Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11985-11989, December 1995 Biochemistry Two alternative processing pathways for a preprohormone: A bioactive form of secretin VALENTINA BONETTO, HANS JORNVALL, VIKTOR MUTT, AND RANNAR SILLARD* Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden Contributed by Viktor Mutt, September 5, 1995 ABSTRACT An N-terminally 9-residue elongated form of a cloned cDNA species (10, 13). This discrepancy was ex- secretin, secretin-(-9 to 27) amide, was isolated from porcine plained in terms of alternative splicing and later the shorter intestinal tissue and characterized. Current knowledge about form of cDNA was also found (14). A proform of secretin peptide processing sites does not allow unambiguous predic- elongated at its C terminus by Gly-Lys-Arg has been described tion of the signal peptide cleavage site in preprosecretin but (15), and it might represent the next product in the C-terminal suggests cleavage in the region ofresidues -10 to -14 counted processing, presumably produced by cleavage with an enzyme upstream from the N terminus of the hormone. However, the specific for a dibasic site. The next, shortened form of the structure of the isolated peptide suggests that the cleavage precursor, secretin extended at its C terminus by glycine, has between the signal peptide and the N-terminal propeptide also been isolated (16). This form is presumably obtained by occurs at the C-terminal side of residue -10. Moreover, the carboxypeptidase action on the Gly-Lys-Arg-extended form. isolated peptide demonstrates that secretin can be fully pro- The mature hormone-C-terminally amidated secretin-is cessed C-terminally prior to the final N-terminal cleavage. produced from the glycine-extended peptide by the combined The results from this report, and those from earlier studies, action of a peptidylglycine a-monooxygenase and a peptidyl- where C-terminally elongated variants were isolated, show amidoglycolate lyase (17). In aHl of these proforms, the N that the processing of the secretin precursor may proceed by terminus starts with secretin itself, and thus cleavage of the one oftwo alternative pathways, in which either ofthe two ends N-terminal flanking peptide has been considered to be the is processed first. The bioactivity of the N-terminally extended initial processing event after removal of the signal peptide (13). peptide on exocrine pancreatic secretion was lower than that In our laboratory, a variant form of secretin in which the of secretin, indicating the importance of the finally processed N-terminal tryptic peptide differs from that of secretin was free N terminus of the hormone for interaction with secretin observed earlier but was not further characterized (18). receptors. The present study demonstrates that an alternative process- ing pathway is possible, where the N terminus remains ex- Secretin, a gastrointestinal hormone, was discovered by Bayliss tended throughout processing of the C terminus, starting with and Starling in 1902 (1), isolated (2), and characterized as a the precursor and ending with arnidation of the C-terminal 27-residue C-terminally amidated peptide (3). It is known to be valine. produced in endocrine S cells of the small intestine (4, 5), pancreatic 13 cells (6), and possibly also in brain (7, 8). Its best MATERIALS AND METHODS known function is stimulation of the secretion of bicarbonate- rich pancreatic fluid (9). Peptide Purification. A concentrate of thermostable intes- The structures of the rat and mouse secretin precursors as tinal peptides, CTIP, was prepared from porcine intestines as deduced from the corresponding cDNA sequences contain a described (19-21). Briefly, the uppermost meter of the intes- signal sequence, an N-terminal flanking peptide, secretin, and tines was boiled for 10 min, frozen, minced, and extracted with a C-terminal extension peptide in that order (10, 11). The 0.5 M acetic acid (20 h at 4°C). Peptides in the extract were amino acid sequence of pig preprosecretin has also been so adsorbed onto alginic acid (pH 2.7 ± 0.1), eluted with 0.2 M deduced, but it is probably lacking a few residues at the N HCl, and precipitated with salt (pH 3.5 ± 0.1) to produce the terminus of the signal peptide (10). The amino acid sequence CTIP. An aqueous solution of CTIP was fractionated with of the precursor is such that current knowledge about signal ethanol as described (22), and the peptides soluble at -20°C peptide cleavage sites (12) does not allow unambiguous pre- were further processed by size-exclusion chromatography on a diction of this site. On the basis of the amino acid sequence, Sephadex G-25 column. A fraction of low molecular weight the cleavage of the