Biosynthesis and post-translational processing of site-directed endoproteolytic cleavage mutants of Pro-CCK in AtT-20 cells.

Site-directed mutagenesis in which individual cleavage site P1 amino acids were changed to Ala was performed to delineate their importance in the processing of pro-CCK in mouse pituitary tumor AtT-20 cells. Individual substitution of cleavage sites on pro-CCK, viz., CCK 58 cleavage site R/A to A/A, CCK 33 cleavage site R/K to A/K, CCK 22 cleavage site K/N to A/N, and CCK 8 cleavage site R/D to A/D, did not inhibit pro-CCK expression or the production of some form of amidated CCK. Wild-type CCK cDNA expression in these cells results in production and secretion of CCK 8 and CCK 22. Substitution of the 58R/A cleavage site with A/A produces only CCK 33; 33A/K and 22A/N produce only CCK 8, whereas 8A/D produces CCK 12 and some CCK 22. Where the GRR residues on the C-terminus of CCK 8 were mutated to GAA, no amidated CCK was produced. Significant amounts of the pro-CCK, C-terminal peptide S9S was found in the medium of cells transfected with GAA mutant cDNA, indicating that this pro-CCK was cleaved at the GAA site probably by a nonprohormone convertase enzyme. Further analysis of the cells expressing the GAA mutant demonstrated that it is not extensively cleaved at other sites to produce CCK 8 GAA or larger peptides. In the mutant where the entire pro-CCK, C-terminal S9S was deleted, CCK 8 is processed and secreted normally. Thus, the cleavage at the C-terminal GRR site is essential for subsequent cleavages, and modification of other cleavage sites (58, 33, 22, and 8) has a major impact on pro-CCK processing. These results suggest that there is a temporal order of cleavages, and the structure of pro-CCK has a strong influence on where and whether pro-CCK is processed.     

Expression and processing of procholecystokinin in a rat medullary thyroid carcinoma cell line.

A rat medullary thyroid carcinoma cell line, CA-77, was shown to express the cholecystokinin (CCK) gene. Measurements using a library of sequence-specific radioimmunoassays before and after enzymic treatment of extracts and chromatographic fractions showed that the cells contained 1.0 pmol of alpha-carboxyamidated cholecystokinins/10(6) cells, 0.4 pmol of glycine-extended intermediates/10(6) cells and 1.0 pmol of further C-terminal-extended pro-CCK/10(6) cells. Gel chromatography and reverse-phase h.p.l.c. revealed both sulphated and nonsulphated CCK-8 in the cells. The growth medium contained in addition alpha-amidated CCK-33, glycine-extended CCK-8 and pro-CCK. Exposure to 0.1 microM-dexamethasone for 6 days increased the cellular content and secretion of all of the described CCK peptides by 2-3-fold. The increase was first noted after 3 days of treatment. Monensin inhibited the synthesis of alpha-carboxyamidated CCK and the secretion of all of the CCK forms measured. Colchicine at a low concentration (0.2 mumol/l) apparently increased the synthesis and secretion of alpha-carboxyamidated CCK, whereas higher concentrations inhibited CCK synthesis. Finally, chloroquine inhibited the alpha-carboxyamidation of CCK. We conclude that the CA-77 cell line is a useful tool for studies of the expression and post-translational processing of pro-CCK.     

Neuronal cell lines expressing PC5, but not PC1 or PC2, process Pro-CCK into glycine-extended CCK 12 and 22.

Endocrine tumor cells in culture and in vitro cleavage assays have shown that PC1 and PC2 are capable of processing pro-CCK into smaller, intermediate and final, bioactive forms. Similar studies have shown that PC5 has the ability to process a number of propeptides. Here, we use GT1-7 (mouse hypothalamic) and SK-N-MC and SK-N-SH (human neuroblastoma) tumor cell lines to study the ability of PC5 to process pro-CCK. RT-PCR and Western blot analysis showed that the cells express PC5 mRNA and protein, but not PC1 or PC2. They were engineered to stably overexpress CCK and cell media was analyzed for pro-CCK expression and cleavage of the prohormone. Radioimmunoassays showed that pro-CCK was expressed, but no amidated CCK was detected. Lack of production of amidated CCK may be due to the lack of the appropriate carboxypeptidase and amidating enzymes. Production of glycine-extended CCK processing products was evaluated by treatment of media with carboxypeptidase B followed by analysis with a CCK Gly RIA. Glycine-extended forms of the peptide were found in the media. The predominant forms co-eluted with CCK 12 Gly and CCK 22 Gly on gel filtration chromatography. The results demonstrate that these cell lines which express PC5 and not PC1 or PC2 have the ability to process pro-CCK into intermediate, glycine-extended forms more closely resembling pro-CCK products in intestine than in brain.     

Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes.

An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.     

Expression of porcine cholecystokinin cDNA in a murine neuroendocrine cell line. Proteolytic processing, sulfation, and regulated secretion of cholecystokinin peptides

The cDNA for porcine preprocholecystokinin (pre-pro-CCK) was engineered for expression in mammalian cells under the control of the Rous sarcoma virus-long terminal repeat promoter. This expression construct was transfected into the murine anterior pituitary cell line, AtT-20. A stable cell line (AtT-20/CCK) was derived that expresses CCK mRNA indistinguishable from the CCK mRNA found in pig brain or gut. The AtT-20/CCK cells carry out proteolytic processing and sulfation reactions to generate authentic sulfated CCK8 from pro-CCK. The cells also store and secrete CCK-immunoreactive peptides. This secretion can be stimulated with corticotropin releasing factor, the natural secretagogue for anterior pituitary cells. In contrast, monkey kidney epithelial cells (COS cells), which are transiently transfected to express CCK, predominantly secrete nonsulfated pro-CCK into the medium. These studies show that a murine neuroendocrine cell line contains the complete processing machinery required to generate authentic porcine CCK8. The processing events include simultaneous proteolytic processing at one and two basic amino acid sites and sulfation of tyrosine residues. The cell line thus duplicates exactly the processing patterns found to occur in pig brain cortex.     

Rat immunoreactive cholecystokinin (CCK): characterization using two chromatographic techniques

Acid and neutral extracts of rat cerebral cortex and upper small intestine were prepared and the endogenous concentrations of cholecystokinin-like immunoreactivity (CCK-LI) measured by three new CCK-specific radioimmunoassays. The characterization of the immunoreactive CCK molecular forms was undertaken using gel permeation chromatography in the presence of 6 M urea to minimise problems relating to peptide adsorption or aggregation. Reverse-phase high-performance liquid chromatography (HPLC) was also performed on the rat tissue extracts. Rat cortex contained 268 +/- 12 pmol/g CCK-LI, and over 90% resembled the sulphated CCK-8, which was preferentially extracted at neutral pH. In contrast, the rat upper small intestine (97 +/- 8 pmol/g of CCK-LI) contained less than 20% CCK-8, the majority of immunoreactive CCK being of larger molecular size and being preferentially extracted at acid pH. In the small intestine the predominant molecular form(s) was intermediate in size between CCK-33 and CCK-8. Large amounts of CCK-33 and of a molecular form larger than CCK-33 were also detected. It is concluded that post-translational cleavage of CCK differs in rat brain and gut.     

Characterization of preprocholecystokinin products in the porcine cerebral cortex. Evidence of different processing pathways

Using gel, ion-exchange, and reverse-phase chromatography monitored by radioimmunoassays specific for five sequences of preprocholecystokinin (prepro-CCK), its processing products were measured in neutral and acid extracts of porcine cerebral cortex before and after incubation with trypsin, carboxypeptidase B, and arylsulfatase. Three categories of peptides were found: biologically active peptides, i.e. peptides with the alpha-amidated COOH terminus Trp-Met-Asp-Phe-NH2, comprising large CCKs, i.e. peptides larger than CCK-58 and peptides eluting like CCK-58, CCK-33, and CCK-22; CCK-octapeptides in sulfated and traces of nonsulfated forms; and small CCKs, i.e. traces of CCK-7, large amounts of CCK-5, and modest concentrations of CCK-4 (the structures of CCK-5 and -4 were confirmed by sequence analysis); four NH2-terminal fragments, of which the two predominant ones correspond to the desnonapeptide fragments of CCK-58 and CCK-33; and COOH-terminal extended peptides corresponding to glycine-extended CCK-58, CCK-33, and CCK-8 in small but significant amounts. Thus, in addition to CCK-8 the porcine cerebral cortex synthesizes larger and smaller active CCK peptides in quantities of an order similar to those of CCK-8. The occurrence of these together with the NH2-terminal fragments and glycine-extended peptides can be explained only by the existence of different processing pathways for preproCCK. Consequently, the results suggest that cerebral CCK neurons are heterogeneous and comprise at least three populations with different biosynthetic machineries.     

Production,purification, and characterization of rat pro-CCK from serum-free adapted Drosophila cells

The precursor of cholecystokinin (pro-CCK) was expressed and purified from media of stably transfected D.Mel-2 cell as an V5-His tagged fusion protein. Its identity was confirmed using SDS-PAGE, immunoblotting, gel filtration chromatography, HPLC, and Mass Spectroscopy. Two major forms of pro-CCK were found with a molecular weight of about 14.4 and 11.3 kDa. The smaller form represents the V5-His tagged pro-CCK after cleavage at a single arginine residue at CCK-58. This cleavage is probably being performed by endogenous proteases in these cells. Purification of the desired larger form of pro-CCK is possible using a nickel column with a recovery of about 20%, yielding 500 microg/L media. The purified protein is stable for several months and can be used for further functional studies of pro-CCK.     

