Messenger RNA Sequence and Expression of Rat ... - Semantic Scholar

Vol. 266,No . 36,Issue of December 25, PP. 24664-24669,1991 Printed in U. S. A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biolow, Inc.

Messenger RNA Sequence and Expressionof Rat Pancreatitisassociated Protein, a Lectin-related Protein Overexpressed during Acute ExperimentalPancreatitis* (Received for publication, March 25, 1991, and in revised form, August 6, 1991)

Juan IovannaS, Beatrice Orelle, Volker KeimpT, and Jean-CharlesDagornll From the Unite 315 Institut National de la Sante et de la Recherche Medicale, 46 bouleuard de la Gaye, F-13009 Marseille, France and the § Uniuersitat Heidelberg, I I Medizinische Klinik, 0-6800 Mannheim, Federal Republic of Germany

Rat pancreatitis-associated protein (PAP) is an adThis observation prompted several investigators to look at ditional protein appearing in pancreatic juice after whether alteration of pancreatic function would beassociated induction of prancreatic inflammation. Its messenger with an abnormal distribution of secretory proteins, in the RNA was cloned and sequenced from pancreas. The hope of finding markers of pancreatic diseases. In addition, deduced amino acid sequence revealed that P A P was characterizing secretory products specifically altered in the synthetized as a preprotein with, inits mature form,a diseased pancreas was expected to provide insight into the predicted molecular weight of 16,630. A search in mechanism leading to pancreatic dysfunction. Chronic calciprotein data bases revealed a marked homology with fying pancreatitis was studied extensively in that respect. The the carbohydrate binding region of animal lectins; no disease was associated with an abnormal distributionof tryphemagglutination activitycould be shownfor PAP, but sinogens ( 5 ) , the presence of lysosomal enzymes in juice (6), the protein induced extensive bacterial aggregation. In healthy rats, the verylow level of P A P expression and a decreased content in pancreatic stone protein (7). By in pancreas could be increased up to 4-fold by physio- contrast, thereis little information on the patternof secretory logical stimuli suchas chronic hormonal or cholinergicproteinsduring acute pancreatitis although the disease is stimulation of pancreatic secretion and adaptation of much more frequent than chronic pancreatitisandhasa rats to a carbohydrate-rich diet. By contrast, induction generally worse prognosis. This is partly because of the bad of acute experimental pancreatitis by retrograde injec- condition of patients suffering from acute pancreatitis,which tion of sodium taurocholate resulted in dramatic over- forbids any attemptof juice collection. Studies have therefore expression. Pancreatic concentration of P A P mRNA relied on different animal models of experimental acute panincreased more than 300 X within 12 h whereas con- creatitis (8).We have shown in the rat (9, 10) that the most centrations of mRNAs encoding major secretory pro- important difference in the pattern of juice proteins was the teins such as amylase decreased. P A P overexpression presence, after induction of pancreatitis, of an additional 2 days of the acute phaseand then protein of about 14 kDa. It was called the “pancreatitispersisted during the returned to the control level during pancreatic recov- associated protein” (PAP).’At the climax of the acute phase, ery. PAP mRNA could not be evidenced inliver, stom- PAP represents about 5% of secreted protein and returns to ach, salivary glands, brain, kidney, or testis. Its pat- undetectable levels when the pancreas has totally recovered tern of expression during severe pancreatic aggression (10). PAP overexpression was observed when pancreatitis was suggests that it might be a stress protein involved in induced by retrograde injection of bile acids (9) or by suprathe control of bacterial proliferation. maximal stimulation by caerulein (ll), suggesting thatit might be a marker of pancreatic inflammation. We report here the cloning and sequencing of its mRNA, from which was deduced the primary structure of the protein andin which In mammals, pancreatic exocrine secretion contains about was found striking homology with animal lectins. Monitoring 20 major secretory products, most of them digestive enzymes PAP gene expression revealed remarkable stimulation after (1). Proportions among them may vary when the pancreas induction of pancreatitis. adapts toa change in diet composition (2) or upon stimulation EXPERIMENTALPROCEDURES by gastrointestinal hormones (3, 4), showing that expression of the corresponding genes is not constitutive but controlled Cloning and Sequencing Rat PAP cDNA-A rat pancreatic cDNA library in the expression vector Xgtll containing 7.6 X lo5 different by nutritional and hormonal factors.