signal peptide has been proposed to occur peptides was extracted with methanol, and the peptides, at the C-terminal side of any of the residues between -10 and methanol soluble at neutral pH, were applied to a CM-22 -14 (10), most likely after residue -12, -13, or -14. Hence, column (14 x 20 cm). From this chromatography step, three for determination of the actual cleavage site, isolation of the large fractions were collected: presecretin, a second fraction corresponding N-terminally elongated form of secretin is that contains mainly secretin, and a more basic fraction necessary. In this report, we show that cleavage of the signal denoted postsecretin normally used for isolation of vasoactive peptide probably occurs at the C-terminal side of residue -10, intestinal peptide (23). This material was then used for further thus suggesting the position of cleavage and the length of the purification by ion-exchange chromatography on carboxy- N-terminal flanking peptide. methyl cellulose (CM-22, Whatman; 5 x 18 cm) in 0.0125 M It has been shown earlier that other secretin proforms also sodium phosphate (pH 6.4). Peptides were eluted with a linear exist in intestinal tissue. A proform, consisting of the secretin gradient of 0-0.3 M NaCl in the same buffer until the part together with its C-terminal flanking peptide, has been absorbance at 280 nm decreased below 0.1 (24). The column isolated, although the length of the latter was found to be 31 was then washed with 0.2 M HCl. NaCl was removed from the residues shorter than the amino acid sequence deduced from eluted peptides by passing the eluate through a column of Sephadex G-25 coarse in 0.2 M acetic acid and lyophilizing the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: TFA, trifluoroacetic acid. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11985 Downloaded by guest on September 28, 2021 11986 Biochemistry: Bonetto et al. Proc. Natl. Acad. Sci. USA 92 (1995) peptide-containing fractions. The fractions eluting between 78 using an uncoated capillary of 75 ,um diameter essentially as and 92 min contained the secretin-(-9 to 27) amide (N- described (25). Two different buffer systems were used (50 mM prosecretin) and were taken for further purification as de- phosphate buffers at pH 2.5 and pH 7.0). scribed below. Structural Analysis. The purified peptide was degraded in Porcine secretin was isolated as described (2) and used as a an Applied Biosystems sequencer (model 470A) and the standard in structural and bioactivity measurements. fractions were analyzed on a Hewlett-Packard HPLC system Reverse-Phase HPLC. The peptide fractions from cation- HP1090. Amino acid compositions were determined with an exchange chromatography were applied to LKB Ultropac TSK LKB Alpha Plus 4151 analyzer. The molecular mass of the ODS (7.8 x 300 mm). The column was eluted with a gradient peptide was measured on a Finnigan-MAT (San Jose, CA) of acetonitrile in 0.1% aqueous trifluoroacetic acid (TFA) Lasermat 2000, using a-cyano-4-hydroxycinnamic acid as the using buffers A (0.1% TFA/water) and B (0.1% TFA/water/ matrix. An aliquot (0.5 ,ul) of the sample, dissolved in 0.1% 80% acetonitrile) with 25-55% buffer B in 40 min. For TFA/70% acetonitrile/water, and of the matrix-saturated cation-exchange HPLC, Vydac (Hesperia, CA) 400VHP552 solution (0.5 ,ul in the same solvent) were mixed on a stainless (5.0 x 25 mm) was used. A NaCl gradient was employed using steel target and allowed to dry at room temperature. Mass buffers A (0.1% TFA/water/25% acetonitrile) and B (1 M spectra were obtained from the average of 10 laser shot NaCl in buffer A) with 0-30% buffer B in 30 min. The peptide recordings at 337 nm. Bovine insulin (Sigma) was used as an fraction from the ion-exchange HPLC step was then applied to internal standard. a Vydac C18 (4.6 x 250 mm) HPLC column and eluted with a Sequence searches were carried out with the FASTA program methanol gradient using buffers A (0.1% TFA/water) and B of the Genetics Computer Group software package (release (0.1% TFA/water/80% methanol) with 60-100% buffer B in 8.0) (26) in the Swiss-Prot (release 30.0) data base. 40 min. Synthetic Peptide. N-prosecretin was synthesized by Scha- Enzymatic Digestion. The isolated prosecretin, its synthetic fer-N (Copenhagen). The crude peptide was purified by re- replica, and secretin were each dissolved (1 mg/ml) in 0.1 M verse-phase HPLC on a Vydac C18 (4.6 x 250 mm) column ammonium bicarbonate (pH 7.9) containing 5% acetonitrile. using 0.1% TFA/water as buffer A and 0.1% TFA/water/80% Endoproteinase Glu-C (analytical grade; Boehringer Mann- acetonitrile as buffer B with a gradient of 35-48% buffer B in heim) was added at an enzyme/substrate ratio of 1:10.