Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates.

Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate.

Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes.

Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation.

Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles.

Occurrence of pancreatic lipase-related protein-2 in various species and its relationship with herbivore diet.

The occurrence of classical pancreatic lipase (PL) and pancreatic lipase-related proteins 1 and 2 (PLRP1s and 2) in the pancreas of ten mammalian species (humans, pig, rat, guinea pig, coypu, rabbit, horse, ox, goat and sheep) and two bird species (ostrich and turkey) was investigated. The lipases were purified from delipidated pancreas and identified based on the results of Western blotting analysis with anti-human PLRP2 serum, the catalytic properties and N-terminal microsequencing data. PLRP2s were detected in the pancreas of monogastric herbivorous animals (guinea pig, coypu, rabbit and horse) but not in that of ruminant herbivorous animals (ox, goat and sheep).

A comparative study on two fungal lipases from Thermomyces lanuginosus and Yarrowia lipolytica shows the combined effects of detergents and pH on lipase adsorption and activity.

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids.

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol).

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.

Human pancreatic lipase-related protein 2: tissular localization along the digestive tract and quantification in pancreatic juice using a specific ELISA.

Human pancreatic lipase-related protein 2 (HPLRP2) was previously found to be secreted by the exocrine pancreas. HPLRP2 shows a high level of activity on galactolipids, and might be involved in the digestion of these common vegetable lipids. Specific antibodies were raised in rabbits using a synthetic HPLRP2 peptide selected for its weak amino acid homology with the corresponding peptides of classical human pancreatic lipase (HPL) and human pancreatic lipase-related protein 1 (HPLRP1).

Immunocytochemical localization of scorpion digestive lipase.

The scorpion hepatopancreas consists of digestive diverticula and interstitial tissue. A digestive diverticulum is composed of two differentiated cell types: the secretory zymogene-like cells and the digestive cells which are the most abundant. The scorpion digestive lipase (SDL) has been previously purified from scorpion hepatopancreas, but its cellular localization has not yet been established.

Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris.

High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter.

Closed and open conformations of the lid domain induce different patterns of human pancreatic lipase antigenicity and immunogenicity.

Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation.

Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice.

Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2).

Critical evaluation of a specific ELISA and two enzymatic assays of pancreatic lipases in human sera.

Background And Aims: Human pancreatic lipases (HPL) include the classical HPL, and two related proteins known as pancreatic lipase-related proteins 1 and 2 (HPLRP1 and 2). The aim of this study was to develop an ELISA for specifically quantifying the classical-HPL level in sera of patients with and without pancreatic disorders.

Methods: The specific activity of various human (including classical-HPL) and microbial lipases was measured using Lipa Vitros and potentiometric (pH-stat) assays.

Human pancreatic lipase-related protein 2 is a galactolipase.

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids).