Monoclonal antibodies to pancreatic stone protein. Radioimmunoassay and immunological comparison with trypsin 1

Biochhnie, 68 (1986) 1109- I 113 © Soci6t6de Chimie biologique/Etsevier,Paris

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Short communication

Monoclonal antibodies to pancreatic stone protein. Radioimmunoassay and immunological comparison with trypsin 1 M. P R O V A N S A L - C H E Y L A N I, M. L U S H E R 2, A. DE CARO l, L. M U L T I G N E R I, G. M O N T A L T O I, H. SARLES I, and M. D E L A A G E 2 t l N S E R M U31, 46, bd de la Gave, 13258 Marseille 9, and 2hnmunotech, Case 915, Luminy, 13288 Marseille, France (Received 22-1-1986, accepted after revision 16-4-1986)

S u m m a r y - Monoclonal antibodies were prepared against pancreatic stone protein, a protein which inhibits calcium carbonate precipitation. Two monoclonal antibodies designated D4 and 2E7 were characterized. Immunoadsorbant columns, obtained by linkage of these monoclonal antibodies to Affigel 10, have been used to isolate immunoreactive forms of pancreatic stone protein from nonactivated human pancreatic juice. These monoclonal antibodies permitted us to test the possible immunological relationship between pancreatic stone protein and human trypsin 1. No immunological similarity was found, in agreement with our previous results, and it was established that pancreatic stone protein is a novel protein and not a degradation product of human trypsin(ogen) 1. pancreatic stone prolein / pancreatic juice / monoclonal antibodies / trypsin 1 R~sum6 - A n t i e o r p s m o n o e l o n a u x dirig~s e o n l r e la prot~ine des calculs paner~atiques. Test r a d i o i m m u n o l o g i q u e et e o m p a r a i s o n i m m u n o l o g i q u e avee la lrypsine 1. Des attticorps monoclonattx ont dtd prdpards contre la protdine des calcttls pancrdatiqttes, ttne protdine qui inhibe la prdcipitation du carbonate de calcitnn. Deux anticorps monoclonattx, nonnnds D4 et 2E7, se sont rdvdlds ~tre caractdristiqttes. Des colonnes d'inmnmoadsorbants, obtenues par fixation de ces anticorps monoclonaux sur A ffigel 10, ont servi p o u r isoler les formes hnnnmordactives de la protdine des calcttls pancrdatiques prdsentes dans le sttc pancrdatique humain non activd. Ces anticorps monoclonattx /torts ont permis de rechercher tree dventttelle relation imnnmologique entre la prot~ine des calculs pancr~atiqttes et la trypsh:e humaine. A uctme identit~ imnnmologique n'a dt~ trouvde, en accord avec nos r~sultats ant&ieurs qui prouvent que la protdine des calculs pancrdatiques est bien une nonvelle protdine sdcrdtoire et non pas un prodttit de ddgradation de la trypsine 1 ott dtt trypsinogbne 1 hnntahzs. prot~ine des calculs pancr~atiqttes I sttc pancr~atiqtte I at:ticorps monoclonattx I trypsine 1

Introduction Chronic calcifying pancreatitis is characterized by the presence of calcium carbonate stones in pancreatic ducts [I]. In 1979, we reported that a major protein exists in human pancreatic stones [2], and named it accordingly, pancreatic stone protein (PSP). A detailed study of some molecular proper-

ties, including the complete amino acid composition, has shown that PSP is an acidic phosphoglycoprotein with an M r of 14000 [3]. Pancreatic stone protein has been found to be present in either normal or pathological pancreatic juice [2]. Its concentration in pancreatic juice rises in response to cholecystokinin-pancreozymin [4]. This is in agreement with the immunocytochemical localization of

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M. ProvansaI-Cheylan et al.

