Site-directed removal of N-glycosylation sites in human gastric lipase.

Human gastric lipase (HGL) is a highly glycosylated protein, as glycan chains account for about 15% of the molecular mass of the native HGL. Four potential N-glycosylation consensus sites (Asn15, 80, 252 and 308) can be identified from the HGL amino acid sequence. We studied the functional role of the individual N-linked oligosaccharide chains by removing one by one all the N-glycosylation sites, via Ala residue replacement by site-directed mutagenesis of Ser and Thr residues from the consensus sequences Asn-X-Ser/Thr. Mutagenized cDNA constructs were heterologously expressed in the baculovirus/insect cell system. Removal of oligosaccharides either at Asn15, 80 or 252 was found to have no significant influence on the enzymatic activity measured in vitro. However, the absence of glycosylation at Asn308, as well as a total deglycosylation, reduced the specific enzymatic activity of recombinant HGL (r-HGL), measured on short- and long-chain triglycerides, to about 50% of normal values. Furthermore, biosynthesis and secretion of r-HGL markedly dropped when all four potential glycosylation sites were mutated. The kinetics of the interfacial adsorption of r-HGL and the completely deglycosylated r-HGL (four-site mutant) were found to be identical when recording the changes with time of the surface pressure either at the air-water interface or in the presence of an egg phosphatidylcholine (PtdCho) monomolecular film spread at various initial surface pressures. This indicates that both recombinant HGLs are identical, as far as recognition of phospholipid film and adsorption on PtdCho are concerned. The N-glycosylation of HGL may contribute to the enzyme stability in the stomach, as under acidic conditions the degradation by pepsin of the unglycosylated r-HGL is increased.

Purification and interfacial behavior of recombinant human gastric lipase produced from insect cells in a bioreactor.

Recombinant human gastric lipase (rHGL) (EC 3.1.1.3) was produced on a large scale (5-13 mg/liter) from recombinant baculovirus-infected insect cells using a bioreactor apparatus. Here an improved procedure is described for purifying rHGL involving the use of cation exchange chromatography followed by immunoaffinity column methods, which gives a total yield of 62% and a purification factor of 464, using 10% isopropanol in all the purification buffers. The presence of isopropanol was necessary to preserve the stability of the enzyme during the chromatographic separation steps. The specific activity of rHGL on tributyroylglycerol (700 U/mg) was lower than that of native HGL (nHGL) (1080 U/mg). The rHGL interfacial adsorption kinetics were studied by recording the changes in the surface pressure with time in the presence or absence of an egg phosphatidycholine monomolecular film spread at the air/water interface at various initial surface pressures. The surface behavior of rHGL was similar to that of nHGL. It can be concluded that the lipid binding affinity of rHGL is identical to that of the native lipase and, consequently, that the presence of detergents and lipids in the insect cell culture media did not affect the interfacial behavior of the purified rHGL. It will be therefore possible to specifically study the binding step of HGL mutants to a lipid monolayer.

Bovine pancreatic preproelastases I and II: comparison of nucleotide and amino acid sequences and tissue specific expression.

Clones encoding bovine preproelastases I and II were isolated from a pancreatic cDNA library and were sequenced in order to define the structural characteristics of these enzymes. The bovine 947- and 884-nucleotide preproelastase I and II cDNAs encode proteins containing a signal peptide of the same length (16 amino acids), but with a slightly different number of amino acids for the activation peptide (10 and 12, respectively) and the mature enzyme (240 and 241, respectively). Considering amino acid sequences, each enzyme shares a high degree of identity (76-86%) within species. In contrast, only 55.3% identity is found between bovine elastases I and II. This difference could explain partly their own specificity. Analysis of the expression of the elastases in various bovine tissues demonstrated that they are specifically expressed in high levels in the pancreatic gland. These two approaches (structure and expression) allowed us to characterize the bovine pancreatic elastases I and II.

