©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Pancreatic Lipase
IMPORTANCE OF THE HINGE REGION BETWEEN THE TWO DOMAINS, AS REVEALED BY MONOCLONAL ANTIBODIES (*)

(Received for publication, August 25, 1994; and in revised form, November 15, 1994)

Mustapha Aoubala (1) Laurence de La Fournière (1) Isabelle Douchet (1) Abdelkarim Abousalham (1) Cécile Daniel (2) Michel Hirn (2) Youssef Gargouri (1) Robert Verger (1) Alain De Caro (1)(§)

From the  (1)Unité Propre de Recherche 9025 du Groupement de Recherche 1000, Laboratoire de Lipolyse Enzymatique du CNRS, 31 Chemin J. Aiguier, 13402 Marseille Cedex 20 and (2)Immunotech, 130 Avenue Maréchal de Lattre de Tassigny, 13009 Marseille, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis.

Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys^1-Phe). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment.

In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C.(1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.


INTRODUCTION

In mammals, the digestion of dietary triacylglycerols is mediated by two main enzymes, a preduodenal lipase that is secreted in the upper part of the digestive system and acts along the whole gastrointestinal tract and a pancreatic lipase that contributes to lipid digestion only in the duodenum(1, 2, 3) . Unlike serine esterases, digestive lipases develop their full activity only on emulsified substrates. Lipase catalysis therefore involves an interfacial activation step occurring at water/lipid interfaces. Moreover, to overcome the inhibitory effects of the bile salts present in the intestinal lumen, pancreatic lipase specifically requires the presence of a small pancreatic cofactor (colipase) that anchors the pancreatic lipase to the bile salt-coated lipid interfaces(4) .

It has been established using biochemical methods that human pancreatic lipase (HPL) (^1)consists of an N-terminal and a C-terminal domain, which are connected together by a hinge region(5, 6) . Crystallographic data have shown that Ser, which is essential for the lipase activity to occur on emulsified substrates, forms with Asp and His the enzyme's catalytic triad(7) . It has also been established that the catalytic site is covered by a loop (the lid) on the surface of the molecule and that it is therefore not accessible to solvent or substrate molecules ((7) ; see Fig. 1). The recently elucidated three-dimensional structures of the complexes formed between lipase and procolipase on the one hand (8) and lipase-procolipase-bile salts-phospholipid on the other hand (9) indicate first that procolipase interacts with the C-terminal domain of lipase. In the presence of mixed bile salt-phospholipid micelles, the lid covering the catalytic site of lipase undergoes a conformational change that leads to the binding of the substrate to the active site of the enzyme(9) . In this new ``open'' conformation of the enzyme, the two lipase domains slightly rotate around the hinge region, and the N-terminal domain of the lipase then interacts with the procolipase via the open lid.


Figure 1: Schematic diagram of the three-dimensional structure of the ``closed conformation'' of HPL described by Winkler et al.(7) . beta-Strands are indicated by arrows, and alpha-helices by coils. The N- and C-terminal domains, the ``lid,'' and the side chains of Phe and Phe, which are the chymotryptic cleavage sites, are indicated.



Using the monolayer technique, it has been established that HPL, in the absence of bile salt and colipase, can efficiently hydrolyze water-insoluble monomolecular films of diacylglycerols(10) . This means that in the presence of an interface, the opening of the lid (interfacial activation) in HPL does not require the presence of colipase or bile salts.

Monoclonal antibodies provide useful tools for investigating the structure-function relationships of proteins. This approach was previously found to be particularly fruitful in the case of colipase and human gastric lipase(11, 12, 13, 14) . We have now produced five mAbs against HPL and examined the effects of these antibodies on the catalytic activity in the presence and absence of bile salts and colipase. By combining immunoinactivation with epitope mapping techniques, we identified the structural regions of HPL that are responsible for catalysis and interfacial binding.


MATERIALS AND METHODS

Proteins

HPL devoid of colipase was purified from human pancreatic juice as described by De Caro et al.(15) . Porcine pancreatic colipase (in the form of procolipase) was a gift from Prof. L. Sarda (Laboratoire de Chimie Biologique, Marseille, France). The specific activities of pancreatic lipase and colipase were measured titrimetrically using standard assays previously described by Gargouri et al.(16) and Rathelot et al.(17) and were estimated to be 8000 and 10,000 units mg, respectively. Bovine serum albumin and anti-mouse immunoglobulin G conjugated to peroxidase or alkaline phosphatase were from Sigma.

Protein Determination

The concentrations of solutions of purified HPL, porcine colipase, and antibodies (IgG isotype) were estimated spectrophotometrically at 280 nm using absorbance coefficients (A) of 13.3, 3.6, and 14, respectively.

Production of Anti-HPL Polyclonal Antibodies

Rabbits were injected subcutaneously and intramuscularly every 3 weeks with 0.5-1 mg of HPL. The first injection included complete Freund's adjuvant, while the last three injections contained incomplete adjuvant. The sera were tested to detect any anti-HPL reactivities by performing ELISA and immunoblotting assays. Finally, the rabbit anti-HPL pAbs were purified on a column of immobilized HPL. For this purpose, 13 mg of HPL were bound to Affi-Gel 10 (Bio-Rad) using the same procedure as that described by Aoubala et al.(18) .

Production of Anti-HPL mAbs

Three young female BALB/c mice were immunized by performing subcutaneous injections of pure native HPL. The first two injections were carried out with 50 µg of HPL emulsified in complete Freund's adjuvant and the third with 120 µg of HPL in 10 mM Na(2)HPO(4)/KH(2)PO(4) (pH 7.4) containing 150 mM NaCl (PBS). The immunizations were carried out at 3-week intervals. Two weeks after the last injection, the anti-HPL specificity of the sera was tested by performing a simple sandwich ELISA. Three days before the fusion, the selected mouse was given a boost immunization with 50 µg of HPL. Fusion was performed as described by Köhler and Milstein(19) . The spleen cells from immunized mouse were fused with X63-Ag8.653 myeloma cells, and cloning of selected hybridomas was achieved by means of the limited dilution technique. The culture supernatants were screened using the ELISA and Western blotting methods described below. For ascites production, 2.5 times 10^6 hybridoma cells were injected intraperitoneally into BALB/c mice. mAbs were purified from mouse ascitic fluids by precipitation with 50% saturated ammonium sulfate followed by affinity chromatography using either immobilized HPL bound to Affi-Gel 10 or protein A-Sepharose CL-4B (Pharmacia Biotech Inc.).

Isotyping of selected mAbs was carried out with a culture supernatant of each clone using the Amersham isotyping kit. The protocol was set up according to the manufacturer's instructions.

ELISAs for Screening Anti-HPL mAbs

The hybridomas producing specific anti-HPL antibodies were screened by performing solid-phase immunoassays using microtiter polyvinyl chloride plates (Maxisorb, Nunc). Two ELISA methods were developed for this study. First, a simple sandwich ELISA was carried out by coating the plates with 500 ng of pure HPL/well using an identical procedure to that previously described by Aoubala et al.(13) . Second, a double sandwich ELISA was performed by coating the plates with 500 ng of pure anti-HPL pAb/well (captor antibody). After saturation of the remaining free sites with bovine serum albumin, HPL was added to the PVC-coated pAb (500 ng/well), which, unlike the simple sandwich ELISA, results in random orientation of the various epitopic regions of the antigen.

Gel Electrophoresis and Western Blot Analysis

Electrophoresis in the presence and absence of SDS was carried out in a Bio-Rad Mini Protein II dual vertical slab gel electrophoresis cell on 10-12% polyacrylamide as described by Laemmli (20) . The immunoreactivity of each mAb with HPL was also studied using the Western blotting technique. Proteins were transferred to a nitrocellulose membrane either electrophoretically from the SDS-polyacrylamide gel or by passive diffusion from native polyacrylamide gels using the procedure described by Gershoni and Palade(21) . After the transfer, nitrocellulose membranes were rinsed three times with PBS. To block the remaining free sites, the blots were incubated with a solution of PBS containing 3% skimmed milk (commercial grade) for 1 h at room temperature. Thereafter, culture hybridoma supernatants or purified mAbs in PBS containing 0.05% Tween 20 were incubated individually for 1 h at room temperature. The strips were rinsed three times with PBS/Tween-20 and incubated for 1 h at room temperature with a 1:2000 dilution of alkaline phosphatase conjugated with goat anti-mouse immunoglobulin (Sigma). The blots were washed three times with PBS/Tween-20 and two times with 0.1 M Tris-HCl (pH 9.5) containing 0.1 M NaCl, and the specific immunoreactivity was revealed with the substrate solution of the alkaline phosphatase (0.1 M Tris-HCl (pH 9.5) containing 0.1 M NaCl, 1 mM MgCl(2), 5 mM 5-bromo-4-chloro-3-indolyl phosphate, and 3.5 mM nitro blue tetrazolium).

