Further biochemical characterization of human pancreatic lipase-related protein 2 expressed in yeast cells.
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 nonproteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All of the enzyme activities were maximum at alkaline pH values and decreased in the pH 5-7 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, and galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on monogalactosyldiglyceride and digalactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2, 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 nonproteolyzed rHPLRP2 by tetrahydrolipstatin and diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media.
Eydoux
Cécilia
C
Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, Centre National de la Recherche Scientifique-Institut de Biologie Structurale et Microbiologie, 13402 Marseille Cedex 20, France.
De Caro
Josiane
J
Ferrato
Francine
F
Boullanger
Paul
P
Lafont
Dominique
D
Laugier
René
R
Carrière
Frédéric
F
De Caro
Alain
A
D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D003089:Colipases / Q000494:pharmacology; D005989:Glycerides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007783:Lactones / Q000494:pharmacology; D008049:Lipase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000502:physiology*; D000077403:Orlistat; D010261:Paraoxon / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D011312:Pressure; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013499:Surface Properties; D013657:Taurodeoxycholic Acid / Q000494:pharmacology
D003089:Colipases; D005989:Glycerides; D007783:Lactones; D011994:Recombinant Proteins; D013657:Taurodeoxycholic Acid; D000077403:Orlistat; D010740:Phospholipases; D002265:Carboxylic Ester Hydrolases; C019262:galactolipase; D008049:Lipase; C075988:pancreatic lipase related protein 2; D010261:Paraoxon