Gastrin biosynthesis in canine G cells

Gastrin requires extensive posttranslational processing for full biological activity. It is presumed that progastrin is cleaved at pairs of basic amino acids by a prohormone convertase to form a glycine-extended intermediate (G-Gly) that serves as a substrate for peptidyl-glycine alpha-amidating monooxygenase (PAM), resulting in COOH-terminally amidated gastrin. To confirm the nature of progastrin processing in a primary cell line, we performed [(35)S]methionine-labeled pulse-chase biosynthetic experiments in canine antral G cells. Radiolabeled progastrin reached a peak earlier than observed for G-Gly or amidated gastrin. G-Gly radioactivity accumulated in G cells and preceded the appearance of radioactivity in amidated gastrin. The conversion of G-Gly to amidated gastrin was enhanced by the PAM cofactor ascorbic acid. To determine whether one member of the prohormone convertase family (PC2) was responsible for progastrin cleavage, G cells were incubated with PC2 antisense oligonucleotide probes. Cells treated with antisense probes had reduced PC2 expression, an accumulation of radiolabeled progastrin, and a delay in the formation of amidated gastrin. Progastrin in antral G cells is cleaved via PC2 to form G-Gly that is converted to amidated gastrin via the actions of PAM.     

Progastrin maturation during ontogenesis. Accumulation of glycine-extended gastrins in rat antrum at weaning.

The post-translational maturation of antral progastrin was studied in the developing rat. While N-terminal proteolysis remained unchanged and tyrosine O-sulphation varied only slightly during ontogenesis, major changes were observed in the degree of alpha-carboxyamidation. In the third week of life the immediate precursor of amidated gastrin, glycine-extended gastrin, accumulated, and at weaning (day 21) the concentrations exceeded those of amidated gastrin. Our results confirm that weaning is accompanied by an increased synthesis of gastrin and imply that alpha-carboxyamidation is the rate-limiting step during the biosynthetic maturation of gastrin.     

Alpha-carboxyamidation of antral progastrin. Relation to other post-translational modifications

Using radioimmunoassays for amidated and glycine-extended gastrin before and after trypsin-carboxypeptidase B cleavage and chromatography, alpha-carboxyamidation of porcine antral progastrin has been related to tyrosine-O-sulfation and proteolytic cleavages. Corresponding to the sequence at the proteolysis and amidation site, -Gly-Arg-Arg-, antrum contained three COOH-terminally extended precursor types. The glycine-extended gastrins were present in the highest concentrations (241 +/- 58 pmol/g). The degree of tyrosine-O-sulfation was identical for amidated and precursor gastrins irrespective of component size, whereas the component size differed for glycine-extended and amidated forms. For instance, gastrin-34-Gly constituted 54% of the glycine-extended gastrins, while gastrin-34 comprised 8% of the amidated gastrins. The results indicate that tyrosine-O-sulfation occurs prior to NH2-terminal cleavages, which again precede carboxyamidation; but a significant correlation between tyrosine-O-sulfation and proteolytic cleavages or alpha-carboxy-amidation of antral gastrin could not be demonstrated. Furthermore, our results suggest that the immediate precursor of the principal hormonal form, gastrin-17, is gastrin-17-Gly rather than gastrin-34 as previously believed.     

Purification and structural characterization of progastrin-derived peptides from a human gastrinoma

Several peptides derived from the gastrin-predicted preprohormone sequence were isolated from a human gastrinoma by gel permeation, anion exchange, and reverse phase chromatography. The peptides were identified and characterized structurally by a combination of radioimmunoassays, mass spectral analysis, and microsequence analysis. The largest peptide, progastrin-(1-35) (cryptagastrin), extends from the putative processing site for the signal peptidase to the double basic residues adjacent to the amino terminus of gastrin 34. A shorter form of this peptide, progastrin-(6-35) (cryptagastrin-(6-35), was also isolated in smaller amounts. In addition, sulfated and nonsulfated gastrin 17 amides (progastrin-(55-71)) and the glycine-extended nonsulfated gastrin 17 (progastrin-(55-72)) were identified by radioimmunoassay, and their structures were confirmed by mass spectral analysis. Isolation of cryptagastrin indicates that the signal peptide of human preprogastrin contains 21 amino acid residues, and progastrin, therefore, contains 80 amino acids. There is minimal processing of the cryptic peptide preceding the sequence of gastrin 34. An amidated gastrin form larger than gastrin 34 could contain 71 amino acids. No evidence was obtained for processing that would produce gastrins containing more than 34 but less than 71 amino acid residues.     

Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells.

BACKGROUND: Since human colon cancers often contain significant quantities of progastrin-processing intermediates, we sought to explore the possibility that the biosynthetic precursor of fully processed amidated gastrin, glycine-extended gastrin, may exert trophic effects on human colonic cancer cells. MATERIALS AND METHODS: Binding of radiolabeled glycine-extended and amidated gastrins was assessed on five human cancer cell lines: LoVo, HT 29, HCT 116, Colo 320DM, and T 84. Trophic actions of the peptides were assessed by increases in [3H]thymidine incorporation and cell number. Gastrin expression was determined by northern blot and radioimmunoassay. RESULTS: Amidated gastrin did not bind to or stimulate the growth of any of the five cell lines. In contrast, saturable binding of radiolabeled glycine-extended gastrin was seen on LoVo and HT 29 cells that was not inhibited by amidated gastrin (10(-6) M) nor by a gastrin/CCKB receptor antagonist (PD 134308). Glycine-extended gastrin induced a dose-dependent increase in [3H]thymidine uptake in LoVo (143 +/- 8% versus control at 10(-10) M) and HT 29 (151 +/- 11% versus control at 10(-10) M) cells that was not inhibited by PD 134308 or by a mitogen-activated protein (MAP) or ERK kinase (MEK) inhibitor (PD 98509). Glycine-extended gastrin did stimulate jun-kinase activity in LoVo and HT 29 cells. The two cell lines expressed the gastrin gene at low levels and secreted small amounts of amidated gastrin and glycine-extended gastrin into the media. CONCLUSIONS: Glycine-extended gastrin receptors are present on human colon cancer cells that mediate glycine-extended gastrin's trophic effects via a MEK-independent mechanism. This suggests that glycine-extended gastrin and its novel receptors may play a role in colon cancer cell growth.     

Gastrin-amidating enzyme in the porcine pituitary and antrum. Characterization of molecular forms and substrate specificity

As is the case with many other peptide hormones of the brain and intestine, the formation of biologically active gastrin from a glycine-extended processing intermediate occurs via the action of a peptidylglycyl alpha-amidating monooxygenase (PAM). The observation that gastrin exists primarily as unamidated precursors in the pituitary but as amidated gastrin in the antrum prompted this study to examine whether the amidating enzymes in the two organs were different in their characteristics. Amidating activity was quantified by measuring the conversion of glycine-extended tridecagastrin (G13-Gly) to amidated tridecagastrin and glycine-extended hexapancreatic polypeptide (PP6-Gly) to amidated hexapancreatic polypeptide by radio-immunoassay. Two molecular forms of amidating activity were identified in both the porcine antrum and pituitary. The first, PAM-A, had an apparent Mr of 51,000 and a net negative charge at pH 7.0, whereas PAM-B was smaller (Mr approximately 30,000) and had a net positive charge at pH 7.0. Both molecular forms were similar in their cofactor requirements (copper, ascorbic acid, and catalase) and pH optima in the antrum and pituitary. The Km was significantly lower and the Vmax higher for PP6-Gly than for G13-Gly in the pituitary and antrum. These data suggest that although there is no difference between antral and pituitary PAM, the selective affinity of PAM for certain substrates may provide a mechanism for the differential amidation of different hormones within a given tissue or cell.     

Molecular analysis of dibasic endoproteolytic cleavage signals.