mAb Affinity Constants for HPL

The measurement of the dissociation constants of each HPLbulletmAb complex was carried out with an ELISA as described by Friguet et al.(22) . Briefly, various concentrations of HPL (ranging from 2.10 to 3.10M) were first incubated in solution with a constant concentration of each mAb (2.10M) until equilibrium was reached. The concentration of free antibody was then determined by performing a simple sandwich ELISA as described above.

HPL Immunoinactivation Studies

To study the ability of each mAb to inhibit HPL lipolytic activity, a fixed amount of enzyme (10-20 µg) was incubated with each mAb at various molar ratios. Incubations were performed in PBS (70-µl final volume) for 1 h at 37 °C. The residual activity of the HPLbulletmAb complexes was measured titrimetrically at pH 8 and 37 °C using the pH-stat method in two different assays. In the first assay (see Fig. 3A), the lipase activity was measured using trioctanoin as substrate without any bile salts or colipase. 0.5 ml of pure trioctanoin (Sigma) was emulsified in 150 mM NaCl to give a final volume of 15 ml. In the second assay (see Fig. 3B), the lipolytic activity of HPL was colipase-dependent in the presence of bile salts. 0.5 ml of pure trioctanoin was emulsified in a solution of 0.3 mM Tris-HCl (pH 8) containing 150 mM NaCl, 1.6 mM CaCl(2), and 5 mM sodium taurodeoxycholate (Sigma) to give a final volume of 15 ml. Colipase was added in a 2-fold molar excess. Another immunoinactivation assay was carried out by preincubating HPL and colipase (1:2 molar ratio) in the presence of sodium taurodeoxycholate (2 mM) and egg phosphatidylcholine (1 mM). After 2 h of incubation at 37 °C, mAbs were added (mAb/HPL molar ratio of 1:1) for an additional 1 h of incubation. The residual activity was measured on trioctanoin in the presence of bile salts as described above.


Figure 3: Effects of mAbs on HPL catalytic activity. The residual activity of the complexes was measured on trioctanoin (TC8) in the absence (A) and presence (B) of bile salts (NaTDC) and colipase. For details, see ``Materials and Methods.''



Epitope Mapping

Epitope mapping studies with native and SDS-denatured HPLs were performed using three different methods. In the first experiment, the epitope specificity of anti-HPL mAbs was first established using the ELISA additivity method described by Friguet et al.(23) . Briefly, microtiter plates were coated with 2.5 ng of native HPL/well, and mAbs were cotitrated in pairs at a molar ratio of 1:1. The ELISA procedure was performed as described previously by Aoubala et al.(13) .

In the second experiment, anti-HPL Fab fragments were prepared from each mAb as described by Parham(24) , and FabbulletHPL complexes were formed by incubating HPL and Fab at a 1:1 molar ratio for 1 h at 37 °C in PBS. FabbulletHPL ternary complexes were formed by mixing HPL with pairs of Fab fragments (HPL/Fab1/Fab2 molar ratio of 1:1:1). The binary and ternary complexes were analyzed by gel filtration chromatography as described by Rugani et al.(25) . Briefly, samples of the binary complex (FabbulletHPL) mixture (0.1 nmol/0.1 nmol) and ternary complex (Fab1bulletHPLbulletFab2) mixture (0.1 nmol/0.1 nmol/0.1 nmol) were applied to a Beckman Spherogel column (7.5 times 600 mm) equilibrated with 0.2 M Na(2)HPO(4)/NaH(2)PO(4) buffer (pH 6.8) and coupled to a Beckman TSK precolumn (7.5 times 7.5 mm). Protein elution was carried out with the same buffer at a flow rate of 1 ml/min and monitored at 230 nm. The retention times (in minutes) of the binary and ternary complexes were determined from the separation chromatographic profile as described by Rugani et al.(25) .

