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

• DOI: 10.1194/jlr.m600486-jlr200
• Publication Date: 2007-03-31T01:17:59Z
• Authors: Cécilia Eydoux; Josiane De Caro; Francine Ferrato; Paul Boullanger; Dominique Lafont; René Laugier; Frédéric Carrière; Alain De Caro
• Keywords: Surface Properties; QD415-436; Biochemistry; Paraoxon; Pichia; Glycerides; Lactones; 03 medical and health sciences; Pichia pastoris; Pressure; Humans; Colipases; Orlistat; Taurodeoxycholic Acid; 0303 health sciences; galactolipase; Lipase; Hydrogen-Ion Concentration; inhibition; Recombinant Proteins; [SDV] Life Sciences [q-bio]; Phospholipases; galactolipid; Carboxylic Ester Hydrolases; constitutive expression; lid domain

Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major with a molecular mass of 50 kDa purified from culture medium using SP-Sepharose and Mono Q chromatography. The found to be highly sensitive proteolytic cleavage peptide bond lid domain. process occurring affected both lipase phospholipase activities rHPLRP2. substrate specificity nonproteolyzed rHPLRP2 investigated pH-stat monomolecular film techniques various substrates (glycerides, phospholipids, galactolipids). All enzyme were maximum at alkaline pH values decreased 5–7 range corresponding physiological conditions duodenum. act preferentially on forming small aggregates solution (monoglycerides, egg phosphatidylcholine, galactolipids) rather than emulsified such as triolein diolein. activity monogalactosyldiglyceride digalactosyldiglyceride films determined compared that guinea pig 2, which shows large deletion presence full-length domain makes it possible for occur higher surface pressures. finding inhibition by tetrahydrolipstatin diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests adopts an open conformation aqueous media. In 1992, Giller et al. (1.Giller T. Buchwald P. Blum-Kaelin D. Hunziker W. Two novel related proteins, hPLRP1 hPLRP2. Differences colipase dependence activity.J. Biol. 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Lastly, cloned total bulbourethral gland (also named Cowper's gland) (9.Sias cleansing genital tract, where role. native (HPLRPs) secreted juice characterized our laboratory. assays are available specifically measure without interference gastrointestinal we developed immunological approach antipeptide antibodies recognizing HPLRPs (HPLRP1 HPLRP2) discriminate more abundant (HPL). procedure made identify monitor their chromatographic behavior (5.De 18.De Barboni (PLRP1) several species.Biochim. 1387: 331-341Crossref (48) recombinant forms pure pastoris cultures (19.Sias Lafont Boullanger Human galactolipase.Biochemistry. 43: 10138-10148Crossref (90) respectively. Some HPLRP2 lipid 19.Sias From results, likely main galactolipase. immunoreactive form detected colon, based Western blot analysis extracted biopsies along tract. colon still remains elucidated, however (20.Eydoux Aloulou Laugier 2: tissular localization tract quantification specific ELISA.Biochim. 2006; 1760: 1497-1504Crossref (21) Using ELISAs, recently quantified HPL levels patients disorders Control subjects significantly (7.7% 1.7% respectively) chronic calcifying pancreatitis (3.7% 0.63% respectively). normal (1.7% proteins), isolate amounts. Therefore, characterization performed To study closely, constitutive improved study, modified purifying produce showing greater purity stability. Further undertaken substrates, including monogalactosyldiglycerides digalactosyldiglycerides. experiments showed structure influence activity. sensitivity proteolysis results (E600) (THL) suggest solution. Pure trioleoylglycerol (triolein) (T-7140) l-α-phosphatidylcholine (P-5394) purchased Sigma-Aldrich Chemical (St. Louis, MO). monoolein diolein commercial low-grade dl-α-monoolein Fluka. Intralipid emulsion (20%, w/w) soybean oil phosphatidylcholine (1.2%, used here, generally parenteral nutrition purposes, Kabi France. cDNA Hoffmann-La Roche, Ltd. (Basel, Switzerland), generous gift Dr. Thomas As described previously induce wild-type (X-33 strain; Invitrogen), pGAPZB vector plasmid integrated electroporation SMD 1168 strain (Invitrogen). transformation resulted under control glyceraldehyde-3-phosphate dehydrogenase promoter medium. After transforming electroporation, plated onto Yeast Peptone Dextrose (YPD) 1% agar containing Zeocin (100 μg/ml) grown 3 days 30°C. clones selected then left 5 ml YPD night agitation. Before production started, preculture step 24 h 250 Erlenmeyer flask inoculate larger media optical density (600 nm) start directly exponential growth phase. 