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Biomedical subjects

M E Lowe

Publications and source records attributed to M E Lowe.

At least 19 recordsLinked to original sources
PubMed2001

Colipase residues Glu64 and Arg65 are essential for normal lipase-mediated fat digestion in the presence of bile salt micelles.

Pancreatic triglyceride lipase (PTL) requires colipase for activity. Various constituents in meals and in bile, particularly bile acids, inhibit PTL. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids. Presumably, colipase functions by anchoring and orienting PTL at the oil-water interface. The x-ray structure of the colipase.PTL complex supports this model. In the x-ray structure, colipase has a hydrophobic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu(45)/Asp(89) and Glu(64)/Arg(65). To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the putative PTL binding domains of human colipase. Each mutant was expressed, purified, and assessed for activity against various substrates. Most of the mutants showed impaired ability to reactivate PTL, with mutations in the Glu(64)/Arg(65) binding site causing the greatest effect. Analysis indicated that the mutations decreased the affinity of the colipase mutants for PTL and prevented the formation of PTL.colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutions. Most mutants stimulated PTL activity normally in monomeric bile salt solutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each mutant colipase bound to tributyrin to the same extent as wild type colipase. These results demonstrate that the hydrophilic surface of colipase interacts with PTL in solution to form active colipase.PTL complexes, that bile salt micelles influence that binding, and that the proper interaction of colipase with PTL requires the Glu(64)/Arg(65) binding site.

Alanine↗
PubMed2000

Properties and function of pancreatic lipase related protein 2.

The lipase gene family includes pancreatic triglyceride lipase and two pancreatic proteins, pancreatic lipase related proteins 1 and 2, with strong nucleotide and amino acid sequence homology to pancreatic triglyceride lipase. All three proteins have virtually identical three-dimensional structures. Of the pancreatic triglyceride lipase homologues, only pancreatic lipase related protein 2 has lipase activity. Like pancreatic triglyceride lipase, related protein 2 cleaves triglycerides, but it has broader substrate specificity. Pancreatic lipase related protein 2 also hydrolyzes phospholipids and galactolipids, two fats that are not substrates for pancreatic triglyceride lipase. The rat-related protein 2 also differs from pancreatic triglyceride lipase in sensitivity to bile salts and in response to colipase. Although the pancreas expresses both lipases, their temporal pattern of expression differs. Pancreatic lipase-related protein 2 mRNA appears before birth and persists into adulthood, whereas PTL mRNA first appears at the suckling-weanling transition. Additionally, intestinal enterocytes, paneth cells and cultured cytotoxic T-cells express mRNA encoding pancreatic lipase related protein 2. A physiological function for pancreatic lipase related protein 2 was demonstrated in mice that did not express this protein. Pancreatic lipase related protein 2 deficient mice malabsorbed fat in the suckling period, but not after weaning. They also had a defect in T-cell mediated cytotoxicity. Thus, pancreatic lipase related protein 2 is a lipase that participates in the cytotoxic activity of T-cells and plays a critical role in the digestion of breast milk fats.

Animals↗
PubMed2000

Discoordinate expression of pancreatic lipase and two related proteins in the human fetal pancreas.

The lipase gene family contains a large number of members. Among the most closely related are pancreatic triglyceride lipase (PTL) and two pancreatic lipase-related proteins (PLRP1 and PLRP2). Previous studies in rodents demonstrated divergent temporal expression of the genes encoding these proteins. PLRP1 and PLRP2 were expressed in fetal pancreas, whereas PTL was not expressed until pups were several weeks old. To determine whether the human pancreas has a similar expression pattern for these genes, we determined the levels of each mRNA in fetal pancreas at various ages. A reverse transcriptase-PCR method was developed and used to quantify the mRNA levels for the three species normalized to the mRNA encoding cyclophillin. The mRNA encoding PLRP1 and PLRP2 was present by 16 wk in the fetal pancreas. In contrast, the mRNA encoding PTL was not present in the fetal pancreas. This pattern of expression suggests that the genes encoding theses proteins have different regulatory elements controlling temporal expression and provides another example of nonparallel expression of genes encoding pancreatic exocrine proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

The open lid mediates pancreatic lipase function.

