HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: M600486-JLR200v1 48/7/1539    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Eydoux, C. Articles by De Caro, A. Search for Related Content PubMed PubMed Citation Articles by Eydoux, C. Articles by De Caro, A. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 48, 1539-1549, July 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

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^1,*

^* Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, Unité Propre de Recherche 9025, Centre National de la Recherche Scientifique-Institut de Biologie Structurale et Microbiologie, 13402 Marseille Cedex 20, France
Laboratoire de Chimie Organique II-Glycochimie, Unité Mixte de Recherche 5181, Université de Lyon, 69622 Villeurbanne Cedex, France

Published, JLR Papers in Press, March 30, 2007.

¹ To whom correspondence should be addressed. e-mail: 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 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.

Supplementary key words constitutive expression • Pichia pastoris • inhibition • galactolipid • lid domain • galactolipase • digestion

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?