In the third experiment, an epitope mapping study was also performed using the peptide mapping method described by Wilson and Smith(26) . The epitopes within specific HPL fragments obtained by limited chymotryptic cleavage were located by Western blotting as described above. Briefly, HPL was subjected to limited digestion with chymotrypsin as described by Abousalham et al.(6) , and the products were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose or Glassybond membranes for Western blotting and N-terminal amino acid sequence analysis(27) , respectively. Automated Edman degradation of the protein was performed using an Applied Biosystems Model 470A gas-phase sequencer. The phenylthiohydantoin-derivatives were identified by HPLC on a Brownlee PTH-C(18) column (2.1 times 220 mm; Beckman Instruments) and were quantified by means of an integration program on a Waters 840 data control station.


RESULTS

Production and Screening of Anti-HPL mAbs

To produce mAbs against functional regions of HPL, the native enzyme was used as antigen. One successful fusion was required to obtain the five anti-HPL producing cell lines of interest. Hybridoma cells secreting specific mAbs were screened by performing two different ELISAs with native HPL and by Western blotting with native and SDS-denatured enzymes. The clones selected and the class of antibodies secreted were as follows: 81-23, 146-40, 315-25, and 320-24, which are of the IgG1 isotype, and 248-31, which is of the IgG2b isotype. The double sandwich ELISA procedure was used to select clone 248-31, while the other clones were selected by performing a simple sandwich ELISA. The results of the Western blot analysis showed that mAbs 81-23, 146-40, 315-25, and 320-24, reacting with the HPL directly adsorbed to the PVC plates, recognized both native and SDS-denatured HPLs, whereas mAb 248-31, which did not react with the HPL adsorbed to the PVC plates, recognized only native HPL.

Immunoreactivity of Purified mAbs with Native and Denatured HPLs Determined by ELISA and Western Blot Analysis

The titration curves given in Fig. 2indicate that all five mAbs showed different reactivities with HPL, whether it was directly adsorbed to the PVC plate (Fig. 2A) or indirectly fixed through the pAb previously adsorbed to the same plate (Fig. 2B). In all the cases studied, the maximal signal obtained with increasing mAb concentrations occurred at concentrations above 0.3 µg/ml (15 ng/well), which corresponds to the saturation of all the accessible epitopes. Fig. 2A also shows that mAb 248-31 does not recognize PVC-adsorbed HPL, as previously observed during the screening procedure. The reactivity of each mAb was also studied in a double sandwich ELISA in which a specific pAb was first adsorbed to the PVC plates. The latter procedure results in the random orientation of HPL. As expected (Fig. 2B), the reactivity of each mAb was higher than previously observed on the basis of the simple sandwich ELISA (Fig. 2A). It is worth noting that mAb 248-31 reacted with HPL only under these conditions. Furthermore, the optical density signals observed with mAb 248-31 were proportional to the amount of HPL added (up to 10 ng/well) in the double sandwich ELISA, whereas no significant signal was detected with the simple sandwich ELISA (data not shown).


Figure 2: Titration curves of the five anti-HPL mAbs. A, immunoreactivity of mAbs with adsorbed HPL in a simple sandwich ELISA; B, immunoreactivity of mAbs with HPL in a double sandwich ELISA. In this case, the plate was first coated with anti-HPL pAb. For details, see ``Materials and Methods.''



The interactions of each mAb with native and SDS-denatured HPLs were also studied using the Western blotting technique. Table 1shows that four mAbs (81-23, 146-40, 315-25, and 320-24) reacted with both native and SDS-denatured HPLs, while mAb 248-31 recognized only native HPL. The values of the dissociation constants determined with the native HPLbulletmAb complexes in solution, using the ELISA procedure described by Friguet et al.(22) , are also given in Table 1.



Effects of mAbs on the Catalytic Activity of HPL

The potentially inhibitory effects of the five mAbs on the lipolytic activity of HPL were tested. The results given in Fig. 3show that four mAbs (81-23, 248-31, 315-25, and 320-24) reduced the hydrolysis of trioctanoin by HPL, while mAb 146-40 had no effect. It is worth noting that comparable inhibition levels were observed in both the presence (Fig. 3B) and absence (Fig. 3A) of colipase and bile salts. We also observed similar inhibitory levels using mAbs or their respective Fab fragments (data not shown).

To investigate the effects of the conformational changes induced by an interface (lid opening) upon the immunoreactivity of HPL with inhibitory mAbs, experiments were carried out to test various mAbs, or their corresponding Fab fragments, with HPL and colipase previously incubated in the presence of mixed bile salt-phospholipid micelles. Under these conditions, identical levels of HPL immunoinactivation were observed (data not shown).

