THE JOURNAL Q 1992 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc.

Vol. 267, No . 28, Issue of October ’ 5, PP. 19829-19832,1992 Printed in U. S. A .

Molecular Cloning of PEC-60 and Expression of Its mRNA and Peptide in the Gastrointestinal Tract and Immune System*

(Received for publication, May 6, 1992)

Madis MetsisSs, Antonio CintralI, Valentina Solfriniq, Patrik ErnforsS, Francesca Bortolottiq, Dante G. Morrasuttin, Claes-Goran Ostensonll, Suad EfendicII, Birgitta Agerberth**, Viktor Mutt**, HHkan PerssonS, and Kjell Fuxell From the Departments of $Medical Chemistry, Laboratory of Molecular Neurobiology, THistology and Neurobiology, 11 Endocrinology, and **Biochemistry, Karolinska Institute, 104 01 Stockholm, Sweden

The peptide PEC-60, structurally related to the pan- creatic secretory trypsin inhibitor, inhibits glucose- induced insulin secretion. Here we report on the struc- ture of a cDNA clone from pig duodenum encoding PEC-60. The cDNA encodes a 86-amino acid long pre- cursor protein containing a 26-amino acid signal se- quence, implying that the mature PEC-60 peptide is secreted from cells. Analysis of porcine duodenum demonstrated a high expression of a 0.6-kilobase long PEC-60 mRNA in this tissue, as well as the presence of strong PEC-60-like immunoreactivity in the cyto- plasm of the majority of the goblet cells of the epithe- lium. High levels of PEC-60 mRNA were also found in the bone marrow and the peripheral blood and mod- erate levels in the spleen. A strong PEC-60-like im- munoreactivity was localized in the monocytes of pe- ripheral blood. Radioimmunoassay revealed high lev- els of pig PEC-60-like immunoreactivity in pig plasma suggesting that the PEC-60 peptide is efficiently re- leased from cells. These findings imply that the gas- trointestinal peptide PEC-60 is formed, stored, and secreted from monocytes present within the bone mar- row and in the peripheral blood, indicating a role of the PEC-60 peptide in the immune system in addition to its function as a gastrointestinal peptide.

An increasing number of gastrointestinal peptides have been isolated and characterized during the last decades (1). Many of these peptides are members of families of structurally related peptides. Although first isolated from the intestine several of these peptides, notably cholecystokinin (2) and vasoactive intestinal peptide (3) have also been found in the brain, where they have been implicated to function as neuro- transmitters/neuromodulators. Within the nervous system these peptides in many instances coexist with classical neu- rotransmitters (4). Molecular clones have been isolated for many of these peptides and nucleotide sequence analyses have

* This work was supported in part by the Swedish Natural Science Research Council Grants 04X-715 and 13X-1010 and Magnus Berg- wall’s Stiftelse. 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/EMBL Data Bank with accession number(s) 67109.

0 Recipient of a visiting scientist fellowship from the Swedish Medical Research Council. To whom correspondence should be ad- dressed: Dept. of Medical Chemistry, Laboratory of Molecular Neu- robiology, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden. Tel.: 46-8-7287658; Fax: 46-8-341960.

shown that they are all synthesized as precursor proteins (5, 6). The use of these clones has permitted the localization of the sites of synthesis of these peptides by in situ hybridization using both rodent and human tissues (7). Thus, from such studies a wealth of knowledge has accumulated during the last decade on the structure, expression, and regulation of these peptides (1).

A 60-residue peptide, initially identified by its inhibition of glucose-induced insulin secretion from the perfused rat pan- creas, has been isolated from the pig intestine (8). This polypeptide was named PEC-60 since it was found to have N- terminal glutamic acid, C-terminal cysteine, and a total of 60 residues. In spite of a partial but distinct amino acid sequence homology to pancreatic secretory trypsin inhibitor (9-11), PEC-60 did not inhibit the activity of trypsin (8). PEC-60 also shows an amino acid sequence similarity to the monitor peptide (12), which has been implicated to stimulate the secretion of cholecystokinin from the intestine into the blood stream. Thus, PEC-60 shows a distant structural similarity to a set of previously described peptides with a range of different functions. Recently, an antiserum against PEC-60 was used to demonstrate PEC-60-like immunoreactivity (IR)’ in almost all catecholamine neurons in the central and pe- ripheral nervous system of rats (13). The biological role of the material showing this immunoreactivity in the function of catecholamine neurons is not known, although the action of PEC-60 on insulin secretion suggests a possible role for it in endocrine regulation also in the nervous system.