* Presented in part at the Annual Meeting of the American Gas-

recombinant cloneswas obtained from Clontech (PaloAlto, CA). The library was screened with purified polyclonal rabbit IgG for rat PAP. Their specificity was controlled by immunodetection of pancreatic proteinsafterseparation by double-dimension gel electrophoresis (data not shown). Immunoscreeningof the library was carried out as described by Huynh et al. (12) using the Protoblot immunoscreening kit (Promega Biotec, Madison, WI). Screening 6 X 10‘ recombinant clones yieldedsixpositiveclones.Asingleclone (4R)remained positive after a second screening of the selected clones in identical conditions except withlower plaque density. Sequence analysis of 4R revealed that it did not extend to the3’ end of the mRNA. No other clone could be found in theXgtll library when screening with the4R

troenterological Association (May 1990). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBLDataBankwith accession number(s) M55149. 4 Supported bya grant from the Fondation pour la Recherche Midicale. 11 To whom reprint requests should be sent. Tel.: 33-91-82-03-15; Fax: 33-91-26-62-19. The abbreviations usedare: PAP, pancreatitis-associated protein; 7 Supported by Grant KE 347/3-1 from the Deutsche ForschungsSDS, sodium dodecyl sulfate; PSP-S, pancreatic stone protein. gemeinschaft.

24664

PAP Messenger RNA insert. It was therefore necessary to construct a library putatively enriched in PAP clones. Acute pancreatitis was induced in rats by retrograde injection of200 pl of 0.4% sodium taurocholate in the main pancreatic duct, as described by Lankisch et al. (13). Total pancreatic RNA was prepared as recommended by Chirgwin et ai. (14) from rats killed 24 h after the induction of pancreatitis. Polyadenylated RNAs were purified by affinity chromatography on oligo(dT)-cellulose (15) and used to direct cDNA synthesis in the presence of reverse transcriptase. DNA polymerase was used in conjunction with RNase H to synthesize the complementary DNA strands. Enzymes and reagents were obtained as a kit from Amersham and were used according to their recommendations. Double-stranded cDNA was ligated into bacteriophage X g t l O DNA with EcoRI linkers and packaged using the Amersham packaging kit. The library contained atotal of 3.5 X IO5independent recombinantclones. 4R insert, 'lP labeled by random priming (16) to a specific activity of lo9 cpm/ pg, wasused as probe to screen 6 X lo4recombinant clones. Screening was performed in duplicate in 6 X SSC (SSC is150 mM NaC1,15 mM sodium citrate), 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.5% SDS, and 200 pg/ml denatured herring sperm DNA at 68 "C. Filters were washed with 0.1 X SSC, 0.1% SDS at 68 "C. Several clones were selected, the positivity of whichwas confirmed by a second screening. Inserts were subcloned in the M13mp18/mp19 system (17) and the single-stranded recombinant phages were sequenced (18) with Taquenase and/or Sequenase (U. S. Biochemical Corp.) following the recommendations of the manufacturer. Sequencing was initiated either by the universal M13 primer or by appropriate synthetic oligonucleotides. Biohazards associated with the experiments described in this publication have been examined previously by the French National Control Committee. Sequence Comparisons-The complete nucleotide sequence of PAP mRNA was compared with the sequences listed in GenBank (19). The search was conducted with the BISANCE system (CITI2, Centre Interuniversitaire d'informatique, Paris) using the program of Goad and Kanehisa (20). The complete amino acid sequence was compared with the sequences listed in GenPro and the National Biochemical Research Foundation data banks, using the FASTPprogram (CITI2). Protein Analysis-Amino acid sequence of the amino-terminal end of PAP purified by ion-exchange chromatography (10) was obtained by 13 cycles of automatedEdman degradation using an Applied Biosystems gas phase analyzer. Erythrocyte Agglutination Tests and Carbohydrate Binding-Agglutination assays were conducted onrabbit, rat,and human(B group) erythrocytes, prepared from freshly collected blood. Erythrocytes were washed and used as such or after further treatment with trypsin and/or glutaraldehyde, according to Nowak et al. (21). Concanavalin A (Sigma) was used as positive control. Carbohydrate binding was tested on agarose affinity columns specific for maltose, D-mannose, D-galactose, N-acetyl-D-galactosamine, D(+) melibiose, L-fructose, or N-acetyl-D-glucosamine (Sigma). The columns were used according to Beyer et al. (22) and Holberg et al. (23). Bacterial Aggregation-Bacterias (Escherichia coli strains KH802 or JM101) were grown to stationary phase in L-broth at 37 "C with constant shaking. Bacteria were pelleted, washed in standard phosphate-buffered saline, and approximately 5 X lo7bacteria were resuspended in 200 pl of phosphate-buffered saline supplemented with 0.5 mM CaC12,in microtitration plates. PAP was added in concentrations ranging from 1 to 50 pg/ml. The presence of macroscopic aggregation was monitored after 3 hof incubation at 25 "C.Albumin and concanavalin A (50 pg/ml) were used as negative and positive controls, respectively. Animal Experiments-Acute pancreatitis was induced as described above in male Sprague-Dawley rats weighing 180-200 g. The animals were killed after 12, 24, 48, 120, or 240 h. Pancreata were removed, quickly trimmed free of fat, and a fragment of the gland processed according to Chirgwin et al. (14) for RNA purification. Nutritional experiments were conducted on male Sprague-Dawley rats. Pancreatic adaptation was obtained by feeding the animals for 2 weeks one of the following diets, the content of which in carbohydrates was respectively low (diet I), intermediate (diet 11), or high (diet 111). Their composition in percent (w/w) in carbohydrate, proteins, and lipids was respectively: diet I, 11, 25, and 58; diet I1,20, 70, and 4, and diet 111, 75, 15, and 4, adequately supplemented in salts and vitamins (24). Total RNA was extracted from a fragmentof the pancreas as described above. Rats submitted to chronic ethanol ingestion received for 3 months 20% ethanol as sole beverage and a balanced commercial diet (dietIV, containing 56% carbohydrate, 20%