Expression in insect cells and purification of a catalytically active recombinant human gastric lipase.

Human gastric lipase (HGL) cDNA was synthesized by RT-PCR amplification and cloned into the PVL 1392 baculovirus transfer vector. The recombinant transfer vector was cotransfected with a modified baculovirus DNA (Baculogold) which contains a lethal deletion. Cotransfection of baculovirus DNA with the recombinant transfer vector rescues the lethal deletion of this virus DNA and reconstitutes viable virus particles inside the transfected insect cells. BTI-TN-5B1-4 insect cells (also called High Five cells) were used to express recombinant HGL. The level of HGL secretion was approximately 32 mg/l of culture medium. The insect cells also accumulated HGL intracellularly, which indicated the existence of rate-limiting steps in the secretion of HGL. Therefore we investigated the effect of replacing the HGL signal peptide (SP) by other SP of secreted proteins. The honeybee melittin SP and the human pancreatic lipase (HPL) SP were tested. The fusion of HGL with HPL SP resulted in a 2-fold increase in the amount of lipase secreted from the insect cells. The recombinant active HGL was not processed at the expected cleavage site of the natural enzyme, however, but at residue +3. On the other hand, High Five cells transfected with the vector encoding HGL fused to the melittin SP did not secrete any detectable active HGL. Recombinant HGL was identified using the Western blot procedure with rabbit polyclonal antibodies. The protein migrated with an apparent molecular mass of 45 kDa under SDS-PAGE analysis (compared with 50 kDa in the case of natural HGL), indicating that the insect cells have only a limited capacity to glycosylate HGL. The maximum specific activities of the recombinant lipase were 434, 730 and 562 units/mg using long-chain (Intralipid), medium-chain (trioctanoylglycerol) and short-chain (tributyroylglycerol) triacylglycerols, respectively.

Caerulein and gastrin(2-17 ds) regulate differently synthesis of secretory enzymes, mRNA levels and cell proliferation in pancreatic acinar cells (AR4-2J).

In order to characterize the biological functions coupled to cholecystokinin (CCK) A and B receptors, the effects of gastrin(2-17 ds) and caerulein were compared. An isolated cell model, the pancreatic acinar cell line AR4-2J, was used and the experiments were carried out in serum-free media. Caerulein was found to evoke no mitogenic effects either alone or in the presence of the CCK antagonists L364,718 and CR1409. Gastrin(2-17 ds) increased cell proliferation by 2-fold with an IC50 of 150 pM, corresponding to the occupancy of the CCK B receptors. CR1409, at concentrations that fully occupied CCK B receptors, inhibited the gastrin(2-17 ds) effects. Caerulein enhanced chymotrypsinogen biosynthesis by 100% and the corresponding mRNA level by 75%; amylase biosynthesis and mRNA level were enhanced by 40% only. Half-maximal increases in chymotrypsin activity and mRNA level were recorded in response to caerulein at concentrations of 100 pM and 50 pM respectively. Gastrin(2-17 ds) at 100 nM enhanced chymotrypsinogen biosynthesis by 26% and its mRNA level by 35%; these responses were lower than those evoked by 0.1 nM caerulein. Furthermore, CR1409 completely inhibited caerulein- and gastrin(2-17 ds)-stimulated chymotrypsinogen synthesis, with similar IC50 (4 microM). These results suggest that both peptides induced the synthesis of the secretory enzyme after occupancy of CCK A receptors.

cDNA cloning and sequence analysis of human pancreatic procarboxypeptidase A1.

Using polyclonal antibodies raised against human pancreatic procarboxypeptidases, a full-length cDNA coding for an A-type proenzyme was isolated from a lambda gt11 human pancreatic library. This cDNA contains standard 3' and 5' flanking regions, a poly(A)+ tail and a central region of 1260 nucleotides coding for a protein of 419 amino acids. On the basis of sequence comparisons, the human protein was classified as a procarboxypeptidase A1 which is very similar to the previously described A1 forms from rat and bovine pancreatic glands. The presence of the amino acid sequences assumed to be of importance for the zymogen inhibition by its activation segment, primarily on the basis of the recently reported crystal structure of the B form, further supports the proposed classification.