In this paper we report on the structure of the PEC-60 precursor protein from pig, deduced from an isolated cDNA clone, as well as on the distribution of PEC-60 mRNA and PEC-60-like IR in pig tissues. Our results suggest that the peptide PEC-60 has functions also in the immune system.

MATERIALS AND METHODS

R N A Preparation, Molecular Cloning, PCR Amplification, and DNA Sequencing-Tissue samples were homogenized in 4 M guanidine isothiocyanate, 0.1 M p-mercaptoethanol, 0.025 M sodium citrate (pH 7.0) for 15 s with a Polytron. Each homogenate was layered over a 4- ml cushion of 5.7 M CsCl in 0.025 M sodium citrate (pH 5.5) and centrifuged at 18 ‘C in a Beckman SW41 rotor at 35,000 rpm for 16 h (14). Polyadenylated RNA (poly(A)+) was prepared by oligo(dT)- cellulose chromatography (15). The recovery of RNA was quantified spectrophotometrically. Porcine duodenal poly(A)’ mRNA (5 pg) was used as a template for the synthesis of single-stranded cDNA using Moloney murine leukemia virus reverse transcriptase (Pharmacia, Sweden). The reaction was primed with oligo(dT)12~1R and single- stranded cDNA was purified on a QIAGEN-tip (DIAGEN Gmbh, Diisseldorf, Germany) according to the manufacturer’s manual. A

The abbreviations used are: IR, immunofluorescence; PCR, po- lymerase chain reaction; bp, base pair.

19829

19830 PEC-60 mRNA and Peptide Tissue Distribution mixture of degenerate oligonucleotides representing all possible co- dons corresponding to the porcine PEC-60 protein sequence MPI- CEHM for the 5"primer and IVKDGEC for the 3"primer with synthetic EcoRI restriction sites on their 5'-ends were synthesized on an Applied Biosystems DNA synthesizer. The two primers were then used to prime the amplification of porcine single-stranded cDNA (25 ng) by the polymerase chain reaction (PCR) (Gene AMP, Perkin Elmer/Cetus). PCR products of the expected size (171 bp, including primers and restriction sites) were purified on a 2% agarose gel and cloned into the pBSKS+ vector (Stratagene, La Jolla, CA). The subcloned PCR products were then subjected to nucleotide sequence analyses using the chain termination method. The sequences were analyzed using a software package from the University of Wisconsin Genetics Computer Group (16). The deduced amino acid sequence from 6 clones out of 8 analyzed showed identity with the amino acid sequence between residues 15 and 53 in the amino acid sequence of the pig PEC-60 peptide (8).

PEC-60 cDNA Cloning-Polyadenylated RNA (5 pg) from porcine duodenum was used as a template for the construction of a cDNA library in phage XgtlO using a cDNA synthesis kit (Pharmacia, Uppsala, Sweden) according to the manufacturer's manual. The insert from one of the subcloned PEC-60 PCR products was purified, labeled with [a-"'PIdCTP by nick-translation, and used to screen a primary library of 4 X lo6 independent clones by conventional procedures (17) under stringent hybridization conditions. Ten randomly picked hy- bridization positive phage clones out of approximately 300 positive clones were purified through 4 cycles of screening. Three clones with the longest inserts were purified and the inserts were subcloned in pBSKS (Stratagene, La Jolla, CA) and sequenced on both strands as described above.

Northern Blot Analysis-Poly(A)' RNA (20 pg) or when indicated total cellular RNA (40 pg) was electrophoresed in a 1% agarose gel containing 0.7% formaldehyde and transferred to a nitrocellulose filter. The filter was then hybridized to the 364-bp long PEC-60 cDNA clone labeled with [ cY-~*P]~CTP by nick-translation to a spe- cific activity of approximately IO9 cpm/pg. Hybridization was carried out in 4 X SSC (1 X SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 40% formamide, 1 X Denhardt's solution, and 10% dextran sulfate at 42 "C. Filters were washed at a high stringency (0.1 X SSC, 0.1% SDS, 60 "C) and exposed to Kodak XAR-5 films.