PSP within the zymogen granules and condensing vacuoles o f acinar cells f r o m normal pancreas [5]. PSP inhibits in vitro calcium carbonate nucleation and crystal growth, a property that is not possessed by other pancreatic secretory proteins [6.] This suggests that PSP may be important in stabilizing supersaturated pancreatic juice. Our observations that the PSP level in pancreatic juice is decreased in calcifying pancreatitis [4] support this idea. In two recent reports, Figarella and coworkers [7, 8] asserted that PSP (also identified by these authors as the so-called precipitate protein or protein X) was a degradation product of trypsin(ogen) 1. This assumption, based on immunological cross-reactivity found between PSP and their preparation of trypsin(ogen) I, is in contrast with our previous results [3]. Recently, we have shown that a m R N A coding for PSP, distinct from the m R N A coding for trypsinogen I, is present in normal h u m a n pancreas, which implies that PSP is a bona fide protein species [9]. In this study, we reinvestigated the question o f an immunological relationship between PSP and trypsin(ogen) 1, using two monoclonal antibodies raised against PSP.

Materials and methods Pure normal human pancreatic juice was collected by endoscopic pancreatic cannulation. Only samples devoid of free proteolytic activity, assessed by undetectable chymotryptic activity, were used. Pancreatic stone protein was isolated from human pancreatic stones after EDTA-demineralization and further purified as already described [3]. Diisopropylfluorophosphate treated human trypsin l (DP-trypsin 1) and rabbit anti DP-trypsin I serum were generous gifts from Dr. O. Guy-Crotte.

Monoclonal antibody OnAb) production and purification BALB/c mice were immunized with a crude extract from pancreatic stones containing at least 85070 PSP [10]. Four onemonth spaced injections of 80 ~g PSP were given subcutaneously. Eight months after the primary injection, a booster injection was given intravenously. Four days later, spleens were removed and spleen cells were fused with X-63 mouse myeloma cells [11]. Positive hybridoma supernates were selected by a solid phase radioimmunoassay using microplates coated with purified PSP and 1251labeled goat antiserum against mouse immunoglobulins. Secreting cells were then cloned and grown as ascitic tumors in BALB/c mice. Monoclonal antibodies were purified from ascitic fluids by protein A-Sepharose chromatography. The purity of mAbs was tested by fast liquid chromatography and by sodium dodecyl sulfate-poly-

acrylamide gel electrophoresis (SDS-PAGE). Determination of the class and subclass of the mAbs was performed by micro-Ouchterlony.

Isolation of PSP from nonactivated human pancreatic juice through use of mAbs Anti PSP mAb was covalently linked to Affigel 10 (Bio Rad). The immunoreactive gel was placed in a column and equilibrated with 150 mM phosphate buffered saline (PBS), pH 7.2, containing 3 mM benzamidine. Lyophilized pancreatic juice proteins were dissolved in PBS containing I mM phenylmethylsulfonylfluoride and added to the gel. After 3 h of incubation at 4°C the column was successively washed with PBS-3 mM benzamidine, 25 mM Tris-HCl, pH 7.5, containing 500 mM NaCI and 0.2070 (w/v) Triton X-t00, and PBS without benzamidine. When the absorbance at 280 nm of the effluents was at baseline, fixed proteins were eluted with 200 mM glycine-HCl, pH 2.8. Glycine fractions containing protein were neutralized by Tris-HCl, pH 8.9, then pooled and dialyzed against water. After lyophilization, PSP was stored at -20°C.

Iodination of proteins and radioinmnmoassay Pancreatic stone protein isolated from pancreatic juice and DP-trypsin 1 were labeled with ~251using a modification of the chloramine-T method in which free iodine was trapped by Gly-Tyr (M. Delaage, personal communication). The proteins were pretreated with 10 mM EDTA in PBS to minimize eventual complexed forms. The labeled proteins were separated from iodinated Gly-Tyr by Sephadex G-50 fine chromatography. Binding experiments and displacement curves were carried out in PBS containing bovine hemoglobin (1 g/I). Dilutions of antibodies were optimized in order to obtain appropriate binding. Antigen-antibody complexes were precipitated by the addition of 20°70 (w/v) PEG 6000. As controls, normal mouse serum (diluted l : 100) and an irrelevant mouse mAb were used.

Other methods SDS-15% PAGE were performed according to the method of Laemmli [12]. Proteins were reduced and alkylated with dithiothreitol and iodoacetamide as described [3]. Protein concentrations were determined by the Lowry method [13], using human serum albumin as the standard.