Mapping of Epitopes on the Surface of Native HPL

To test whether the anti-HPL mAbs each recognized different epitopes on HPL, the ELISA double antibody binding test (ELISA additivity test) developed by Friguet et al.(23) was used. Competition between two antibodies for the HPL adsorbed to the PVC plate was expressed by means of ELISA additivity indexes (AI(e)) using the following formula: AI(e) = ((2A/(A(1) + A(2))) - 1) times 100, where A is the absorbance obtained in the ELISA with the mixture of two mAbs at a 1:1 molar ratio, and A(1) and A(2) are the absorbances obtained with each mAb, respectively. This index (expressed as a percentage) makes it possible to evaluate the simultaneous binding of two mAbs to the antigen. Four mAbs (81-23, 146-40, 315-25, and 320-24) were studied in all the possible pairs, and the AI(e) values are given in Table 2. Unfortunately, mAb 248-31 could not be tested because of its lack of immunoreactivity with HPL adsorbed to PVC plates (Fig. 2A). Based on the values of the additivity index given by the ELISA (see above the diagonal in Table 2), these mAbs can be classified into two groups of antibodies directed against two different antigenic determinants: Group I (AI(e) < 50%) includes three mAbs (81-23, 315-25, and 320-24), and Group II (AI(e) > 50%) consists of mAb 146-40.



Another strategy employed recently in epitope mapping studies by Rugani et al.(25) involved incubating the antigen with all the possible pairs of Fab fragments prepared from each mAb. The Fab-antigen complexes were then separated by gel filtration chromatography, and the retention times of binary and ternary complexes were characterized (see below the diagonal in Table 2). In practical terms, this amounts to assessing the molecular size of the Fab-antigen complexes formed. To normalize the results of the gel filtration chromatography, a new additivity index (AI(c)) has been defined for a pair of Fab fragments as follows: AI(c) = ((T(1) - T(x))/(T(1) - T(2))) times 100, where T(x) is the retention time for a given pair of Fab fragments, T(2) is the minimal retention time of a ternary complex (HPL and two Fab fragments), and T(1) is the maximal retention time of a binary complex (HPL and one Fab fragment). If the two Fab fragments bind to the same site, T(x) will be equal to T(1), and AI(c) will be equal to zero. If, on the contrary, the two Fab fragments bind independently at separate sites, T(x) will be equal to T(2), and AI(c) will be equal to 100%. Using this new index (see data below the diagonal in Table 2), we confirmed the previous classification of four mAbs into two groups (I and II) based on the ELISA additivity test of Friguet et al.(23) . Furthermore, with this separate chromatographic method, it was possible to identify mAb 248-31 as constituting a distinct group (III) since it forms ternary complexes (AI(c) > 50) with HPL and Fab fragments belonging to either Group I (mAbs 81-23, 315-25, and 320-24) or Group II (mAb 146-40).

Mapping of Epitopes along the Primary Sequence of HPL

To carry out epitope mapping along the primary sequence of HPL, four fragments resulting from the limited chymotryptic cleavage of HPL (14, 26, 30, and 36 kDa) were characterized by Western blotting (Fig. 4) and N-terminal amino acid sequence analysis. Based on the latter analysis, these fragments were located along the primary amino acid sequence of HPL (Fig. 5). However, from the known amino acid sequence of HPL, as shown in Fig. 5, a fifth peptide of 12 kDa (Pro-Phe) is expected to be generated by the chymotryptic cleavage of the lipase at the two cleavage sites (Phe and Phe). In fact, this peptide contains 13 other potential chymotryptic cleavage sites (Phe and Tyr). This suggests that this peptide (Pro-Phe) is highly sensitive to chymotryptic proteolysis, which might explain why we did not detect it using either SDS-PAGE or the Western blotting technique.


Figure 4: Immunoreactivity of HPL fragments produced by limited chymotryptic cleavage. HPL was subjected to limited digestion with chymotrypsin(6) , and the products were separated by SDS-PAGE. The molecular masses of the peptides were estimated by a linear interpolation from a semilogarithmic plot of the molecular masses of the markers versus their relative migration. Lane1, uncleaved HPL revealed by the pAb or mAb; lanes 2-6, immunoreactivity of HPL cleavage peptides with pAb and mAbs 146-40, 81-23, 315-25, and 320-24, respectively. The migration of the molecular mass markers is indicated by arrows on the right.