24665

protein, and18%lipid, w/w) ad libitum. Their pancreas was processed as described above. Chronic pancreatic stimulation was obtained as described previously (25) by intraperitoneal injection of cholecystokinin (120 units/ kg, 20% pure, Kabi-Vitrum), caerulein (6 pglkg, Farmitalia, Milano), pilocarpine (60 mg/kg, Sigma) or saline (control) twice daily for 7 days. RNA Analysis by Filter Hybridization-RNA preparations were controlled for the absence of degradation by electrophoresis on agarose-methyl mercury gel (26). Northern blot analysis was performed by blotting RNAs onto Biodyne nylon membrane (PALL BioSupport, East Hill, NY) after electrophoretic separation on agarose-formaldehyde gels(27) then incubating the filter in 50% formamide, 5 X SSPE (SSPE is 180 mM NaC1, 1 mM EDTA, 10mM NaH2P04,pH 7.5), 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.5% SDS, and 200 pg/ml denatured herring sperm DNA at 42 "C in the presence of the appropriate 3ZP-labeledprobe. Then the filters were washed four times for 5 min at room temperature in 2 X SSC, 0.1% SDS, twice for 15 min at 50 "C in 0.1 X SSC, 0.1% SDS, and once for 30 min in 0.1 X SSC. Probes were cloned inserts that were labeled by random priming as described above. Rat amylase cDNA used in control experiments was a 1,100-base pair insert from clone pcXP100 (28) generously provided by Dr. R. J. MacDonald. Quantitative analysis of RNAs was performed according to Whiteand Bancroft(29), as described previously (30). RNA samples (5, 2.5, 1.25, and 0.625 pg/sample) were denatured, dotted ontonitrocellulose filters, and hybridized to the 32P-labeled insert of clone4R. The autoradiographs of the blots were scanned with a Multiscan apparatus (LKB instruments) and the results analyzed as described previously (30), the slopes of the regression lines giving an estimate of the mass of the probed mRNA/pg of total RNA. RESULTS

