Pancreatic lipase-related protein 1 (PLRP1) is present in the pancreatic juice of several species

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Abstract

Pancreatic lipase-related protein 1 (PLRP1) was purified from human, canine, porcine and rat pancreatic juices. The four PLRP1s were identified using microsequencing methods after performing gel filtration on Ultrogel AcA-54 followed by chromatography on Heparin–Sepharose cation-exchanger. Polyclonal antibodies specific to human PLRP1 (HPLRP1) were raised in the rabbit using a synthetic decapeptide from HPLRP1. The results of Western blotting analysis showed that these antibodies recognized native HPLRP1 and recombinant HPLRP1 produced by insect cells, and cross-reacted only with rat PLRP1 (RPLRP1). No significant lipolytic activity was observed with native canine PLRP1 and recombinant HPLRP1 on various glycerides, phospholipid and vitamin esters, or on cholesterol esters. It was established for the first time that this protein is secreted in variable amounts by the adult exocrine pancreas of several species.

Introduction

In 1992, Giller et al. [1] described the isolation of cDNAs coding for two novel human pancreatic lipase-related proteins (PLRP), referred to as HPLRP1 and HPLRP2, showing 68% and 65% amino acid identity, respectively, with the classical human pancreatic lipase (HPL). Based on sequence comparisons with previously described pancreatic lipases (PL) from other animal species, Giller et al. [1] suggested that the PL family should be divided into three sub-groups: classical PL, PLRP1 and PLRP2. Since that time, several new pancreatic lipase sequences have been identified which fit these three sub-groups. The above system of classification has been corroborated on the basis of the kinetic characteristics of several members of the three sub-groups. (i) The classical PLs are highly active and selective for triglyceride substrates. (ii) The guinea-pig [2, 3, 4], coypu [5] and rat [6] PLRP2s display both lipase and phospholipase A1 activities. The biochemical properties and the physiological role of PLRP2s have by now been well documented [1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13]. (iii) No significant enzymatic activity has been reported to occur up to now with recombinant PLRP1s from humans [1] or rats [14] and their physiological role is still unknown.

The cDNA sequence of a dog pancreatic lipase was first published by Kerfelec et al. [15]. This sequence was later identified by Giller et al. [1] as the dog PLRP1 cDNA sequence (DPLRP1). Using this cDNA, a 15.5 kb gene was sequenced by Mickel et al. [16] and referred as to the dog pancreatic lipase gene. Comparisons between this DPLRP1 gene and the structure of the classical HPL gene [17] have shown that the exon pattern (13 exons) is similar in both cases, whereas the lipoprotein lipase (LPL) and hepatic lipase (HL) genes only possess ten exons [18]. These data suggest that the PLRP1 gene diverged from the PL gene after the divergence of the LPL and HL genes. The human LPL and HL genes are located on chromosomes 8 [19] and 15 [19, 20], respectively. The PLRP1 and PL genes have been co-located on chromosomal region 10q24–q26 in humans [17, 21], which is syntenic with chromosome 19 in the mouse [22]. This gene cluster probably results from the duplication of an ancestral gene.

The PLRP1 gene is characterized by two sequences showing homology with a 20 bp enhancer consensus sequence described as pancreas-specific by Boulet et al. [23]. The presence of an intron immediately upstream of the initiation codon ATG suggests, however, that alternative promoters and differential splicing processes might serve in other tissues to express PLRP1 [16]. Up to now, the PLRP1 mRNA has been found only in the pancreas [1, 14, 24]. The pattern of mRNA expression has been studied during development in the rat, and low PLRP1 mRNA levels were recorded in the foetal rat pancreas. These levels reach a maximum just before birth before decreasing again 12 h after birth, and they are low in adults, whereas the PL mRNA levels are low at birth, increase rapidly during the suckling-to-weaning transition phase, and are still high in adults. These findings indicate that PL and PLRP1 genes are subject to separate regulatory systems of control [14]. The PLRP1 gene is also characterized by two glucocorticoid receptor binding sequences [16]. Using the AR42J pancreatic cell line, Kullman et al. [25] observed that the PLRP1 mRNA level decreased twofold when the cells were grown in the presence of dexamethasone, whereas the PL mRNA level remained unchanged under these conditions.