Results Two m A b s against PSP, designated D4 and 2E7, have been prepared. Both mAbs were identified as IgG I. Immunoreactive forms o f PSP were isolated from h u m a n nonactivated pancreatic juice using Affigel 10 bound m A b 2E7. S D S - P A G E o f the

Monoclonal antibodies to pancreatic stone protein

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eluted proteins led to the resolution o f five c o m ponents ( M r 14000, 16000, 16800, 18000 and 18 800). A similar pattern was obtained by affinity c h o m a t o g r a p h y using Affigel 10 b o u n d m A b D4 (Fig. 1 and [10]). The isolated P S P was employed in the following experiments.

Gel filtration of 125Llabeled protehls As can be seen in Fig. 2a, x2SI-PSP emerges as a symmetric peak consistent with a h o m o g e n e o u s protein sample. In contrast, the ~25I-DP-trypsin 1 elution peak has shoulders, suggesting a heterogeneous protein p o p u l a t i o n (Fig. 2b).

Binding data *-13 *-12.3 *-11

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Results o f cross-reactivity studies between P S P and DP-trypsin I are shown in Table I. Anti P S P m A b s D4 and 2E7 reacted with xzSI-PSP but there was no reactivity with ~2SI-DP-trypsin 1. O n the other hand, the rabbit anti DP-trypsin 1 serum reacted with 125I-DP-trypsin 1, as expected; and to a l e s -

4

a

Fig. 1. SDS-PAGE of the immunoreactive forms of PSP from human pancreatic juice. The four different samples used were eluted from mAb D4 immunoadsorbant (lanes l and 2), and from mAb 2E7 immunoadsorbant (lanes 3 and 4). Molecular weight markers are: 34.7K: pepsin (34700 M); 24K: bovine trypsinogen (24 000 M,); 13K and 11K: bovine chymotrypsin A (chain B 13000 M, and chain C 11000 31¢r) ; 12.3K: cytochrome c (12300/~,I,).

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Table I. Binding data of m I - D P trypsin and m I - P S P with respectively rabbit anti DP-trypsin 1 serum and anti PSP monoclonal antibodies D4 and 2E7. Antibody (dilution) Anti DP-trypsin 1 (1 : 100) Anti DP-trypsin 1 (1 : 1000) D4 mAb (1 : 100) 2E7 mAb (I : 100) Non specific binding

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Fig. 2. Elution profiles a: SZSl-PSP and b: ~z~I-DP trypsin I from gel filtration chromatography. (Amnes column: 45 × l cm ; Sephadex G-50 fine; flow rate: 9.6 ml/hr; elution buffer: PBS containing bovine hemoglobin (1 g/I), 10 mM Na azide).

M. Provansal-Cheylan et al.

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ser extent with 125I-PSP. A 10-fold decrease in the amount o f this antiserum decreases the binding o f 125I-DP-trypsin 1 about 15070, whereas the binding of 125I-PSP shows a drop o f about 42°7o. These results suggest that the rabbit anti DPtrypsin 1 serum contains additional antibodies against PSP.

Displacement curves Pancreatic stone protein and DP-trypsin 1 were tested for their abilities to inhibit the binding of 125I-PSP to mAb D4 and 2E7 (Fig. 3). When increasing amounts of unlabeled PSP were added to the reaction mixture, the capacity to competitively inhibit the binding of 125I-PSP started at 1/zg.ml-l of PSP. The bound tracer reached the level o f non specific binding with PSP concentrations o f more than 25 tzg.ml-1 In contrast, DP-trypsin 1 did not decrease 125I-PSP binding by more than 6070, even for concentrations exceeding 100 t~g.ml-1.

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Fig. 3. Displacementcurves of n~I-PSP bound to mAb D4 (a) and mAb 2E7 (b), in the presence of increasing quantities of competing Iigands: PSP (o), DP-trypsin i (e).