Biologically active peptide hormones are synthesized from larger precursor proteins by a variety of post-translational processing reactions. To characterize these processing reactions further we have expressed preprogastrin in two endocrine cell lines and examined the molecular determinants involved in endoproteolysis at dibasic cleavage sites. The Gly93-Arg94-Arg95 carboxyl-terminal processing site of progastrin must be processed sequentially by an endoprotease, a carboxypeptidase, and an amidating enzyme to produce bioactive gastrin. For these studies the dibasic Arg94-Arg95 residues that serve as signals for the initiation of this processing cascade were mutated to Lys94-Arg95, Arg94-Lys95, and Lys94-Lys95. In the GH3 cells the Lys94-Arg95 mutation slightly diminished synthesis of carboxyl-terminally amidated gastrin, whereas in the MTC 6-23 cells this mutation had no effect on amidated gastrin synthesis. In contrast, both Arg94-Lys95 and Lys94-Lys95 mutations resulted in significantly diminished production of amidated gastrin in both cell lines. A specific hierarchy of preferred cleavage signals at this progastrin processing site was demonstrated in both cell lines, indicating that cellular dibasic endoproteases have stringent substrate specificities. Progastrins with the Lys94-Arg95 mutation in GH3 cells also demonstrated diminished processing at the Lys74-Lys75 dibasic site, thus single amino acid changes at one processing site may alter cleavage at distant sites. These studies provide insight into the post-translational processing and biological activation of not only gastrin but other peptide hormones as well.     

Amidation of joining peptide, a major pro-ACTH/endorphin-derived product peptide

Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.     

Identification and characterization of glycine-extended post-translational processing intermediates of progastrin in porcine stomach

We developed a radioimmunoassay specific for glycine-extended progastrin processing intermediates (G-Gly) using antisera generated against the synthetic peptide Tyr-Gly-Trp-Met-Asp-Phe-Gly. Distribution of immunoreactivity in the porcine gastrointestinal tract obtained with this antibody paralleled that of gastrin with the mucosa containing the highest quantity, 116 +/- 22 pmol/g, wet weight (mean +/- S.E., n = 5), or roughly 4% of gastrin concentration. This immunoreactivity was localized specifically to antral mucosal G-cells by immunohistochemistry. On Sephadex G-50 column chromatography of porcine antral mucosal extracts glycine-extended progastrin processing intermediates were separated into three principal molecular forms, each corresponding to known molecular forms of gastrin, component I, tetratriacontagastrin (G34) and heptadecagastrin (G17). Following purification by antibody-coupled affinity chromatography, one molecular form corresponding to G17 in size was shown to have an amino terminus identical to that of G17. Another molecular form corresponding to G34 in size could be converted to the molecular form corresponding to G17 by tryptic digestion. Our findings indicate that glycine-extended progastrin processing intermediates may serve as immediate precursors for each molecular form of gastrin, thus suggesting an alternative pathway for gastrin biosynthesis more complex than that previously conceived.     

Rat progastrin processing yields peptides with altered potency at the CCK-B receptor

Details of prohormone processing patterns are revealed by purification and characterization of molecular forms stored in the tissues where the hormones are expressed. Molecular forms of rat gastrin were purified from antral extracts by gel permeation, anion exchange, and reverse-phase HPLC. Amidated and glycine-extended gastrins were detected with specific antisera and their structures determined by mass spectrometry. In rats, the only form shorter than gastrin-17 observed contained 16 amino acids. These data suggest that two enzymes process the amino terminus of gastrin-17. Pyrrolidone carboxylic acid peptidase removes the amino terminal pyrrolidone carboxylic acid (pyroGlu), forming gastrin-16. In mammals other than rat, gastrin-16 is then cleaved by dipeptidyl peptidase IV to form gastrin-14. In rat, this reaction does not take place because of proline residues Pro(2)-Pro(3)- in gastrin-16. Gastrin-16 is found in sulfated and nonsulfated forms and comprises 28% of the total gastrin immunoreactivity. Glycine-extended forms of gastrin-16 and gastrin-17 comprises 45% of the total gastrin immunoreactivity. The sulfated forms of gastrin-16 and gastrin-17 bind to the CCK-B receptor transfected into CHO cells with 10-fold higher affinity than the nonsulfated forms of these peptides. Therefore, processing of rat progastrin may modulate the expression of gastrin biological activity.     

Post-translational processing of gastrin in neoplastic human colonic tissues.