Pancreatic triglyceride lipase (PTL) and the homologous pancreatic lipase related protein 2 (PLRP2) provide a unique opportunity to understand the molecular mechanism of lipolysis. They differ in substrate specificity, sensitivity to bile salts, and colipase dependence despite their close amino acid and tertiary structure identity. One important structure, present in both lipases, is the lid which occupies different positions in the inactive and active forms of PTL. We investigated the role of the lid in lipase function by site-specific mutagenesis. By exchanging the lids between PTL and PLRP2, we created two chimeric lipases. Additionally, we made multiple substitution mutations in the PTL lid. PLRP2 with the PTL lid had kinetic properties similar to PLRP2. PTL with the PLRP2 lid was greatly impaired and had no activity at micellar bile salt concentrations even in the presence of colipase. Both chimeras showed interfacial activation suggesting that the closed lid position was maintained. A series of substitution mutations were made in positions Arg257 and Asp258. These mutations demonstrated the importance of these two residues to maintaining the normal activity, triglyceride acyl chain specificity, and colipase interaction of PTL. The preserved interfacial activation in the chimeras, the similar crystal structure of the two lids in the closed position, and the importance of Arg257 and Asp258 in mediating the open conformation of the lid argue that the position of the open lid influences the differences in activity against triglycerides, in sensitivity to bile salts, and in colipase dependence between PTL and PLRP2.

Amino Acid Sequence↗
PubMed1998

Decreased neonatal dietary fat absorption and T cell cytotoxicity in pancreatic lipase-related protein 2-deficient mice.

The pancreas secretes several different lipases. The most abundant is pancreatic triglyceride lipase (PTL). The pancreas also synthesizes two homologues of PTL, the pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Cytotoxic T-lymphocytes also express PLRP2 under certain conditions. We sought to determine the role of PLRP2 in fat absorption and in T-cell cytotoxicity by creating a PLRP2-deficient mouse. Adult PLRP2-deficient mice had normal fat absorption. In contrast, suckling PLRP2-deficient mice had fat malabsorption evidenced by increased fecal weight, increased fecal fats, and the presence of undigested and partially digested dietary triglycerides in the feces. As a result, the PLRP2-deficient pups had a decreased rate of weight gain. To assess T cell cytotoxicity, we immunized PLRP2-deficient mice with a mastocytoma cell line, P815, and determined the ability of splenocytes from the immunized mice to kill P815 cells in a 51Cr release assay. PLRP2-deficient cells had deficient killing activity in this assay, and PLRP2-deficient splenocytes released fewer fatty acid from the target cells than did control cells. Our results provide the first evidence of a physiological function for PLRP2. PLRP2 participates in T cell cytotoxicity, and PLRP2 performs a crucial role in the digestion of dietary fats in suckling animals.

Age Factors↗
PubMed1998

Purification and characterization of human procolipase expressed in yeast cells.

We report the successful, efficient, and large-scale expression of recombinant human procolipase in yeast. Using the full-length cDNA of human procolipase, constructs were made using either the native human procolipase signal peptide sequence or the signal peptide sequence of yeast. These constructs were used to transform yeast cells, and expression was followed. Only minimal expression was seen with the procolipase using the native human signal peptide. Robust secretion of the procolipase occurred when the yeast signal peptide was exchanged for the native signal peptide. Expression yielded more than 30 mg/liter. The recombinant protein was purified from the medium by immunoaffinity chromatography. The highly purified procolipase was free of proteolytic degradation and displayed activity and binding characteristics that were indistinguishable from those of tissue-purified human pancreatic colipase. Expression in yeast cells provides a useful tool for expressing intact, unprocessed recombinant wild-type and mutated procolipase.

Chromatography, Affinity↗
PubMed1998

Human pancreatic triglyceride lipase expressed in yeast cells: purification and characterization.

A cDNA clone encoding human pancreatic triglyceride lipase was cloned into a yeast expression vector so that the yeast PHO1 signal peptide replaced the native signal peptide. Pichia pastoris cells were transfected with the vector, and clones expressing human pancreatic triglyceride lipase were isolated. Recombinant human pancreatic lipase was expressed in broth cultures and was purified from the medium by DEAE blue Sepharose and hydroxyapatite chromatography. The highly purified lipase had specific activities for various triglyceride substrates identical to those of tissue-purified human pancreatic triglyceride lipase; it was inhibited by bile salts, required colipase for activity, and demonstrated interfacial activation. This expression system is suitable for the rapid, efficient production of human pancreatic triglyceride lipase in amounts adequate for biophysical studies.