Cloning Rat PAP Messenger RNA and Analysis of the Sequence-Only one positive clone (4R) could be selected in the cDNA library constructed in Xgtll, after two successive immunoscreenings. Further evidence of the immunological relationship of the protein encoded by 4R and PAP was obtained by inserting the4R insert into theexpression plasmid pEX2, having the encoded protein expressed in E. coli as a hybrid and immunodetecting the hybrid by Western blot with the PAP antibody (not shown). The 4R insert comprised 765 nucleotides but did not include the 3' end of the mRNA. Complete sequence from the 5' end of PAP mRNA was obtained from 4R in two steps, with the help of a synthetic oligonucleotide in position 476-493. Sequence in the opposite direction was obtained by sequencing the 3' end of 4R and was completed by sequencing clones 10-5 and 10-3, selected from a cDNA library constructed inX g t l O with rat pancreatic RNA obtained after induction of pancreatitis. Concatenation of the sequences yielded a mRNA sequence of 781 nucleotides, exclusive of the poly(A) tail (Fig. 1). A putative polyadenylation signal (AATAAA) waspresent 16 nucleotides upstream from the poly(A) extension. The 4R insert was used as aprobe to analyze by Northern blotting RNAs obtained from rat pancreas. A single band was observed, after induction of pancreatitis or in healthy controls (see Fig. 5 ) , corresponding to a transcript of about 920 nucleotides. Sequence Analysis of the Encoded Protein-A single open reading frame was found in the cDNA sequence. To see whether it encoded PAP, we determined by microsequencing the amino-terminal sequence of the mature protein purified from pancreatic juice. That sequence, EDSPKKIPSARIS, was actually found encoded from nucleotide 140 (Fig. 1).Six in-frame methionines were present upstream from the amino terminus of mature PAP. The only one complying with the structural requirements of prepeptides, which include a net positive charge near the amino-terminal end followed by a cluster of hydrophobic residues (31), was in position 62 of the cDNA sequence (Fig. 1).The predicted prepeptide and the mature protein comprised respectively 26 and 149 residues.

PAP Messenger RNA

24666

~ ~ ~ ~ ~ ~ ~ t ~ ~ ~ a ~ t ~ g c c c g c a ~ g ~ ~ 50 a g ~ t ~ a g g ~ g g ~ g ~ ~ g ~ ~ ~ g ~ t g ~ tgagagttaat ATG TTG CAT CGC TTG GCC TTC CCA GTC ATG

-

9

1 16

91 10

-

147

-

54

TCC TGG ATG CTG CTC TCC TGC CTG ATG CTC TTA TCA 130 CAG

44

1 23

-

158

.53

GTG C M GGA G M GAC TCT CCGM G hM ATA CCC TCT GCA 169 Glu A s p Ser Pro Lys Lys Ile Pro Ser Ala 36

408

1 96

CGC ATT AGT TGC CCCA U GGC TCC CAG GCA TATGGC TCC 200 Arg I l e Sex CYS Pro LYJ Gly Ser Gln Ala Tyr Gly Ser 49 TAC TGC TAT GCC CTG TTT CAG ATA CCA CAG ACC TGG TTT 247 Tyr Cy3 TYr Ala Leu Phe Gln Ile Pro Gln Thr Trp Phe 62

BLVSVLN B L Ira1 D s

.1:

GAT GCAG M CTG GCC TGC CAG AAG AGA CCT GAA GGA CAC 286 Aap Ala Glu Leu Ala Cy8 Gln Lys Aeg Pro Glu GlyH i s 75

HIOPR Q L V HCSCP E L T

CTT GTA TCT GTG CTC AAT GTA GCT GAA GCT TCA TTC TTG 325 k U Val Ser V a l Leu ASn Val Ala Glu Ala Ser Phe Leu 88 GCA TCC ATG GTCAAG AAC ACT GGA AAC AGC TAC CAA TAT Ala Ser Het Val L ~ SAS^ Thr oly AS^ ser T Y ~ G I TYX ~

PAPR

HTY CBL

364 101

8 I S

186 93 a3 197 492 441 36 134 -

IIS

RXCR

B L A S V T S

SPL DPSA

Q L V T l E S R I A A P H S

CCC AAT GGA GGT GGA TGG GAG TGG MAGT C AAT GAC ATA 442 Pro A m Gly GIY Gly Trp Glu Trp Ser A9n As" A$p Ile 127

TTA GAC CGC GGA TTC TGT GGC AGC TTG TCA AGA TCT TCT Leu ASP Arg Gly Phe Cy:, Gly Sex Leu Ser Arq Ser Ser

520 153

G GGA TTT CTA AGA TGG AGA GAT ACC ACA TGT GAA MGTG Gly Phe Leu Arg Trp Arg Asp Thr Thr Cy3 G1u Val Lys

559 166

TTG CCC TAC GTCTGC AAA TTT ACA GGT taaaattaccagaca Leu PI0 Tyr V a l Cy3 Lyl Phe Thr Gly 175

87

119

601

9 = a a = c = q c t t t ~ q t t t g t c c t g ~ ~ q = ~ ~ ~ t = ~ t g 653 t ~ ~ ~ g g q q ~ ~ ~ ~ ~ t ~ t g a a g a = t t q c q t = g a a a a a g t g t ~ t t c t = t = t ~ = ~ q t = = = t = t t ~ g ~705 g~t=t=

."