The 1.8 kb mRNA encoding HPLRP1 was isolated from the total RNA of human pancreas and the results of Northern blot analysis showed that the rate of expression of HPLRP1 is fourfold lower than that of HPL [1]. The exact cellular location of PLRP1 is unknown, however, and this protein has never been directly identified in the exocrine pancreatic secretion. In fact, a protein corresponding to PLRP1 was accidentally co-purified with the classical PL from dog pancreatic juice by Cudrey et al. [26] but this finding was overlooked. In the course of the dog pancreatic lipase (DPL) purification, the N-terminal amino acid sequence analysis yielded a double pattern of two sequences amounting to 20% and 80% of the initial amount of protein. The first amino acid sequence (53 residues) was identical to the DPLRP1 sequence described by Kerfelec et al. [15]. The second amino acid sequence, showing 70% homology with the previous one, was identified based on sequence comparisons as the classical DPL: the complete cDNA sequence of this lipase is still unknown.

The PLRP1 mRNA encodes for a 465-amino-acid polypeptide, including a 15-residue signal peptide, and the theoretical molecular mass of the mature polypeptide (450 residues) is 49.6–49.9 kDa. The deduced amino acid sequence of HPLRP1 does not contain any potential N-glycosylation sites, whereas both DPLRP1 and RPLRP1 possess one potential glycosylation site. In a recent study using mouse acinar cell cultures, the existence of a sulfated protein analogous to PLRP1 was observed [27]. The primary sequence of PLRP1 is closely related to the classical PL, since it includes the Ser–Asp–His catalytic triad and several amino acid residues involved in colipase binding, but the recombinant HPLRP1 expressed in COS cells was reported to be inactive towards triolein [1]. No lipolytic activity of RPLRP1 was detectable in a medium obtained from Sf9 insect cells when triolein was used as the substrate [14]. These findings suggest that PLRP1 may act on other substrates, or that another protein cofactor analogous to colipase may be required for complete PLRP1 activity to be possible, or that this protein may be intrinsically inactive. Recently, the three-dimensional (3-D) structure of dog PLRP1 (DPLRP1) was solved at 2.10 Å [28] and showed that two amino acid residues (Val 178, Ala 180) might impair the ability of the substrate to bind to the active site due to a steric hindrance mechanism. In all the active enzymes from the PL gene family studied, these residues are Ala and Pro. The double mutation Val 178 Ala and Ala 180 Pro introduced into the HPLRP1 cDNA yielded an active recombinant enzyme with triglycerides as the substrate.

The aim of the present study was to identify and characterize native PLRP1 purified to homogeneity from pancreatic juices of several mammalian species including man, dog, rat and pig. The recombinant HPLRP1 was also expressed in insect cells and purified to homogeneity. A specific anti-peptide antibody against HPLRP1 was produced for a use with a specific HPLRP1 immunodetection procedure.

Section snippets

Pancreatic juice samples

Human pancreatic juice was obtained from patients devoid of pancreatic disease by performing endoscopic retrograde catheterization on the main pancreatic duct, at the Sainte-Marguerite Hospital (Marseille), under the supervision of Prof. R. Laugier. Canine and rat pancreatic juices were collected under caerulein (68 ng/kg per h) and secretin (1.3 units/kg per h) stimulation. All the pancreatic juices were mixed with a solution of protease inhibitors, PMSF and benzamidine, each at a final

Production and purification of the recombinant HPLRP1 expressed in insect cells

The amount of the recombinant HPLRP1 (rHPLRP1) secreted by insect cells was estimated to be about 12 mg/l after 3 days of infection according to the procedure of Thirstrup [30]. The recombinant protein was purified in a single cationic exchange chromatographic step. A single 50 kDa band corresponding to the expected molecular mass of the mature protein was observed by SDS–PAGE (Fig. 2), and the results of the microsequencing procedure confirmed that the cleavage of the signal peptide had taken

Discussion

The presence of PLRP1 observed here in the pancreatic secretion of four mammalian species shows that this lipase is synthesized by the exocrine pancreas. It is worth noting that in all amino acid sequences performed in the present study, we found no indication of the presence of a PLRP2. Various amounts of PLRP1 were detected in the pancreatic juice of the four species investigated. Similar amounts of PL and PLRP1 were isolated from canine pancreatic juice (Table 1); whereas the levels of PLRP1

Acknowledgements

We thank Daniel Campèse and Stéphane Canarelli for performing the protein sequencing, and Per F. Nielsen (Novo Nordisk) for performing mass spectrometry analysis. Our thanks are also due to Jessica Blanc for correcting the English. This work was carried out in the framework of the EC Biotech-G Lipase projects (BIO2-CT94-3041 and BIO2-CT94-3013).

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