Our long-term goal is to elucidate the molecular mechanisms of dietary fat digestion by pancreatic lipases. In this application, we will focus on the steps in lipolysis that influence substrate specificity. Lipases differ from esterases that act on water soluble substrates in that lipases must absorb to the interface of the substrate emulsion before substrate binds in the active site. In addition, the archetype of pancreatic lipases, pancreatic triglyceride lipase (PTL), requires colipase to absorb to interfaces. Importantly, mutations in the absorption surface of colipase can change the substrate preference of the PTL-colipase complex underscoring the importance of absorption in determining substrate specificity. Thus, three distinct steps--colipase binding, interfacial absorption and substrate binding in the active site--all influence substrate specificity. To investigate the molecular elements that mediate the interaction of PTL with substrate, we will take advantage of a close PTL homologue, pancreatic lipase related protein 2 (PLRP2). These lipases have highly conserved primary structures, share a common three-dimensional structure and share common catalytic machinery;yet, they have quite different enzymatic properties. PTL prefers triglycerides whereas PLRP2 also cleaves galactolipids and phospholipids. Furthermore, PTL clearly requires colipase whereas the PLRP2 requirement for colipase remains controversial. Our central hypothesis is that specific structural domains govern each step of the interaction between pancreatic lipases and substrate. To identify domains involved in determining the substrate specificity of PTL and PLRP2, we will utilize available information on structure and kinetic properties of these lipases. We base this approach on multiple observations from our published and preliminary data. 1) Structural studies and our published studies of the PTL-colipase complex have guided the identification of distinct domains that influence colipase binding to PTL. 2) Analysis of structural data and our preliminary studies have identified critical residues in PTL-colipase and PLRP2 that influence the absorption of these lipases to lipid emulsions. 3) Comparison and preliminary analysis of the structures of PTL and PLRP2 identified domains that can mediate the differences in substrate specificity. Completion of our Specific Aims will provide molecular details about each step in the interaction of pancreatic lipases with substrate. Ultimately, we will be able to rationally engineer the enzymatic properties of lipases to meet specific therapeutic or industrial needs.

Public Health Relevance

Pancreatic lipases are critical for the efficient digestion of dietary fats, which provide a major source of energy, essential fatty acids, a vehicle for fat-soluble vitamins, important components of cell membranes and precursors for prostaglandins, thromboxanes, and leukotrienes. Because of this role pancreatic lipases figure prominently in nutritional therapy and as targets for therapy of obesity and hepatosteatosis. A thorough understanding of the structures that mediate the functions of pancreatic lipases will enhance our understanding of dietary fat digestion, result in safer lipase inhibitors and bring us closer to the rational design of lipases with specific properties for nutritional therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK080820-04
Application #
8281683
Study Section
Clinical and Integrative Gastrointestinal Pathobiology Study Section (CIGP)
Program Officer
Serrano, Jose
Project Start
2009-07-01
Project End
2013-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$322,956
Indirect Cost
$109,784
Name
University of Pittsburgh
Department
Pediatrics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Johnson, Karin; Ross, Leah; Miller, Rita et al. (2013) Pancreatic lipase-related protein 2 digests fats in human milk and formula in concert with gastric lipase and carboxyl ester lipase. Pediatr Res 74:127-32
Xiao, Xunjun; Ferguson, Michael R; Magee, Kelsey E et al. (2013) The Arg92Cys colipase polymorphism impairs function and secretion by increasing protein misfolding. J Lipid Res 54:514-21
Xiao, Xunjun; Ross, Leah E; Sevilla, Wednesday A et al. (2013) Porcine pancreatic lipase related protein 2 has high triglyceride lipase activity in the absence of colipase. Biochim Biophys Acta 1831:1435-41
Ross, Leah E; Xiao, Xunjun; Lowe, Mark E (2013) Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles. Biochim Biophys Acta 1831:1052-9
Xiao, Xunjun; Mukherjee, Amitava; Ross, Leah E et al. (2011) Pancreatic lipase-related protein-2 (PLRP2) can contribute to dietary fat digestion in human newborns. J Biol Chem 286:26353-63
Xiao, Xunjun; Ross, Leah E; Miller, Rita A et al. (2011) Kinetic properties of mouse pancreatic lipase-related protein-2 suggest the mouse may not model human fat digestion. J Lipid Res 52:982-90