115

VPS

114

". ". ". ". ". ". ". ." ".

a t = ~ t t c ~ t t a ~ o C = a t t t t g t s t a a g t t g t q t c c t c a 757 t g

35 133

19

228 122 419 75 165

ATG AAT TAT GTCM C TGG GAG AGG EAC CCA TCT ACT GCC 4 8 1 net ASn TYr V11 A m Trp G1U Arg AsnPro Ser Thr Ala 140

185 92 82 196 491 446

49

88 80 116 211 123

ACC TGG ATT GGA CTC CAT GAC ccc ACT CTT GGT GGA GM 403 Thr Trp I l e Gly Leu H i s ASP Pro Thr Leu Gly Gly Glu 114

39

40

115

H S

40

114

114

241

150 147 254 549

512 105

191

-

. --

~

.

--

145

216 122 118

221

521 178 14

164

114

113

173 216 109 116

253 548

511 104 190

148 152 204 278 184 119 285

sa1 540

137 220

FIG. 3. Alignment of PAP with selected members of the lectin family and related proteins. Human thrombomodulin FIG. 1. Nucleotide sequence of rat PAP cDNA and deduced ( H T M ) (45), chicken hepatic lectin (CHL) (46), rat hepatic lectin sequence of the encoded preprotein. Sequences obtained from (RHL) (471, ratcartilage proteoglycan (RCAR) (48), human tetranecclones 4R, 10-3,and 10-5 were aligned. The 5'- and 3'-noncod~ng tin ( H T N ) (49), human IgE receptor (HZGER) (50), human chonregions are in lowercase letters. The putative prepeptide is underlined. droitin sulfate core protein (HCSCP) (51),rat Kupffer ceII receptor The amino-terminal residueofmature PAP obtained by microse- (RKCR) (52), Sarcophaga peregrina lectin ( S P L ) (42), and dog pulquencing the protein is the glutamic acid in position 27 of the monary surfactant apoprotein (DPSA) (53) were aligned. Symbols 192

g t a a f a = a C C t C ~ 4 t C t C t C t t ~ q ~ ~ ~ ~ ~ = ~ ~ ~ = =

preprotein sequence.

a; : : ~

are as in the legend to Fig. 2.

: 1 ::

