(Received for publication, August 25, 1994; and in revised form, November 15, 1994)
From the
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-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.
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) ()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) . -Strands are
indicated by arrows, and
-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.
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.
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.''
In the
second experiment, anti-HPL Fab fragments were prepared from each mAb
as described by Parham(24) , and FabHPL complexes were
formed by incubating HPL and Fab at a 1:1 molar ratio for 1 h at 37
°C in PBS. Fab
HPL 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 (Fab
HPL) mixture (0.1
nmol/0.1 nmol) and ternary complex (Fab1
HPL
Fab2) mixture
(0.1 nmol/0.1 nmol/0.1 nmol) were applied to a Beckman Spherogel column
(7.5
600 mm) equilibrated with 0.2 M Na
HPO
/NaH
PO
buffer
(pH 6.8) and coupled to a Beckman TSK precolumn (7.5
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 column (2.1
220 mm; Beckman
Instruments) and were quantified by means of an integration program on
a Waters 840 data control station.
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 HPLmAb
complexes in solution, using the ELISA procedure described by Friguet et al.(22) , are also given in Table 1.
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).
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) has been defined for a pair of Fab fragments
as follows: AI
= ((T
- T
)/(T
- T
))
100, where T
is
the retention time for a given pair of Fab fragments, T
is the minimal retention time of a ternary complex (HPL and two
Fab fragments), and T
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
will be equal
to T
, and AI
will be equal to
zero. If, on the contrary, the two Fab fragments bind independently at
separate sites, T
will be equal to T
, and AI
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
> 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).
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
-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).
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
-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 Fab
HPL 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.