.
Highlights
Type 2 diabetes mellitus is a multifactorial and polygenic disorder with increasing prevalence
To determine the effect of the Arg92Cys substitution on colipase function, we first measured the ability of cysteine 92 (Cys92) and arginine 92 (Arg92) colipase to reactivate sodium taurodeoxycholateinhibited pancreatic triglyceride lipase (PTL) over a range of colipase concentrations in the presence of excess tributyrin and a constant amount of PTL
The nearly identical concentrations of 210.5 6 6.3 for Arg92 and 249.6 6 28.5 for Cys92 to restore half-maximal activity indicate that the two colipases activated PTL with similar efficiency
Summary
Construction of Cys colipase All manipulations of DNA were done by standard methods unless noted otherwise [10]. A substitution of Arg with Cys was accomplished by site-directed mutagenesis using the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The Arg and Cys plasmids were transformed into Pichia pastoris strain GS115 and expressed as described previously [11]. The sample was applied to two 5 ml HiTrap Phenyl HP columns (Amersham Biosciences, Uppsala, Sweden) connected in tandem and equilibrated in 50 mM NaPO4 containing 1 M ammonium sulfate, pH 7.0. The fractions were pooled and concentrated over an Amicon Ultra-15 5,000 MWCO centrifuge filter according to the parameters described by the manufacturer (Millipore). The buffer was exchanged by gel filtration over a Superdex 75 HR 10/30 column equilibrated in 50 mM Tris-Cl, pH 8.0, and 150 mM NaCl. Fractions containing colipase were pooled and concentrated over the centrifugation filter as above. The homogeneity of the purified colipase was confirmed by SDS-PAGE and staining with GelCode Blue according to the manufacturer’s instructions (Pierce, Rockford, IL) [12]
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