l : ~ l ; ; $ J ; $ E l : E ~ ~

lectin andhuman thrombomodulin. Homologous domains, appearing as clusters of amino acid identities andconservative PIDP * C Q L K P E C n t V B V L N V A E n S ~ t ~ ~ ~ " ~ ~ ~ ~ ~ ~ ~ U ~ ~ ~ ~ G ~ included the conserved amino acids of the PSPR N 5 f l Y [ t z \ S U ~ N ~ R ~ :i P : i: ~replacements, R consensus carbohydrate binding domain of Ca2+-dependent PAPR H O P T L G G E P N G C G Y E Y 9 N N O l M N I V N U E R N P S T E L D R C F C lectins (35). Attempts to characterize a carbohydrate binding PsPR - ~. sJl&Js G s u T G y p N s L 1 :i 1 failed. PAP did not induce erythrocyte agglutination, even PAPR G S L S R S S C F ~ R Y R D T T C L V K L P ~ V C K ~ T G 1 2 1 - 199 after trypsin andglutaraldehyde treatments. In addition, PAP 116 - 1 6 a PSPR V a s N F d K ( I ( Y 1 N r S C l R Q R S L l L M FIG. 2. Amino acid sequence comparison between PAP and did not adsorb to affinity columns specific for maltose, DPSP-S. Identical amino acids are indicated in bold type; areas with mannose, D-galaCtOSe, N-acetyl-D-glucosamine, N-acetyl-Dconserved sequences (amino acid identities and conservative replace- galactosamine, D(+)-melibiose, or L-fucose. Bacterial Aggregation-The lowest concentration of PAP ments according to Goad and Kanehisa (20) are boxed. Numbers refer to positions in the mature peptide chain. resulting in visible bacterial aggregation after formation of a pellet was 7.5 pg/ml (0.5 p ~ ) .With higher concentrations Computer analysis of the sequence indicated that mature PAP aggregation proceeded more rapidly and was almost immediate with 50 pg/ml. Fig. 4 shows the aspect of bacterial aggrehad a molecular weight of 16,630. Sequence Comparison with Other Proteins-A search for gates obtained with a PAP concentration of 20 pg/ml. Albuhomology between PAP and proteins listed in the NBRF and min was ineffective, and aggregation induced by concanavalin GenPro data banks was conducted as described under "Ex- A was similar to thatobtained with PAP. The following sugars perimental Procedures." Among pancreatic proteins, asignif- were tested, at a concentration of 100 mM, as possible inhibicant degree of similarity was observed only with the regen- itors of aggregation by PAP: N-acetyl-D-mannosamide; a - D erating protein (reg) from rat islets of Langerhans (32), which galactose 1-phosphate; a-D(+)-mannOSe 1-phosphate; D-frucis identical to the pancreatic stone protein (PSP-S) (32, 33) tose 1-6 diphosphate; D-glucosamine 6-phosphate; D-fructose (Fig. 2). PSP-S is 5 amino acids shorter than PAP. When 6-phosphate; D(+)-mannose; D-glucose 6-phosphate; D(-)those amino acids were deleted as a cluster in position 85-89 arabinose; D(+)-galaCtOSe; D-mannose 6-phosphate; N-acetylof mature PAP, PSP-S and PAP sequences were 63% similar D-galactosamine; N-acetyl-D-glucosamine; a-D(+)-melibiose; (amino acid identities and conservative replacements). Cys- D-galactose 6-phosphate; D-mannosamine; a-D(+)-fucose; ateines 14, 25, 42, 120, 137, and 145 were conserved in relative D-glUCOSe 1-phosphate; D-fructose 1-phosphate. None of them positions, suggesting a relationship with lectins (34). Th'IS was could prevent aggregation. Regulation of PAP mRNA Expression during the Course of investigated further by comparing sequences of PAP, nine animal lectins, and human tetranectin (Fig. 3). The amount Acute Pancreatitis-Pancreatic RNA was obtained from rats of similarities between PAP and the other proteins ranged at different times after induction of acute pancreatitis by from 16 to 26%, highest values being obtained with rat hepatic retrograde injection of sodium taurocholate. Northern blot

2;::

FDN

M

N V

- O N R

tq

a

::z

PAP Messenger RNA

24667

TABLE I PAP and amylase concentrations in pancreas after induction ofacute pancreatitis Messenger RNA concentrationswere measured by dot-blot hybridization on appropriate dilution of total RNA preparations.Expression is in arbitrary unitsluaof total RNA (meanf S.E.; n = 4). PAP

Time days

0 1,200 0.5 1 2 5

10

Amylase units/+g

unitslpg

3.8 25.6 f 1.2 f 270 1,45010.8 f 360 1,51015.0 k 350 22 f 15 25 f 11

f 3.7 11.1 f 2.2 f 2.0 f 2.5 15.5 f 3.0 27.5 f 5.8

TABLE I1 FIG. 4. Aggregation of E. coli KH802 by PAP. The bacterial suspension (5 X IO' cells in 200 pl of phosphate-bufferedsaline Effect of diets and ethanol on PAP and amylase mRNA incubated with 20 pg of PAP for 3 h aggregated and formed a pellet concentration9 at the bottom of the well. A, examination under a light microsco~e Dietscontained, respectively, 11% (I), 20% (II), and 75% (111) ( X 320) of thepellet after resuspension. B, bacterialsuspensionin carbohydrate (w/w). ~i~~ IV was a balanced commercial chow. the absence of PAP. Ethanol was given ad libitum as a 20% solution in drinking water. Messenger RNA concentrationswere expressed as arbitrary units/pg of total pancreatic RNA (mean f S.E.; n = 4). Experiments on diets 1-111 and IV were conducted separately. PAP Tvve of diet kb

-0.9

I I1 111 IV

A

H20 EtOH ND. not determined.

00.51 2

-

5m

FIG. 5. Northern blotanalysis of rat pancreaticRNAs durIdentical ing the course of acute experimental pancreatitis. amounts of totalRNAfromrats killed a t differenttimesafter pancreatitis induction were blotted after electrophoresis on agaroseformaldehyde gels. The filter wasprobed with "P-labeled PAP cDNA. After autoradiography (panel A ) , the same filter was washed and hybridized with '"P-labeled amylase cDNA (panel B ) . The size of the RNAs revealed by hybridization is indicated on theright in kilobases (kb).