30°C orbital agitation rate 100 rpm liter 200 devoid Zeocin. Cell stopped after 40 h, dithiothreitol phenylmethylsulfonyl fluoride added final concentration mM limit oxidation sulfhydryl groups rHPLRP2, monitored measuring tributyrin pelleted centrifugation 5,000 min. (1.5 liters) collected lipase. Cells removed centrifuging twice 20 min 4°C Beckman J-25 centrifuge. adjusting 5.0, supernatant incubated batch Performance (Pharmacia) gentle stirring. gel first equilibrated 10 MES buffer, 5.0. More 95% bound when per used. washed extensively 5.5, packed column (Pharmacia; 1.6 × 7.5 cm) connected ÄKTA explorer™ device (Amersham Pharmacia Biotech). washing buffer above, successive linear NaCl gradients (0–0.3 0.3–0.5 M NaCl) applied 120 15 min, active fractions pooled, dialyzed against Tris-HCl 8.0, benzamidine, concentrated Amicon PM 30 ultrafiltration membrane. resulting HR 5/5 dialysis buffer. eluted 0.25 gradient 0 0.5 flow adjusted ml/min, collected. ∼3–4 mg/ml, aliquots, stored −20°C. performing quantitative amino acid 6300 analyzer. measured potentiometrically 37°C (718 STAT Titrino; Metrohm). reaction carried out vigorous stirring vessel μl monoolein, diolein, or triolein, trioctanoin, olive (10%, gum arabic (GA; 10%, 0.1 CaCl2, giving volume ml. Under standard fatty acids released titrated directly. down free back-titrated end incubation period (5 min) shifting value up 9.0 (21.Gargouri Piéroni Rivière Saunière J-F. P.A. Sarda Kinetic assay gastric short- long-chain triacylglycerol emulsions.Gastroenterology. 1986; 91: 919-925Abstract (211) Porcine tissue (22.Chapus Desnuelle Fogglizzo Stabilization C-terminal part carboxypeptidase trypsin inhibitors.Eur. 1981; 115: 99-105Crossref (66) Phospholipase porcine A2 (P-0861; Sigma) (23.Abousalham Egg yolk lipoproteins lipases.Biochim. 1485: 56-62Crossref (42) substrate. Specific international units (1 unit = μmol minute) milligram enzyme. [1,2-di-O-dodecanoyl-3-O-β-d-galactopyranosyl-sn-glycerol (MGDG sn-3) 2,3-di-O-dodecanoyl-1-O-β-d-galactopyranosyl-sn-glycerol sn-1)] [3-O-(6-O-α-d-galactopyranosyl-β-d-galactopyranosyl)-1,2-di-O-dodecanoyl-sn-glycerol (DGDG)] substrates. MGDG DGDG synthesized chemical procedures, Refs. Scholar 24.Lafont Syntheses α[email protected]@ce:[email protected]@[email protected]@/ce:[email protected]@-Gal-(1→6)-β[email protected]@-Gal diglyceride, substrate.Carbohydr. 341: 695-704Crossref kinetics hydrolysis KSV-2200 barostat (KSV, Helsinki, Finland) "zero order" Teflon trough (25.Verger De Haas G.H. Enzyme reactions model. new technique monolayers.Chem. Phys. Lipids. 1973; 10: 127-136Crossref (332) products lipolysis (monododecanoyl-galactopyranosyl-glycerol, dodecanoic acid, monolauroyl-galactopyranosyl-glycerol, lauric acid) soluble water, equipped mobile barrier keep pressure constant desorption products. Surface Wilhelmy plate (perimeter, 3.94 attached electromicrobalance microprocessor controlling movements barrier. room temperature. subphase composed Tris-HCl, NaCl, 21 EDTA, 8.0. (0.1 μg; nM concentration) injected through film. area compartment cm2, reservoir 27.9 cm long 14.8 wide. estimated covered known molecule. areas versus compression isotherm (Fig. 1). moles hydrolyzed (cm2) minute referred overall molarity initially (mol·cm−2·min−1·M−1). Inhibition E600 sodium acetate (pH 6.0) CaCl2 (26.Moreau Moulin Gargouri Noel J.P. Inactivation diethyl p-nitrophenyl phosphate.Biochemistry. 1991; 30: 1037-1041Crossref (76) (5.2 10−5 M) (2.0 25°C [stock 4.16 (D-9286, Sigma)] lipase-inhibitor molar ratio 1:85 absence 4 taurodeoxycholate 1:1. THL 8.0) NaCl. (stock solutions ethanolic solution) 1:100 NaTDC Residual times trioctanoin 2-fold excess optimize measurements. Electrophoresis 7.5% polyacrylamide gels SDS Laemmli (27.Laemmli U.K. Cleavage assembly head bacteriophage T4.Nature. 1970; 680-685Crossref (207537) blotting Gershoni Palade (28.Gershoni J.M. G.E. Electrophoretic transfer dodecyl sulfate-polyacrylamide positively charged filter.Anal. 1982; 124: 396-405Crossref (306) Membranes anti-HPLRP2 rabbit serum diluted 1:1,000 anti-rabbit immunoglobulins conjugated phosphatase (diluted 1:2,000; A-0418; Quantitative Scion Image software program (Scion Corp.). sugars detection blots labeling digoxigenin (DIG Glycan Detection Kit, 142 372; Roche Diagnostics). determine N-terminal fragments generated partial proteolysis, material separated means SDS-PAGE electroblotted polyvinylidene difluoride membranes (Bio-Rad). bands Ponceau S staining cut subjected Applied Biosystem 476 sequencer. Matrix-assisted laser-desorption ionization time flight (MALDI-TOF) spectrometry samples