Figure 5: Diagram of the primary sequence of HPL. The arrows show the chymotryptic cleavage sites (ChT). The N-terminal domain (emptyframe) as well as the C-terminal domain (dottedframe) are indicated. The various HPL fragments are labeled with their corresponding experimental molecular masses (14, 26, 30, and 36 kDa). The dashedline indicates the undetermined C-terminal end of the 36-kDa fragment. Sequenced peptides, determined using the Edman degradation technique, are indicated in boldfaceletters, and those deduced from the cDNA sequence are indicated in italicletters. Numbering in parentheses has been used to indicate the mAbs that recognized their corresponding fragments. The approximate location of the hinge region is indicated. CHO is the carbohydrate moiety linked to Asn.



As shown in Fig. 4, the anti-HPL pAbs reacted with the four HPL fragments (lane2), while individual mAbs exhibited differential staining patterns. mAb 146-40 reacted with the 30- and 36-kDa fragments (lane3). These two fragments did not include the C-terminal domain of HPL (14-kDa fragment from Ala to Cys; see Fig. 5). These results indicate that the epitope recognized by mAb 146-40 is present in the N-terminal domain of the enzyme (Lys^1-Phe). On the other hand, mAbs 81-23, 315-25, and 320-24 react only with the 26-kDa fragment (Fig. 4, lanes 4-6), which includes the entire C-terminal domain of HPL (Ala-Cys) and part of the N-terminal domain of HPL (Pro-Phe) ( Fig. 1and Fig. 5). The latter mAbs do not immunoreact, however, with either the C-terminal domain generated by the chymotryptic cleavage at Phe-Ala or the chymotryptic fragments of the N-terminal domain of HPL (30 and 36 kDa).


DISCUSSION

An obvious advantage of mAbs as compared with polyclonal antisera is the selectivity with which they interact with the antigen. When the antigen is a multifunctional protein such as a lipase, mAbs provide a useful approach for correlating specific structural characteristics with functional features of the protein. The aim of this study was to produce mAbs that specifically recognize native HPL. These antibodies are of special interest since they can be used to identify the various regions of the lipase molecule involved in the hydrolysis of lipid substrates and are complementary to the three-dimensional structure of HPL. Five mAbs against native HPL were produced, and their interactions with the native and SDS-denatured enzymes as well as their effects on the lipolytic activity of HPL were investigated. Four mAbs (81-23, 146-40, 315-25, and 320-24) were found to react with the HPL adsorbed to the PVC plate (simple sandwich ELISA), while mAb 248-31 did not react. All five antibodies interacted, however, with HPL in the double sandwich ELISA. These results can be explained by the fact that the epitope recognized by mAb 248-31 is in a hydrophobic region adsorbed to the PVC plate and is therefore not accessible to the antibody. An alternative explanation might be that a conformational change (denaturation) may have occurred during HPL adsorption to the PVC plate, resulting in the loss of the recognition site. In the case of proteins with functional hydrophobic regions such as lipolytic enzymes, the double sandwich ELISA involving specific polyclonal antibodies adsorbed as the first layer on PVC plates therefore yields randomly oriented antigenic regions and preserves the second antibody recognition.

As regards the effects of the five mAbs on the lipolytic activity of HPL, it was observed that four mAbs (81-23, 248-31, 315-25, and 320-24) had inhibitory effects on the activity of the enzyme under standard assay conditions in both the presence and absence of bile salts and colipase (Fig. 3). It is worth noting that the presence of bile salts and phospholipid did not significantly affect the antigenic recognition, probably because the affinity of HPL for mAb is higher than that of lipids. This situation is highly favorable for ELISA determination of HPL under in vivo conditions(3, 18) .