analysis of those RNAs with a PAP cDNA probe (Fig. 5A) revealed a strong signal at 12, 24, and 48 h after induction but not in healthy control animals nor at days 5 and 10. When the same filter was dehybridized then probed witha rat amylase cDNA, a strong signal was observed incontrols, which decreased during the acute phase of pancreatitis and then increased back to control level during the recovery phase (Fig. 5B). Patterns similar to that of amylase were obtained when probing for trypsinogen I and chymotrypsinogen B (not shown). Quantification of the increase in PAP mRNA concentration occurring during the acute phase of pancreatitis was performed by dot-blot hybridization (Table I). The concentration at 12 h was 320 times higher than in controls and slightly increased to 380 and 400 times a t 24 and 48 h. It decreased to 6-fold the control level a t day 5 and did not decrease significantly a t day 10. Regulation of PAP mRNA Expression by Nutritional and Hormonal Stimuli-PAP mRNA concentration was measured in the pancreas of rats adapted to diets containing different amounts of carbohydrates (Table 11). When compared with the low carbohydrate diet (diet I, 11% carbohydrate, w/w),

Amylase unitslpg

unitslpg

6.2 f 2.0 11.5 f 2.8 23.5 f 6.2

26.4 f 4.7 95.2 f 8.2 225.8 f 25.4

12.0 f 3.2 28.4 f 4.5

ND" ND

TABLE 111 Effect of chronic secretagogue treatment on PAP and amylase mRNA concentrations Secretagogues andNaCl(ascontrol) were administeredasdescribed under "Experimental Procedures." Messenger RNA concentrations were expressed as arbitrary units/pg of total pancreatic RNA (mean f: S.E.. n = 4). PAP

Amylase unitslug

NaCl CCK" Caerulein Pilocamine CCK, cholecystokinin.

14.4 f 3.1 60.8 f 15.2 43.5 f 10.8 35.1 f 9.7

64.3 f 12.1 60.4 f 15.6 68.9 f 22.1 42.5 f 13.8

PAP mRNA concentration was, respectively, 2 and 4 times higher in diets containing 20 and 75% carbohydrate (diets I1 and 111, respectively). Inthe same animals, the amylase mRNA concentration increased, respectively, 2 and 9 times. In rats fed a commercial chow (dietIV), chronic ethanol consumption (20% ethanol in water for 3 months) was associated with a2.5 times increase inPAP mRNA concentration, compared with control animals drinking water (Table 11). In rats submitted to chronic pancreatic stimulationby hormonal or cholinergic secretagogues, PAP mRNA concentration was found higher than in control (NaC1-treated) animals (Table 111). After treatment with pancreozymine, caerulein, or pilocarpine the increase relative to control was respectively 4, 3, and 2.5 times. Amylase mRNA concentration did not change significantly with pancreozymin or caerulein and decreased with pilocarpine. PAP mRNA Expression in Other Tissues-Total RNA extracted from salivary glands, liver, brain, kidney, stomach, and testes obtained from control rats was probed with PAP

