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

Abstract

Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease Adeficient 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, C., J.

Kinetic properties of mouse pancreatic lipase-related protein-2 suggest the mouse may not model human fat digestion

“…This characterization is critical for interpreting any results in mice because the kinetics of PLRP2 varies considerably among species. For instance, human PLRP2 (hPLRP2) has almost no activity in the presence of bile salt micelles and is not reactivated by colipase (10)(11)(12). The same is true for horse PLRP2 ( 13 ).…”

“…The same is true for horse PLRP2 ( 13 ). It has been suggested that the major physiological role of hPLRP2 is as a galactolipase ( 10,12,14,15 ). In contrast, rat PLRP2 has activity in the presence of bile salt micelles even in the absence of colipase.…”

“…Furthermore, in the presence of colipase, BSA substantively inhibited mPTL lipase activity, but it had no apparent impact on mPLRP2 lipase activity. Collectively, these gum arabic in the assay signifi cantly increased the activity of hPLRP2 against triolein, compared with emulsifi cation with bile salts ( 10 ). The activity of hPLRP2 in the duodenal lumen with a test meal is unknown at present.…”

Bioinformatics and Evolution of Vertebrate Pancreatic Lipase and Related Proteins and Genes

“…PTL functions in the presence of colipase in the hydrolysis of emulsified fats in the small intestine following secretion from the pancreas [1][2][3][4][5]; PLR1 is also found in pancreatic secretions but has an as yet unknown function and exhibits no detectable triacylglycerol, cholesterol ester or galactolipase activities [6,8]; PLR2 functions as a pancreatic secretion galactolipase but may also contribute to T cell cytotoxicity in the body [6,25,65]; while PLR3 serves a related, but as yet unknown, lipase function.…”

“…Structural and biochemical studies of mammalian PLR2 have reported strong similarities with PTL although differences were observed in kinetic and colipase binding properties and significant species differences were also reported [4][5][6][7][23][24][25]. Significantly, this enzyme has been shown to be predominantly responsible for pancreatic lipase-like neonatal fat digestion in rats and mice [26] and participates in the dietary fat digestion in human newborn children [27,28].…”