Immunocytochemistry-Small pieces of pig and human duodenum were immersion fixed overnight in a fixative consisting of 4% para- formaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (18). Smears of pig blood were prepared and fixed by dipping slides on the same fixative during 5 min and rinsed in phosphate-buffered saline. The blocks of tissue were rinsed in buffered 10% sucrose solutions for 2 days. Fourteen-pm thick sections were prepared on a cryostat and incubated with a rabbit antibody against PEC-60 (number 2510) which had previously been characterized by means of a standard radioimmunoassay procedure (19). Blood smears were incubated in a similar way. The incubation with the primary antibody was performed overnight at 4 "C in a humidity chamber. After washing, the sections and smears were incubated with a fluorescein isothiocyanate-conju- gated goat anti-rabbit secondary antibody (Amersham, Amersham, United Kingdom), mounted in an antifading medium, and studied in a Nikon Microphot FX microscope with an appropriate combination of excitation and barrier filters for fluorescence microscopy. In control experiments the preparations were incubated with the diluent for the

1 ~ " ~ ~ ~ C r r ~ R R Q P R I S Y A V R L W V V A L A L A

61 --- A L F I V D R E V P V S A E K Q V F S R

121 AxmXa" M P I C E H M T E S P D C S R I Y D P V

ATAU'IACCCAGT

181 ~ ~ T ~ T X A A G C T C ~ T ~ C G T D G V T Y E S E C K L C L A R I E

241 A-CCTT

=a

N K Q D I Q I V K D G E C *

301 PTXG'C4TTW

361 AEAA

FIG. 1. Nucleotide sequence and deduced amino acid se- quence of porcine PEC-60. The putative initiation codon for synthesis of the precursor protein (ATG) is shown in bold. The arrow indicates the start of the mature PEC-60 peptide. Star indicates stop codon. A hexanucleotide sequence used for polyadenylation is under- lined.

primary antibody or a PEC-60 antiserum which had been absorbed with 50 pg/ml of purified PEC-60. No or a very faint staining could be observed in these control sections.

Radioimmunoassay of Plasma PEC-60-like IR-From four healthy pigs blood samples were drawn into heparin tubes kept on ice, centrifuged 1500 X g for 10 min, and the plasma separated and stored at -20 "C. PEC-60-like IR was measured by radioimmunoassay in nonextracted plasma as well as after extraction, either on octadecyl- silyl cartridges (Sep-Pak C18, Waters Co, Inc., Milford, MA) as previously described (20), or after extraction with 0.5 M acetic acid at 4 "C overnight after boiling plasma for 10 min. Before radio- immunoassay, extracted plasma samples were lyophilized and recon- stituted in assay buffer solution (20). The radioimmunoassay of PEC- 60-like IR was performed as described (19) using PEC-60 antiserum 2510, '251-PEC-60, and, as standard, purified pig PEC-60 (8). The sensitivity of the assay was 12 pM.

To determine the molecular size of extracted plasma PEC-60-like IR, a sample was run on an Ultropac G3000SW column (LKB, Stockholm, Sweden) as described (20). The column was calibrated with porcine PECBO (M, SSOO), and the fractions obtained were lyophilized and redissolved in assay buffer for measurements of PEC- 60-like IR.

