In vivo and in vitro studies on the stereoselective hydrolysis of tri- and diglycerides by gastric and pancreatic lipases.

The stereoselectivity of dog gastric and dog pancreatic lipases was investigated both in vitro, under simulated physiological conditions, and in vivo, during the digestion of a liquid test meal. In vitro it was observed that although both lipases had a stereopreference for the sn-3 position in triglycerides, it was about three times higher in the case of the gastric lipase. On the other hand, both lipases clearly showed a comparable enantioselectivity for the sn-1 position when a racemic diolein was used as the substrate. In the case of pancreatic lipase, the enantiomeric excess of 1,2-sn-diolein generated in vitro by the hydrolysis of triolein was found to decrease significantly, and even to be slightly reversed, at high rates of hydrolysis (above 50%) due to the further stereoselective hydrolysis of diglycerides into monoglycerides. This finding may explain the low enantiomeric excess of the diglycerides observed in vivo during the early phase of intraduodenal digestion when pancreatic lipase plays a predominant role and the rate of triolein hydrolysis is already high. On the other hand, a large enantiomeric excess of 1,2-sn-diolein generated from triolein was always the fingerprint of the gastric lipase in vitro even at high hydrolysis rates. This fingerprinting of gastric lipase was observed during both the intragastric phase and the late intestinal phase of lipolysis. This feature was therefore taken as an index to determine the respective roles of gastric and pancreatic lipases during in vivo lipolysis. To the best of our knowledge, this is the first time that stereoselectivity has been used as a tool to discriminate between the activities of two enzymes hydrolyzing the same substrate in vivo.

Assignment of microsatellite sequences to the region duplicated in CMT1A (17p12): a useful tool for diagnosis.

Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent form of the peripheral hereditary neuropathies, has been associated with a duplication of a genomic segment of 1.5 Mb, located in 17p11.2. Recently, the same segment has been found to be deleted in patients with another peripheral neuropathy, hereditary neuropathy with liability to pressure palsies (HNPP). Highly polymorphic markers are rare in this area, rendering the diagnosis highly dependent either on invasive examinations (like nerve biopsy) or not totally reliable (like gene dosage). Thus, we used a contig of YACs, including the whole region duplicated in CMT1A, to map highly polymorphic microsatellite loci, designed in Genethon. We showed that four of these loci are located in the duplicated region, allowing us to propose them as diagnostic markers for CMT1A and HNPP.

Assignment of a Polycomb-like chromobox gene (CBX2) to human chromosome 17q25.

A human clone corresponding to the homologue of the murine Polycomb-like gene M33 has been used to map this gene (CBX2) to human chromosomes. Both somatic cell hybrid panels and FISH on metaphase chromosomes have been used. These techniques gave a consistent localization, at the tip of the long arm of chromosome 17 (17q25). This localization, as well as the potential role of a mammalian Polycomb-like protein, suggests a potential involvement in two different pathologies: the campomelic syndrome, an inherited disorder, and neoplastic disorders linked to allele loss already described in this region.

Cutinase, a lipolytic enzyme with a preformed oxyanion hole.

Cutinases, a group of cutin degrading enzymes with molecular masses of around 22-25 kDa (Kolattukudy, 1984), are also able to efficiently hydrolyse triglycerides (De Geus et al., 1989; Lauwereys et al., 1991), but without exhibiting the interfacial activation phenomenom (Sarda et al., 1958). They belong to a class of proteins with a common structural framework, called the alpha/beta hydrolase fold (Martinez et al., 1992; Ollis et al., 1992). We describe herein the structure of cutinase covalently inhibited by diethyl-p-nitrophenyl phosphate (E600) and refined at 1.9-A resolution. Contrary to what has previously been reported with lipases (Brzozowski et al., 1991; Derewenda et al., 1992; Van Tilbeurgh et al., 1993), no significant structural rearrangement was observed here in cutinase upon the inhibitor binding. Moreover, the structure of the active site machinery, consisting of a catalytic triad (S120, H188, D175) and an oxyanion hole (Q121 and S42), was found to be identical to that of the native enzyme, whereas the oxyanion hole of Rhizomucor lipase (Brzozowski et al., 1991; Derewenda et al., 1992), like that of pancreatic lipase (van Tilbeurgh et al., 1993), is formed only upon enzyme-ligand complex formation. The fact that cutinase does not display interfacial activation cannot therefore only be due to the absence of a lid but might also be attributable to the presence of a preformed oxyanion hole.

Digestive lipases: inactivation by phosphonates.

Phosphonates mimicking the transition state which occurs during carboxyester hydrolysis were synthesized and investigated as potential inactivators of human pancreatic (HPL) and gastric (HGL) lipases. Their efficiency as inactivators was studied on the basis of the alkyl chain length, the nature of the leaving group and the influence of the ester substituent. In each case, HGL was found to be more sensitive than HPL towards these phosphonates. The released p-nitrophenol to enzyme ratio indicates that a 1:1 complex was formed. In the absence of substrate, the most powerful inactivator was O-methyl O-(p-nitrophenyl) n-pentylphosphonate (4A), which has a short alkyl chain, a small methoxy substituent and a good leaving group.

