J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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A more recent version of this article appeared on July 1, 2007

Papers In Press, published online ahead of print March 30, 2007
J. Lipid Res., doi:10.1194/jlr.M600486-JLR200
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Submitted on November 14, 2006
Revised on March 30, 2007
Accepted on March 30, 2007

Further biochemical characterization of human pancreatic lipase-related protein 2 expressed in yeast cells

Cécilia Eydoux, Josiane De Caro, Francine Ferrato, Paul Boullanger, Dominique Lafont, René Laugier, Frédéric Carrière, and Alain De Caro

SDV, CNRS, Marseille 13402

Corresponding Author: caro{at}ibsm.cnrs-mrs.fr

Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major protein with a molecular mass of 50 kDa was purified from the culture medium using SP-Sepharose and Mono Q chromatography. The protein was found to be highly sensitive to the proteolytic cleavage of a peptide bond in the lid domain. The proteolytic cleavage process occurring in the lid affected both the lipase and phospholipase activities of rHPLRP2. The substrate specificity of the non-proteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All the enzyme activities were maximum at alkaline pH values and decreased in the 5-7 pH range corresponding to the physiological conditions occurring in the duodenum. rHPLRP2 was found to act preferentially on substrates forming small aggregates in solution (monoglycerides, egg phosphatidylcholine, galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on mono- and di-galactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2 (GPLRP2), which shows a large deletion in the lid domain. The presence of a full length lid domain in rHPLRP2 makes it possible for enzyme activity to occur at higher surface pressures. The finding that the inhibition of non-proteolyzed rHPLRP2 by tetrahydrolipstatin (THL) and diethyl-p-nitrophenyl phosphate (E600) does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media.


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