Processing of prosecretin: isolation of a secretin precursor from porcine intestine.

Abstract

A precursor to the gastrointestinal hormone secretin has been isolated. The starting material for the purification of the precursor was a peptide fraction purified from pig intestinal extracts, containing peptides with a molecular weight higber than that of secretin. The purification could be followed by measurement ofsecretin bioactivity (alkali secreted in the pancreatic juice of anesthetized cat). Sequence analysis of the isolated secretin precursor revealed a 71-amino acid residue polypeptide that contained the sequence of secretin N terminally, followed by a Gly-Lys-Arg sequence and a Cterminal extension of 41-amino acid residues. With the exception of an arginine residue, which occurs directly after the Gly-Lys-Arg sequence, the remalnder of the C-terminal residues in this precursor are identical to the 40 C-terminal residues predicted by the recently described cDNA sequence for porcine preprosecretin. Compared to the deduced preprosecretin sequence, a stretch of 32 amino acid residues directly following the Gly-Lys-Arg sequence is missing in the now purified secretin precursor. This implies that differential splicing may occur when the secretin gene transcript is processed to mRNA.After its discovery in 1902 by Bayliss and Starling (1), almost 60 years elapsed before secretin (the porcine form of it) was isolated (2) and another 10 years elapsed before its primary structure was finally deduced (3). Pig secretin is a 27-amino acid residue peptide with a C-terminal valine amide. Cow and guinea pig secretin were found to be identical to that of the pig form (4, 46), whereas the human (5), dog (6), rat (7), and rabbit (8) secretin all differ slightly from each-other and from the porcine/bovine/guinea pig form. Chicken secretin is different from all the mammalian forms. However, it is, like the others, composed of 27-amino acid residues and is C-terminally amidated (9). The main action of secretin is to stimulate secretion of bicarbonate-rich pancreatic juice, but it also exerts other actions (10). Although secretin immunoreactivity, as well as bioactivity, has been reported in the central nervous system (11-13), secretin has so far only been isolated from intestine, where it is known to be localized in endocrine cells, called S cells, in the duodenum and upper jejunum (14,15).Until recently, the structure of the secretin precursor was unknown. This was probably due to secretin being composed of amino acids with degenerated codons, making it difficult to synthesize appropriate probes. However, using the polymerase chain reaction technique, Kopin et al.. (16) were able to isolate cDNA encoding pig and rat preprosecretin. The deduced amino acid sequence of the porcine secretin precursor (16) includes a signal peptide, an N-terminal flanking peptide ('10 amino acid residues), secretin, a Gly-Lys-Arg cleavage and amridation sequence, and a 72-amino acid residue C-terminal flanking peptide.Because of the difficulties in isolating the secretin precursor by conventional cDNA techniques and to investigate the ...

Two alternative processing pathways for a preprohormone: a bioactive form of secretin.

“…However, we do not know the amounts of the peptide distributed between side fractions throughout the purification. For comparison, 16 mg of highly purified secretin is usually obtained from 1000 kg of intestine under optimal conditions and 450 Ag of C-terminally elongated variant of secretin was obtained from 17,500 kg of intestinal tissue (13). Thus, the N-prosecretin yield is higher than that for the C-terminally extended form by a factor of 10.…”

“…§1734 solely to indicate this fact. a cloned cDNA species (10,13). This discrepancy was explained in terms of alternative splicing and later the shorter form of cDNA was also found (14).…”

“…The mature hormone-C-terminally amidated secretin-is produced from the glycine-extended peptide by the combined action of a peptidylglycine a-monooxygenase and a peptidylamidoglycolate lyase (17). In aHl of these proforms, the N terminus starts with secretin itself, and thus cleavage of the N-terminal flanking peptide has been considered to be the initial processing event after removal of the signal peptide (13). In our laboratory, a variant form of secretin in which the N-terminal tryptic peptide differs from that of secretin was observed earlier but was not further characterized (18).…”

Expression of Secretin in Porcine and Rat Central Nervous System

“…Rat secretin corresponds to prosecretin amide. Four bioactive proforms of secretin have been isolated from the porcine intestine: secretin-Gly, 7) secretin-Gly-Lys-Arg, 8) a C-terminally extended form (secretin-Gly-Lys-Arg followed by 41 amino acid residues) 9) and an N-terminally extended form (secretin-(-9 to 27) amide). 10) The distribution of secretin-producing cells is well established in the intestinal tract.…”

Secretin

“…Its cDNA (56) and gene sequences (57,116) have been determined and the gene consists of four exons with the mature peptide in a single exon. A precursor for secretin was isolated from porcine and rat intestine which has additional N-terminal and C-terminal peptides that are cleaved to result in the mature peptide which is then amidated at the C-terminal (36,56). The domain structure of the precursor and the amino acid sequence of secretin is shown in Fig.…”