To localize epitopes within the structure of the lipase, three epitope mapping procedures were carried out. From the two additivity tests, it was concluded that the five mAbs can be assigned to three different groups of antibodies that are directed against three different antigenic regions on HPL. Group I contains three mAbs (81-23, 315-25, and 320-24) that recognize closely overlapping epitopes in the same antigenic region; Group II contains mAb 146-40; and Group III contains mAb 248-31. These results were complemented with peptide mapping data and served to localize two antigenic regions in the primary sequence of HPL that are recognized by two of the three groups of antibodies. mAb 146-40, which does not inhibit HPL, recognized the N-terminal domain of HPL, and its epitope is located between the N-terminal residue and residue 227. Since this region contains the catalytic Ser, which is covered by the lid, this mAb is probably directed toward a site that is distinct from the active center of the enzyme. As shown in Fig. 5, the three mAbs (81-23, 315-25, and 320-24) that inhibit lipolytic activity reacted only with the 26-kDa HPL fragment containing part of the N-terminal domain (Pro-Phe) and the entire C-terminal domain (Ala-Cys). The lack of immunoreactivity of the above antibodies against the C-terminal domain (14 kDa) and against the 36-kDa peptide suggests two possibilities. (i) The antigenic region of HPL, which was recognized by the three inhibitory mAbs, might be cleaved by chymotrypsin at the bond between Phe and Ala, inducing a loss of these antibodies' immunoreactivity against the two separated domains of HPL; or (ii) the 36-kDa peptide might result from a chymotryptic cleavage at the C-terminal end of the N-terminal domain of HPL (Lys^1-Phe). Since HPL is a glycoprotein having a carbohydrate moiety with a molecular mass of 3 kDa (15) that is linked to Asn(28) , it is likely that the N-terminal domain of HPL may be truncated at its C-terminal end, to the extent of 20-30 amino acid residues. This truncation is located at the hinge region and probably contains the epitopes recognized by the three inhibitory mAbs (Fig. 5). In both cases, the above results clearly indicate that these mAbs recognized closely overlapping epitopes that were included in the hinge region between the N- and C-terminal domains of HPL. Fig. 1is a schematic diagram of the three-dimensional structure of HPL indicating the location of the two chymotryptic cleavage sites. From this schematic three-dimensional structure, the accessibility of the two cleavage sites (Phe and Phe) might explain the loss of immunoreactivity of the three inhibitory mAbs toward the three characterized proteolytic fragments of HPL (36, 30, and 14 kDa). The results of the immunoinactivation and epitope mapping studies support the hypothesis that the binding of mAbs 81-23, 315-25, and 320-24 to native HPL, in both the presence and absence of a substrate, affects the essential rearrangement of the enzyme recently described in the context of studies on the three-dimensional structure of the HPL-procolipase-phospholipid complex(9) . It has been demonstrated that the open lid is stabilized by many new hydrogen bonds and salt bridges, partly with the core of the protein and partly with the N-terminal region of procolipase. When mAbs, or their respective Fab fragments, interact with the hinge region in the absence of the substrate, they may inhibit all the conformational changes in the lid that are essential for the interfacial activation to occur. It is worth noting that in the case of inhibitory Fab fragments that are as large as the HPL molecule, steric hindrance may be a critical step in the expression of lipase activity when the FabbulletHPL complexes are formed.

Previously published results of in vitro studies have shown, however, that HPL can hydrolyze acylglycerides in the absence of bile salts and colipase. This fact supports the idea that the movement of the lid also occurs without any colipase. The present immunoinactivation and epitope mapping data suggest that the movement around the hinge region between the N- and C-terminal domains of HPL is indeed important for the proper locking of the lid, which is necessary to the positioning of the functional catalytic machinery. This conclusion is in good agreement with crystallographic data showing that a rigid body rotation occurs around the hinge region, resulting in a shift of the lipase C-terminal domain toward the catalytic N-terminal domain(9) . The three mAbs 81-23, 315-25, and 320-24 provide potentially useful tools for future studies on the essential rearrangement occurring during the interfacial activation and the movement of the lid upon the adsorption of the enzyme to the water/lipid interface.


FOOTNOTES

*
This work was supported by the BRIDGE-T-lipase Program of the European Communities under Contract BIOT-CT910274 (DTEE) and by BIOTECH G-Program BIO 2-CT94-3041. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-91-16-44-88; Fax: 33-91-71-58-57.

(^1)
The abbreviations used are: HPL, human pancreatic lipase; mAb, monoclonal antibody; pAb, polyclonal antibody; ELISA, enzyme-linked immunosorbent assay; PVC, polyvinyl chloride; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. N. Rugani and Prof. L. Sarda for permission to use the HPLC equipment at their laboratory. Our thanks are also due to J. Bonicel for performing the amino acid sequence analysis. Help by Dr. F. Carrière in modeling the HPL three-dimensional structure is acknowledged. English revisions by Drs. J. Blanc and R. Lehner are acknowledged.


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