RESULTS

Molecular Cloning and Structure of PEC-60-A porcine PEC-60 cDNA clone containing a 364-bp long insert was isolated. Nucleotide sequence analysis of the clone revealed an open reading frame coding for 93 amino acids (Fig. 1). An in-frame initiation codon for translation (ATG) was found 23 bp from the 5'-end of the cDNA clone. The nucleotide se- quence surrounding this ATG triplet showed a clear resem- blance to consensus sequences around ATG codons used for initiation of translation, including a G at position +4, C a t position -1, A at position -3, and repeated GCC motifs upstream of the ATG codon (21). Assuming that this ATG triplet is used for initiation of translation of PEC-60 mRNA, the PEC-60 protein is synthesized as a 86-amino acid long precursor protein. The N-terminal of this precursor protein contains a 26-amino acid long hydrophobic sequence which resembles the general structure of previously identified signal sequences (23). The deduced amino acid sequence following the signal peptide shows complete identity with the pig PEC- 60 amino acid sequence reported earlier (8). A hexamer se- quence (AATAAA) specifying polyadenylation (23) is present in the 3'-end of the isolated cDNA clone, 70 bp downstream of the translational termination codon (TGA) (Fig. 1). DNA sequencing of other cDNA clones with the same size inserts revealed the same nucleotide sequence as the one shown in Fig. 1. To ensure that no other PEC-60 cDNA inserts with a longer size was present in the library, the 3'-PCR primer was used in combination with X g t l O sequencing primers to amplify PEC-60 inserts from the library. The products of the PCR reactions with the longest size were then subcloned and se- quenced. Since 6 out of 13 cDNA clones analyzed had the same 5'-end as the clone shown in Fig. 1 (all other clones had shorter cDNA inserts) it is likely that the sequenced cDNA clones represent full or close to full length clones. All full length cDNA clones, as well as 3 clones with shorter inserts, had the same 3'-end strongly arguing that the sequence shown in Fig. 1 includes the entire 3'-noncoding region.

Expression of PEC-60 mRNA in Different Pig Tissues- Northern blot analysis of mRNA prepared from different pig tissues revealed a 0.6-kilobase PEC-60 mRNA in the duo- denum (Fig. 2). The same size PEC-60 mRNA was also detected at similar levels in bone marrow, and in total cellular RNA from peripheral blood. A lower level of PEC-60 mRNA was found in spleen, while the levels of PEC-60 mRNA were below the detection limit in the pig thymus, lung, and adrenal gland. Hybridization of filters containing mRNA from differ-

PEC-60 mRNA and Peptide Tissue Distribution

0,

0.6 kb

B - "

aclin-

FIG. 2. Expression of PEC-60 mRNA in adult porcine tis- sues. A, poly(A)' RNA ( 20 pg/slot) or total cellular RNA (40 pg), in the case of peripheral blood, was electrophoresed in a formaldehyde- containing agarose gel, transferred to a nitrocellulose filter, and hybridized to the porcine PEC-60 cDNA. The filter was washed at high stringency followed by exposure to x-ray film. B, to ensure equal amounts of RNA loaded in each slot, the same filter was also hybrid- ized under reduced stringency to a cDNA probe for mouse a-actin. Note the similar levels of actin mRNA in all slots except peripheral blood where total cellular RNA was used instead of poly(A)+ RNA.

FIG. 3. Immunohistochemical detection (indirect immuno- fluorescence technique using fluorescein isothiocyanate as fluorophore) of PEC-60-like IR in pig and human duodenum (primary antiserum diluted 1/400). In a, strong cytoplasmatic PEC-60 IR is localized in goblet cells (arrows) of the duodenal pig mucosa (duod). b shows an adjacent section of pig duodenum stained with PEC-60 antiserum (1/400) preincubated with the PEC-60 pep- tide (50 pglml). No specific immunofluorescence appears. In the pig blood smear (c), cytoplasmatic PEC-60 IR is shown in a monocyte with a kidney shaped nucleus. d is a control section where the PEC- 60 antiserum has been preincubated with the PEC-60 peptide (50 pg/ ml). No immunofluorescence appears. In e the blood smear has been stained, omitting the primary PEC-60 antiserum. No specific fluores- cence is seen.

ent rat tissues at reduced stringency showed a tissue distri- bution of rat PECBO similar to the distribution in the pig (data not shown).

19831

FIG. 4. Strong cytoplasmatic PEC-60-like IR (a) is ob- served in goblet cells of human duodenum (arrows) using the PEC-60 antiserum and a fluorescein isothiocyanate-conju- gated secondary antibody. After absorption with the PEC-60 peptide (50 pg/ml), no PEC-60-like IR can be observed (b) .

Immunocytochemical Demonstration of PEC-60-like IR- As shown in Figs. 3 and 4 immunohistochemical stainings demonstrated a strong cytoplasmatic PEC-60-like IR within the goblet cells of the columnar epithelium of both the pig and human duodenum. This strong PEC-60-like IR was not seen after incubation with PECSO antisera which had been absorbed with PEC-60 peptide (Figs. 3 and 4). In pig periph- eral blood, strong cytoplasmic PEC-60-like IR was demon- strated in monocytes identified by Giemsa staining (Fig. 3). This IR was also abolished after the antiserum had been preincubated with the PEC-60 peptide. No immunoreactivity was seen after incubation with the secondary antiserum (Fig. 3).