PAP Messenger RNA

24668

be attributed to PAP2 and their physiological significance remains unknown. The high percentage of similarities (63%) between PAP and PSP-S (Fig. 2), the inhibitor of CaC03 crystal growth inpancreatic juice (33, 36), suggested that PSP-S/reg and PAP genes might originate from a common ancestor gene. Promoter regions of the two genes might be related since PSP-S also increases during inflammation (37), although to a smallerextent, whereas other secretory products decrease. They differ, however, in that PSP-S expression in healthyanimals is abundant, contrary toPAP,andthat changes infood composition alter theirexpression in opposite directions (37). Functional analogy with PSP-S was unlikely since PSP-S inhibitory activityis borne by its amino-terminal A B C D E F G undecapeptide (36), of which functionally important acidic FIG. 6. Expression of PAP in various rat tissues. 25 pg of residues are not conserved in PAP. Purified PAP was tested RNA from pancreas obtained 2 days after induction of pancreatitis for calcium carbonate crystal growth inhibition in uitro and ( A ) was analyzed by Northern blotting in parallel with RNA from was found inactive, as expected (not shown). kidney ( E ) ,stomach (F), and salivary gland ( B ) ,brain (C), liver (D), What makes PAP very peculiar among pancreatic secretory testes (G). The probe was '*P labeled PAP cDNA. To demonstrate similar loading RNA/line, 25-pg aliquots of the same RNA samples proteins is its dramatic overexpression after induction of used in the top panel were electrophoresed in a agarose-formaldehyde inflammation (Fig. 5), when synthesis of most secretory progel, stained with ethidium bromide, and photographed under ultra- teins is repressed (38). The magnitude of the increase in PAP violet light (bottom panel). kb, kilobases. mRNA concentration, more than 300-fold, is unprecedented in pancreatic protein regulation. The highest increments recDNA in Northern blot analysis (Fig. 6). No signal could be ported previously were 9 times for amylase after adaptation and 14 times for P23, a minor detected. RNAs from the same tissues obtained from rats 2 to a carbohydrate-rich diet (30) days after induction of acute pancreatitis also gave negative trypsinogen, in response to caerulein (39). PAP increase is too rapid to be attributed tochanges in mRNA turnover rate results (not shown). and must be a consequence of gene overexpression. Such a DISCUSSION response to inflammation is reminiscent of acute phase reacWe have cloned and sequenced (Fig. 1)the messenger RNA tants synthetized by the liver (40) which are induced up to encoding rat PAP,a protein appearingin ratpancreatic juice several hundredfold whereas proteins normally synthesized after induction of pancreatic inflammation (9). A putative in theabsence of inflammation, such as albumin, apolipoproPAP cDNA clone was selected in the pancreatic library by tein AI, or transferrin, are reduced. Some acute phase reacimmunoscreening with monospecific antibodies, and the im- tants such as the human mannose-binding protein (41) are lectins, to which PAP is structurally related (Fig. 3). Another munological relationship to PAP of the encoded protein was lectin,presentin the flesh-fly Sarcophagaperegrim (42), confirmed after expression of the cDNA in E. coli (not shown). resembles PAP in that itis virtually not expressed in normal On the other hand, the amino-terminal sequence of PAP conditions and appears upon injury of the body wall. It was obtained by microsequencing the protein purified from juice shown more recently that receptors implicated in the adhesion was found starting at residue 27 of the encoded preprotein. of circulating leucocytes to the vascular endothelium (LECThe size of the protein encoded by the PAPcDNA (16.6 kDa) CAMS) were lectins expressed shortly after the onset of the was compatible with the apparent molecular weight of PAP inflammatory response (43, 44). Yet, contrary to acute phase estimated by SDS-polyacrylamide gel analysis. In addition, reactants which are released in blood and LEC-CAMSwhich expression of the mRNA corresponding to theselected cDNA are membrane receptors, PAP is an exocrine protein. If its increased dramatically in the hours after pancreatitis induc- overexpression is part of a defense mechanism against tissular tion (Fig. 5 and Table I),along a pattern very similar to that injury, its siteof action should be either within the secretory obtained previously by monitoring PAP concentration in juice pathway or in the intestinallumen. We demonstrated (Fig. 4) (11).These results demonstrate the proposal by Keim et al. that PAP was able to aggregate bacterias invitro, which prevented their proliferation. The amount of PAP required (9, 10) thatPAP is a novel pancreaticsecretoryprotein becoming detectable during pancreatic inflammationbecause for activity is small compared with the amount available in of increased synthesis and not a degradation product accu- juice during the acute phase of pancreatitis (10). A possible role for PAP would therefore be to protect the tissue against mulating during the disease. Small amounts of PAP mRNA are presentin the pancreas bacterial infection during the period in which inflammation of healthy animals. Whether thisis because small, undetected and necrosis increase the risk of bacterial contamination. The spectactular overexpression of PAP during pancreatitis inflammation foci are present in tissue or because PAP is normally expressed a t low levels in acinar cells is unknown, and itsproperties of controlling bacterialproliferation suggest although demonstration that PAP mRNA concentration is that thisprotein is an importantcomponent of the mechanism modulated by nutritional factors (Table 11), as already de- of defense against pancreatic aggression. In addition, PAP might prove very interesting as a biological marker of panscribed for many secretory proteins (2), is in favor of the creatic inflammation if its equivalent was present in human latter. PAPis to date the only pancreatic protein withamylase pancreas. whose mRNA concentrationincreases markedly with the proportion of carbohydrate in diet. PAP mRNA concentration Acknowledgment-We wish to thank Dr. Schmidt for performing also increased in response to chronic pancreatic stimulation microsequencing experiments and P. Garrido for excellent technical (Table 111), thereby resembling serine proteases (25). These assistance. results cannot be discussed in terms of pancreatic adaptation to diet since no enzymatic activity on dietarysubstrates could * V. Keim, unpublished data. kb

..-)

*.

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