Gastrin has been postulated to stimulate proliferation in colorectal neoplasms. Although gastrin mRNA has been demonstrated to be present in colon cancer cell lines, the intact peptide had not been recovered from human colorectal neoplasms. We demonstrate that gastrin and its precursors are present in both colorectal neoplasia and adjacent normal-appearing colonic mucosa. In colonic tissue, the glycine-extended precursor form of the peptide is over 10-fold more abundant than the amidated gastrin, and progastrin is more than 700-fold more abundant. In contrast, amidated gastrin in the human antrum is the predominant form of gastrin by a factor of 10. Furthermore, the ratio of gastrin precursors to gastrin is significantly increased in neoplastic colonic mucosa when compared with normal colonic tissue. These data suggest that the processing of gastrin is unique in the human colon and that further differences in processing occur in neoplastic colonic tissue.     

Probing the production of amidated peptides following genetic and dietary copper manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM(+/-)) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM(+/-) mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM(+/-) mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.     

Characterization of gastrin amidation in the rat and porcine antrum: comparison with the pituitary

The formation of biologically active gastrin from glycine-extended processing intermediates occurs via the action of a peptide alpha-amidating enzyme. The observation that gastrin exists primarily as unamidated precursors in the pituitary but as amidated gastrin in the antrum prompted these studies to examine whether the amidating enzymes in the two organs were different in their characteristics. Furthermore, the amidating enzyme in the stomach has not previously been characterized in extensive detail. Amidating activity was quantified by measuring the conversion of Tyr-Gly-Trp-Met-Asp-Phe-Gly (glycine-extended hexagastrin) to Tyr-Gly-Trp-Met-Asp-Phe-NH2 (amidated hexagastrin) by radioimmunoassay. The activity of the antral enzyme in both the rat and hog had a similar apparent molecular weight (45,000-60,000), cofactor requirements (copper, ascorbic acid, and catalase), pH optima (5.5-8.5), and Km (12 microM) as the pituitary enzyme. These data suggest that antral and pituitary peptide alpha-amidating enzymes are the same enzyme, thus it is unlikely that differences in amidating enzymes can account for the observed differences in the tissue specific processing of gastrin.     

Metorphamide, a C-terminally amidated opioid peptide, in human adrenal and human phaeochromocytoma

There appears to be only one possible site for the production of an amidated peptide in the human proenkephalin sequence; this will give rise to the peptide named metorphamide. Since amidation of peptides is commonly an activation step in the synthesis of regulatory peptides, we have examined the levels and form of immunoreactivity to metorphamide in human post-mortem adrenal and phaeochromocytoma extracts. In three out of four post-mortem adrenal extracts, and in each of the two phaeochromocytoma extracts examined, there was 3-4 times more immunoreactivity to the carboxy-terminus of pro-enkephalin, Met-enkephalin(Arg6,Phe7), than to metorphamide. The metorphamide immunoreactivity was shown in each extract to measure only the amidated octapeptide according to gel exclusion and reverse-phase chromatography data. The implications for processing of proenkephalin in human adrenal are indicated.     

PPARalpha agonists stimulate progastrin production in human colorectal carcinoma cells

The three subtypes of peroxisome proliferator activated-receptors (PPARalpha, delta and gamma) control the storage and metabolism of fatty acids. Treatment of rats with the PPARalpha ligand ciprofibrate increases serum gastrin concentrations, and several lines of evidence suggest that non-amidated gastrins act as growth factors for the colonic mucosa. The aim of the present study was to investigate the expression of PPARs and the effect of PPAR ligands on gastrin production and cell proliferation in human colorectal carcinoma (CRC) cell lines. mRNAs for all three PPAR subtypes were detected by PCR in all CRC cell lines tested. The concentrations of progastrin, but not of glycine-extended or amidated gastrin, measured by radioimmunoassay in LIM 1899 conditioned media and cell extracts were significantly increased by treatment with the PPARalpha ligand clofibrate. Similar increases in progastrin were seen following treatment with the PPARalpha ligands ciprofibrate and fenofibrate, but not with bezafibrate, gemfibrozil or Wy 14643. The PPARgamma agonist rosiglitazone had no significant effect on progastrin production. The PPARalpha ligand clofibrate also stimulated proliferation of the LIM 1899 cell line. We conclude that some PPARalpha ligands increase progastrin production by the human CRC cell line LIM 1899, and that clofibrate increases proliferation of LIM 1899 cells. These studies have revealed a relationship between PPARs and gastrin, two regulatory molecules implicated in the pathogenesis of CRC.     