Gastric and pancreatic enzyme activities and their relationship with some gut regulatory peptides during postnatal development and weaning in calves.

Changes in the activities of three gastric and nine pancreatic enzymes plus colipase were determined during postnatal development and weaning in calves. In calves exclusively milk-fed for 2, 7, 28, 56, 70 and 119 d, the enzyme activities per kilogram of empty live weight increased with age for chymotrypsin, elastase, carboxypeptidases A and B, ribonuclease and alpha-amylase, decreased for chymosin, lysozyme and colipase but showed no change in the case of pepsin, trypsin, lipase and phospholipase A2 compared with animals at birth. The greatest increase was that in alpha-amylase activity (about 50-fold between d 2 and 119). In calves weaned between d 28 and 56, all the activities were higher than in milk-fed animals, except that of chymosin (which was slightly lower) and that of colipase (which did not change). At 119 d of age, chymotrypsin, carboxypeptidase A, alpha-amylase and lipase were 1.6- to fourfold higher in ruminants than in preruminants. Thus, most enzyme activities were modified first by colostrum and milk intake, and again upon weaning by development of the forestomachs and ingestion of solid food. These ontogenic patterns might be under the control of many gut regulatory peptides, the plasma concentrations of which changed simultaneously. Some gastric and pancreatic enzymes were correlated to plasma concentrations of these gut regulatory peptides.

Primary structure of rat pancreatic lipase mRNA.

The sequence of rat pancreatic lipase mRNA was determined. The data have been assigned the following accession number, X61925, in the EMBL data library. The total length of the messenger is 1531 nucleotides, plus a poly(A) stretch of about 60 nucleotides. A 72-nucleotides 5'-noncoding region is followed by a 1419-nucleotides open reading frame which encodes a protein of 473 amino acids, including the 17 amino acid signal peptide. The mature enzyme (456 residues) has 6 additional C-terminal amino acids, as compared with the amino acid sequence of pig (direct amino acid sequence), dog, man and rat isoenzyme from Genbank, M58369 (all deduced from the nucleotide sequence). A higher degree of homology exists between the amino acid sequence of rat mature enzyme with those of dog (88%), pig (75%) and man (75%) than with that of rat isolipase (74%).

Regulation of amylase and chymotrypsinogen expression by dexamethasone and caerulein in serum-free-cultured pancreatic acinar AR4-2J cells. Influence of glucose.

The direct effects of dexamethasone and caerulein on two pancreatic enzymes, amylase and chymotrypsin, were determined in AR4-2J cells cultured under serum-free conditions at two glucose concentrations (1.0 and 4.5 g/l). In the absence of any hormone, the higher glucose concentration resulted in a 1.6-1.8-fold increase in the basal levels of amylase and chymotrypsinogen. Dexamethasone (50 nM) increased the biosynthesis and mRNA levels of both enzymes at both glucose concentrations. However, dexamethasone had a more pronounced effect on amylase biosynthesis (5-fold induction) than on chymotrypsinogen biosynthesis (1.8-fold induction). The parallel increases in mRNA and protein indicated the existence of pre-translational regulation. This is in contrast with what was observed in serum-containing media, where a translational regulation of amylase biosynthesis took place, probably under the control of both glucose and some serum factors. By contrast, caerulein (10 nM) exerted a more specific action on chymotrypsinogen. The increases in chymotrypsinogen mRNA were 2.2- and 2.1-fold, and increases in chymotrypsin activity were 1.6- and 2.9-fold at 1.0 and 4.5 g of glucose/litre respectively. Thus the regulation by caerulein occurred mainly through the enhancement of chymotrypsinogen transcription and/or mRNA stabilization.