Discussion In order to clarify the great confusion caused by the various terminologies encountered in the literature, it should be specified that: a) pancreatic stone protein (PSP) is a 14000 M r polypeptide initially discovered in the pancreatic stones of patients suffering from chronic calcifying pancreatitis [2] ; b) precipitate protein is a 14 000 M r polypeptide isolated from intraductal pancreatic plugs (or tiny stones) obtained from the same group o f patients. This protein presents complete immunological identity with PSP [14]; c) normal exocrine pancreatic secretion contains an array o f immunoreactive forms o f PSP having

MrS of 14000, 16000, 16800, 18000, and 18800 [10]; d) the so-called protein X corresponds to a 14 000 M r polypeptide present in normal pancreatic secretion and is immunologically identical to the protein extracted from pancreatic plugs (precipitate protein) [71. All these above mentioned proteins were recognized by the same monospecific polyclonal anti PSP antibody [7, 10, 14]. Using this antibody, only one mRNA coding for PSP was detected in human pancreas. Immunoprecipitation of translation products o f poly A + RNA yielded a single product with an M r of 16000, as expected for pre-PSP [9]. This supports that all the 14 000 M r polypeptides mentioned above are the same and unique molecular entity. We believe that the higher M r forms secreted in pancreatic juice are related to post-translational modifications, such as glycosylation. Moreover, all these immunoreactive 1 4 0 0 0 M r polypeptides, as well as the higher M r forms, were recognized by our t w o anti PSP mAbs D4 and 2E7. Since we previously reported that no immunological identity existed between PSP and any other human pancreatic secretory protein [3], our aim in this study was to reinvestigate these data by the use of anti PSP mAbs. And since "protein X was found to be immunologically identical to the protein extracted from precipitate.., a n d shown to be a degradation product of trypsinogen 1" [7], we especially investigated a possible relationship between PSP and trypsin(ogen) 1. In this paper, we confirm that there is no immunological c r o s s - r e a c t i o n between P S P and trypsin(ogen) 1. Using our two distinct anti PSP mAbs D4 and 2E7, we cannot reveal a common epitope between PSP and DP-trypsin I. Anti PSP mAbs do not present any reactivity with 125I-DP-trypsin 1 and DP-trypsin 1 is unable to inhibit the binding o f 125I-PSP to mAbs D4 and 2E7. However, the presence in the rabbit anti DP-trypsin 1 serum o f a slight binding activity for 125I-PSP could be due to PSP contamination o f the DP-trypsin 1 preparation used as antigen. This is not surprising, since we have routinely observed in our laboratory that pure trypsinogen 1 or trypsin 1 was very difficult to obtain without the presence o f low molecular weight contaminants. These data underline once again the individuality o f PSP and demonstrate that PSP is a previously unrecognized secretory protein distinct from any other pancreatic secretory protein. Overall, PSP appears clearly not to be a degradation product o f trypsin(ogen) 1.

Monoclonal antibodies to pancreatic stone protein

References 1 Sarles H. (1974) Gastroenterology 66, 604-616 2 De Caro A., Lohse J., Sarles H. (1979) Biochenl. Biophys. Res. Commtm. 87, 1176-1182 3 De Caro A., Multigner L., Lafont H., Lombardo D., Sarles H. (1984) Biochenl. J. 222, 669-677 4 Multigner L., Sarles H., Lombardo D., De Caro A. (1985) Gastroenterology 89, 387-391 5 Lech~ne de la Porte P., De Caro A., Lafont H., Multigner L., Sarles H. (1984) Digestion 30, 81 6 Multigner L., De Caro A., Lombardo D., Campese D., Sarles H. (1983) Biochem. Biophys. Res. Comntttn. 110, 69-74 7 Guy-Crotte O., Amouric M., Figarella C. (1984) Biochem. Biophys. Res. Comntttn. 125, 516-523

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8 Figarella C., Amouric M., Guy-Crotte O. (1984) Biochem. Biophys. Res. Commun. 118, 154-161 9 Giorgi D., Bernard J.P., De Caro A., Multigner L., Lapointe R., Sarles H., Dagorn J.C. (1985) Gastroenterology 89, 381-386 10 Montalto G., Lusher M., De Caro A., Multigner L., Sarles H., Delaage M. (1985) C.R. Acad. Sci. Paris t300, Ser. III, 6, 199-202 I 1 K6hler G., Milstein C. (1975) Nature (London) 256, 495-497 12 Laemmli U.K., (1970) Nature (London) 227, 680-685 13 Lowry O.H., Rosebroug N.J., Farr A.L., Randall R.J. (1951) J. BioL Chem. 193,265-271 14 Guy O., Robles-Diaz G., Adrich Z., Sahel J., Sarles H. (1983) Gastroenterology 84, 102-107