Amino Acid Sequence↗
PubMed1998

Rat gastric procolipase: sequence, expression, and secretion during high-fat feeding.

BACKGROUND & AIMS: Procolipase, the cofactor for pancreatic lipase, was recently found in the rat stomach using immunohistochemistry. The aim of this study was to determine the sequence of rat gastric procolipase, to evaluate the expression and secretion during high-fat feeding, and to find out the conditions for activation of gastric procolipase to form colipase and enterostatin. METHODS: Gastric procolipase was cloned from a rat complimentary DNA (cDNA) library using a 32P-labeled pancreatic procolipase probe for screening. For the expression of gastric procolipase, rats were fed a high-fat diet for 0, 1, 2, 5, and 14 days. Gastric mucosa was collected for isolation of RNA and gastric juice for measurement of procolipase. After treatment with pepsin, HCl, and trypsin, gastric juice was analyzed on high-performance liquid chromatography for identification of enterostatin. RESULTS: The cDNA sequence for gastric procolipase was identical to that of pancreatic procolipase. High-fat diet decreased the expression of gastric procolipase. Enterostatin was present in the gastric juice, with pepsin and acid involved in the cleavage of gastric procolipase. CONCLUSIONS: Gastric procolipase is activated to release colipase and enterostatin. The role of gastric colipase may be to prepare lipase-catalyzed fat digestion already in the stomach. Gastric enterostatin may be involved in the onset of early satiety.

Amino Acid Sequence↗
PubMed1998

The hydrophobic surface of colipase influences lipase activity at an oil-water interface.

The interaction of pancreatic triglyceride lipase and colipase at an oil-water interface is required for efficient digestion of dietary fats and provides a model system for the interaction of proteins at biological membranes. Colipase has two important surfaces, a hydrophilic surface that interacts with lipase and a hydrophobic surface that presumably interacts with substrate. To begin our investigations into the role of the hydrophobic surface in the function of colipase, we replaced three neighboring tyrosine residues at positions 55, 58, and 59 in the hydrophobic surface with aspartic acid. Two of the three residues, Tyr55 and Tyr59, influenced the activity of colipase. Introducing aspartic acid at either position decreased the activity with long-chain triglycerides, but not with a short-chain triglyceride. Decreased ability of the mutants to anchor lipase to long-chain triglycerides did not explain the altered activity of the mutants. A mutant containing aspartic acid at positions 55 and 59 had no activity with any substrate and did not anchor lipase to either short- or long-chain triglycerides. These results identify the two tyrosine residues that interact with substrate and suggest that the hydrophobicity of the surface containing these tyrosines influences colipase function and the substrate selectivity of pancreatic triglyceride lipase.

Aspartic Acid↗
PubMed1997

Colipase stabilizes the lid domain of pancreatic triglyceride lipase.

Pancreatic lipase is characterized by increased activity against water-insoluble substrates and by dependence on another protein, colipase, for binding to the substrate interface. In most models of pancreatic lipase activity, colipase functions to anchor lipase on the substrate interface. Recent studies of the x-ray crystal structure of the complex between colipase and lipase suggest another function for colipase in maintaining the active conformation of lipase. We tested this hypothesis by introducing mutations into colipase at position 15, a residue that contacts the lid domain lipase in the open conformation. Multiple mutant colipases were expressed and shown to have decreased activity. To further investigate the function of the interaction between Glu15 of colipase and lipase, we examined one mutant, E15R, in detail. This mutant had 175-fold less activity compared with wild-type colipase. Although E15R had decreased activity, it was as effective as wild-type lipase in anchoring lipase to mixed emulsions of bile salt and tributyrin. The importance of the interaction with the lid domain was tested by determining the activity of E15R with lid deletion mutants of lipase. E15R was as active as wild-type colipase with these mutant lipases. These results indicate that Glu15 is critical for activity of the colipase-lipase complex at an interface and that colipase has a function in lipolysis in addition to anchoring lipase to an interface. We propose that this function is to stabilize the lid domain of lipase in the open conformation, thereby facilitating lipolysis.