Inactivation of pancreatic lipases by amphiphilic reagents 5-(dodecyldithio)-2-nitrobenzoic acid and tetrahydrolipstatin. Dependence upon partitioning between micellar and oil phases.

We have reported previously that Cys103 (SHII) of human pancreatic lipase (HPL), unlike the nonessential Cys181 (SHI), was buried and inaccessible to classical water-soluble sulfhydryl reagents. The lipolytic activity of HPL was lost after the labeling of the above two SH groups with the amphiphilic sulfhydryl reagent, 5-(dodecyldithio)-2-nitrobenzoic acid (C12-TNB), suggesting that the SHII residue may play an important role in the hydrolytic process [Gargouri, Y., Cudrey, C., Medjoub, H., & Verger, R. (1992) Eur. J. Biochem. 204, 1063-1067]. For the present experiments, we selected dog pancreatic lipase (DPL), purifying it for the first time, and recombinant guinea pig pancreatic lipase (r-GPL), which both contain a buried SHII group but no accessible SHI group. The single SHII of DPL and r-GPL reacted only with the amphiphilic SH reagent (C12-TNB), and its labeling was correlated with a rapid lipase inactivation. Although it is spatially remote from the catalytic triad, the SHII group of pancreatic lipases, when chemically labeled, was found to be responsible for the loss of their lipolytic activity. The presence of a bulky dodecyl chain, linked by a disulfide bond to the SHII, may have prevented the critical beta-5 loop (residues 76-85) movement by steric hindrance and consequently disturbed the formation of the oxyanion hole. Thus, pancreatic lipase inactivation by the amphiphilic sulfhydryl reagent can be said to be due to the prevention of a productive induced fit. Tetrahydrolipstatin (THL) is an amphiphilic inactivator reacting with the essential serine of the lipase active site.(ABSTRACT TRUNCATED AT 250 WORDS)

A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase.

Typically pancreatic lipases are characterized by the following properties: (1) they are activated by lipid/water interfaces (interfacial activation), (2) they are inhibited by bile salts but reactivated by colipase (a small activator protein), and (3) they do not hydrolyze significantly phospholipids. A cDNA clone encoding a guinea pig pancreatic (phospho)lipase (GPL) has been sequenced and expressed. The enzyme (recombinant as well as native) differs from other pancreatic lipases in that (1) it is not interfacially activated, (2) its activity is unaffected by the presence of bile salts and/or colipase using tributyrin as substrate, and (3) it exhibits equally phospholipase A1 and lipase activities. The amino acid sequence of GPL is highly homologous to that of other known pancreatic lipases, with the exception of a deletion in the so-called lid domain that regulates access to the active centers of other lipases. We propose that this deletion is directly responsible for the anomalous behavior of this enzyme. Thus GPL challenges the classical distinction between lipases, esterases, and phospholipases.

Stereoselective hydrolysis of triglycerides by animal and microbial lipases.

In the present paper, a study on the stereoselectivity of 25 lipases of animal and microbial origin towards homogeneous prochiral triglycerides is presented. All the lipases tested catalyse the hydrolysis of the chemically alike but sterically nonequivalent ester groups in trioctanoin and triolein with different degrees of stereobias, depending on the fatty acyl chain length of the substrate (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990). Hydrolysis of the sn-2 ester group is catalysed by very few lipases and only Candida antarctica A shows a clear preference for this position. Most of the lipases investigated (12 with trioctanoin and 16 with triolein) showed a preference for the sn-1 position. Using trioctanoin as substrate we observed a total stereoselectivity for position sn-1 with Pseudomonas sp. and Pseudomonas aeruginosa and for position sn-3 with Candida antarctica B. This was not the case with triolein as substrate. Among the 23 lipases studied here and the other two lipases described previously (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990), 17 show a higher stereoselectivity with trioctanoin than with triolein. With guinea pig pancreatic lipase and with three mold lipases (Geotrichum candidum M, Geotrichum candidum A, and Candida antarctica B), the preference switches from sn-3 to sn-1 when the acyl chain length increases from eight to 18 carbon atoms. The main conclusion to emerge from the present study is that the specific stereopreference of each lipase for a given substrate under given lipolytic conditions can be said to be its fingerprint.

Inactivation of human pancreatic lipase by 5-dodecyldithio-2-nitrobenzoic acid.

Both thiol groups of native human pancreatic lipase can react with the new hydrophobic sulfhydryl reagent 5-dodecyldithio-2-nitrobenzoic acid (Dod-S-NbS) in the absence of a denaturing agent. Here we describe for the first time the covalent and stoichiometric modification of the inaccessible SHII group of native pancreatic lipase, using a 16-fold molar excess of this hydrophobic sulfhydryl reagent. A direct correlation was found to exist between the covalent modification of this SHII group and the loss of lipase activity. The question has not yet been answered, however, as to how Dod-S-NbS reaches the SHII-containing residue, whereas classical hydrophilic sulfhydryl reagents are unable to do so. This difference in reactivity may be attributable to the hydrophobic character of Dod-S-NbS and its potential capacity to form aggregates inducing a conformational change in the lipase molecule.