Levels of Secreted PEC-60 in Pig Plasm-In nonextracted plasma, PEC-60-like IR was 714 k 22 pM (n = 4). After extraction on Sep-Pak C18 or with acetic acid, these levels decreased to 429 f 105 and 410 f 72 PM, respectively (both p < 0.05 versus nonextracted plasma). In extracted plasma, more than 85% of the PEC-60-like IR eluted in the fractions corresponding to the pig PECSO standard (data not shown).

DISCUSSION

In the present study, a cDNA clone encoding the peptide PEC-60 has been isolated from pig duodenum. The 5'-end of the cDNA clone contains an in-frame ATG codon for the translation of a 86-amino acid long precursor protein. The 60-amino acid mature PEC-60 protein is preceded by a 26- amino acid long hydrophobic sequence with a structural sim- ilarity to previously identified signal sequences present in secreted proteins (22). The presence of this signal sequence suggests that the PECBO peptide is secreted from cells, which is in line with its proposed function as a gastrointestinal peptide that inhibits glucose-induced insulin secretion. How- ever, in contrast to many other gastrointestinal peptides, the PEC-60 peptide is processed from a preprotein and not from a preproprotein. The substantial levels of PEC-60-like IR found in porcine plasma further support the notion that PEC- 60 is released into circulation, although it is unclear to what extent different PECSO producing cells contribute to these levels. In this context, it is of interest that the measured concentrations of plasma PEC-60-like IR are comparable to

19832 PEC-60 mRNA and Peptide Tissue Distribution

those of insulin during intravenous glucose stimulation in healthy pigs (20).

Analysis of the porcine duodenum demonstrated high levels of a 0.6-kilobase PEC-60 mRNA in this tissue. The size of this mRNA is in good agreement with the length of the isolated cDNA clone adding a polyadenylation tail of approx- imately 200 bp. A strong PEC-60-like IR was seen in the cytoplasm of the vast majority of the goblet cells of the pig mucosa and the same result was also seen in the human duodenum. These results clearly indicate that PEC-60 may be formed and secreted from the goblet cells. The action of the secreted PEC-60 is unknown but it could reflect a role of this peptide in maintaining blood glucose levels as suggested by its ability to inhibit glucose-induced insulin secretion. PEC-60 has not been found to inhibit trypsin activity (8), although it is structurally related to the pancreatic secretory trypsin inhibitor. Moreover, 14 of the 18 residues between cysteines 20 and 39 in pig PEC-60 are identical to residues between positions 22 and 39 in the 61-amino acid long se- quence of the mature rat monitor peptide (12) and an overall amino acid identity of 43% is seen between these two proteins. The monitor peptide has been shown to stimulate release of cholecystokinin from the gut mucosa to the blood circulation (12). It remains to be established if PEC-60, based on its structural similarity to the monitor peptide, could also play a role in regulating the release of other gastrointestinal pep- tides.

An abundant expression of PEC-60 mRNA was also dem- onstrated within the bone marrow and in peripheral blood. In agreement with this, strong PEC-60-like IR was selectively demonstrated in the monocytes of the peripheral blood. These findings clearly suggest that PEC-60 may be formed, stored, and secreted from monocytes present within peripheral blood. It seems possible that also the low levels of PEC-60 mRNA in spleen may be derived from monocytes. The spleen was, however, difficult to evaluate immunocytochemically in view of the abundant unspecific fluorescence. The high level of PEC-60 mRNA in the bone marrow could indicate that PEC- 60 is also expressed in promonocytes which are precursor cells

of monocytes. These results open up the possibility of a role of PEC-60 within the immune system. It should be noted that neuropeptide Y and its mRNA are present in rat megakary- ocytes and thrombocytes emphasizing a role of peptides also in other types of blood cells (24,25). In conclusion, high levels of PEC-60 mRNA have been demonstrated within cells of the duodenum (goblet cells) and peripheral blood (monocytes), suggesting multiple and novel biological roles for PEC-60.

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