Animals↗
PubMed1997

Molecular mechanisms of rat and human pancreatic triglyceride lipases.

Dietary fats affect health and disease. The assimilation of dietary fats into the body requires that they be digested by lipases. One lipase, pancreatic triglyceride lipase, is essential for the efficient digestion of dietary fats. Pancreatic triglyceride lipase is the archetype of the lipase gene family that includes two homologues of pancreatic triglyceride lipase, pancreatic lipase-related proteins 1 and 2. In recent years, important advances have been made in delineating the mechanisms of lipolysis. The cDNA sequences encoding pancreatic triglyceride lipase and the related proteins have been described. The tertiary structure of human pancreatic triglyceride lipase has been determined alone and in a complex with colipase, a pancreatic protein required for lipase activity in the duodenum. This structural information has allowed the rational design of site-specific mutants of pancreatic triglyceride lipase. Together with the structural information, these mutants have greatly advanced our understanding of the molecular details governing lipolysis. This review describes these studies, which will eventually provide the background for the rational design of nutrition therapy in patients with pancreatic insufficiency and fat malabsorption.

Amino Acid Sequence↗
PubMed1997

Structure and function of pancreatic lipase and colipase.

Dietary fats are essential for life and good health. Efficient absorption of dietary fats is dependent on the action of pancreatic triglyceride lipase. In the last few years, large advances have been made in describing the structure and lipolytic mechanism of human pancreatic triglyceride lipase and of colipase, another pancreatic protein that interacts with pancreatic triglyceride lipase and that is required for lipase activity in the duodenum. This review discusses the advances made in protein structure and in understanding the relationships of structure to function of pancreatic triglyceride lipase and colipase.

Binding Sites↗
PubMed1996

Pancreatic lipase-related protein 2 but not classical pancreatic lipase hydrolyzes galactolipids.

The pancreatic lipase family contains three subfamilies, the 'classical' lipases and the pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2). Galactolipids are present in membranes of leaves and vegetables and consist of digalactosyldiacylglycerol (DGalDG) monogalactosyldiacylglycerol (MGalDG) and sulfoquinovosyldiacylglycerol (SQDG). These lipids were incubated with PLRP2 from guinea-pig (GPLRP2) and rat (RPLRP2). In the presence of bile salts DGalDG was efficiently hydrolyzed by GPLRP2 and, although less efficiently, by RPLRP2 to digalactosylmonoacylglycerol (DGalMG), free fatty acids and water-soluble galactose-containing compounds. Also, MGalDG and SQDG were hydrolyzed by GPLRP2 and RPLRP2. These data suggest a possible role of PLRP2 in the digestion of dietary galactolipids.

Animals↗
PubMed1996

Mutation of the catalytic site Asp177 to Glu177 in human pancreatic lipase produces an active lipase with increased sensitivity to proteases.

The catalytic mechanism for members of the lipase gene family incorporates a serine-histidine-acidic group triad. In general, the acidic group is an aspartate, Asp177 in human pancreatic lipase, but glutamate is found in some lipases. Previously, we demonstrated that site-specific mutagenesis of Asp177 to Glu177 produced a mutant human pancreatic lipase with near normal activity against triolein, thereby, raising questions about the role of Asp177 in the catalytic triad and about the evolutionary pressure which selected Asp over Glu in the catalytic mechanism. To address these questions, we constructed and expressed mutants of Asp177 and Asp206, another acidic residue that could participate in the catalytic triad. The Glu177 mutant had a substrate specificity, specific activity, pH profile, colipase dependance, and interfacial activation comparable to the native lipase, Asp177. Several mutants of Asp206 were normally active, thus, confirming the important role of Asp177 in pancreatic lipase function. Additionally, we found that the Glu177 mutant had increased susceptibility to proteases and to urea denaturation. These findings demonstrated decreased conformational stability of the mutant lipase and provided an explanation for the preference of aspartate in the catalytic triad of human pancreatic lipase.

Amino Acid Sequence↗