Glycine-extended processing intermediate of proVIP: a new form of VIP in the rat

The biosynthesis of many peptides including vasoactive intestinal polypeptide (VIP) requires enzymatic alpha-carboxyamidation via a glycine-extended intermediate form. In an effort to identify and quantify glycine-extended VIP in rat tissue extracts a radio-immunoassay specific for this peptide was developed. The concentrations of glycine-extended VIP ranged from 1.3 pmol/g in the brain to 83.9 pmol/g in the small intestine. The identity of the peptide was substantiated by cation-exchange HPLC. The ratio of glycine-extended VIP to amidated VIP varied considerably being highest (63%) in the small intestine. The natural occurrence of glycine-extended VIP in connection with our recent demonstration of its biological activity suggest a physiological role for this biosynthetic intermediate VIP form.     

Patterns of prohormone processing. Order revealed by a new procholecystokinin-derived peptide.

An 83-amino acid cholecystokinin peptide with a sulfated tyrosine and an amidated carboxyl terminus (CCK-83) was purified from human intestinal mucosa. The purified peptide was chemically characterized, and its bioactivity was compared to CCK-8. Several post-translational processing steps such as cleavage at basic residues, sulfation, and amidation are necessary to form biologically active cholecystokinin from its nascent prepropeptide. The discovery of CCK-83 gives new insight into the order of preprohormone processing. The processing of prepro-CCK appears to be in the order of: 1) signal peptidase cleavage, 2) tyrosine sulfation, 3) cleavage after a carboxyl-terminal pair of basic residues, 4) carboxypeptidase B-like cleavage of these basic residues, 5) amidation (which results in the formation of CCK-83), and 6) cleavage at monobasic residues by endopeptidases (which results in the smaller molecular forms of cholecystokinin). The characterization of biologically active CCK-83 with a sulfated tyrosine and an amidated carboxyl terminus establishes the site of signal peptidase action and suggests an order of post-translational modifications that give rise to the various molecular forms of cholecystokinin.     

Mitogenic effects of both amidated and glycine-extended gastrin-releasing peptide in defunctioned and azoxymethane-treated rat colon in vivo

Although there is abundant evidence that gastrin-releasing peptide acts as a mitogen in various carcinoma cell lines, the effect of administration of gastrin-releasing peptide on the colorectal mucosa in vivo has not been reported. The aims of this study were to determine whether continuous infusion of gastrin-releasing peptide stimulated proliferation or accelerated carcinogenesis in the rat gastrointestinal tract and other organs. The possible requirement for C-terminal amidation for mitogenic activity in vivo was also investigated. Proliferation was measured in the colon by metaphase index and by immunostaining for the proliferation marker Ki-67, and in other tissues by immunostaining alone. Acceleration of colorectal carcinogenesis was assessed by counting aberrant crypt foci after treatment with the carcinogen azoxymethane. Defunctioning of the rectum reduced both the proliferative index and the crypt height of the rectal mucosa of untreated rats. Treatment with amidated or glycine-extended gastrin-releasing peptide for 4 weeks using implanted mini-osmotic pumps resulted in a two- to three-fold increase in proliferation, and an increase in crypt height, in the defunctioned rectal mucosa (p<0.001), with smaller but significant increases in the caecum and distal colon. No changes in proliferation were detected in lung, pancreas or gastric mucosa. The numbers of aberrant crypt foci in the mid-colon, distal colon and rectum following treatment with azoxymethane were also significantly increased by infusion with amidated or glycine-extended gastrin-releasing peptide. We conclude that administration of gastrin-releasing peptide to mature rats stimulates proliferation and accelerates carcinogenesis in the colorectal mucosa, and that C-terminal amidation is not essential for either effect. Gastrin-releasing peptides could thus potentially act as promoters of colorectal carcinogenesis.