Differential bile-pancreatic secretory effects of CCK-58 and CCK-8

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCK(A) and CCK(B) receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCK(A) and CCK(B) receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity.     

NMR evidence for different conformations of the bioactive region of rat CCK-8 and CCK-58

Sulfated CCK-58 and CCK-8 have identical bioactive C-terminal primary sequences but distinct C-terminal solution structures and different bioactivities. To examine structural differences in greater detail, rat CCK-58 and -8 were synthesized with isotopic enrichment of C-terminal residues with (15)N at alpha-amino nitrogens. Proton and nitrogen chemical shift assignments of peptide solutions were obtained by homo- and heteronuclear NMR methods. These data show that the tertiary structure ensembles of C-terminal CCK-8 and CCK-58 differ significantly. Thus, distinct solution conformations may explain differences in CCK(A) and CCK(B) receptor interactions of large and small molecular forms of CCK.     

The subcellular distribution of peptides immunoreactive for the amino-terminal of pro-cholecystokinin in rat brain

Antiserum 1942 raised against the synthetic peptide V-9-M is specific for the amino-terminus of pro-cholecystokinin (pro-CCK). It detects three major peptides in whole rat brain extracts with molecular weights of about 13 000 (peak 1), 8000 (peak 2) and 2700 (peak 3), of which the major one is peak 3. Rat brain was found to contain large quantities of these V-9-M-like peptides. Subcellular fractionation of whole rat brain was performed to determine what cellular component was enriched in these peptides. The molecular weight of the V-9-M-like and CCK-8-like peptides enriched in various subcellular fractions has been determined by Sephadex G-50 chromatography. Primary subcellular fractionation experiments indicated a significant enrichment of V-9-M-like peptides in the mitochondrial pellet (P2), a lesser amount in the microsomal pellet (P3), and a slight enrichment in the soluble fraction (S3). Further purification of the P2 fraction demonstrated an increase of V-9-M-like immunoreactivity in purified synaptosomes. With the exception of the enrichment in the soluble fraction, V-9-M-like peptides follow a similar distribution to that of CCK-8-like peptides. Sephadex chromatography of P2 and P3 fractions indicates that the major form of V-9-M present is the peak 3 (2700) form. This V-9-M-like peptide may represent an intermediate in the processing of CCK, and its presence in synaptosomes may indicate that the proteolytic cleavage of pro-CCK into CCK 58 and peak 3 takes place in synaptic vesicles.     

Increased synthesis but decreased processing of neuronal proCCK in prohormone convertase 2 and 7B2 knockout animals

In addition to its role as a gut hormone, cholecystokinin (CCK) is a widespread and potent neurotransmitter. Its biosynthesis requires endoproteolytic cleavage of proCCK at several mono- and dibasic sites by subtilisin-like prohormone convertases (PCs). Of these, PC1 and PC2 are specific for neuroendocrine cells. We have now examined the role of PC2 and its binding protein, 7B2, in the neuronal processing of proCCK by measurement of precursor, processing-intermediates and bioactive end-products in brain extracts from PC2- and 7B2-null mice and from corresponding controls. PC2-null mice displayed a nine-fold increase of cerebral proCCK concentrations, and a two-fold increase in the concentrations of the processing-intermediate, glycine-extended CCK, whereas the concentrations of transmitter-active (i.e. alpha-amidated and O-sulfated) CCK peptides were reduced (61%). Chromatography showed that O-sulfated CCK-8 still is the predominant transmitter-active CCK in PC2-null brains, but that the fraction of intermediate-sized CCK-peptides (CCK-58, -33 and -22) was eight-fold increased. 7B2-null brains displayed a similar pattern but with less pronounced precursor accumulation. In contrast with the cerebral changes, PC2 deficiency was without effect on proCCK synthesis and processing in intestinal endocrine cells, whereas 7B2 deficiency halved the concentration of bioactive CCK in the intestine. The results show that PC2 plays a major neuron-specific role in the processing of proCCK.     

Receptor binding of cholecystokinin analogues in isolated rat pancreatic acini

The receptor binding of CCK analogues was determined in terms of the inhibition of [125I]CCK binding in isolated rat pancreatic acini. The inhibition curve produced by CCK-8 showed the same feature as that produced by synthetic human CCK-33. The relative potency values of CCK analogues to half-maximally inhibit specific CCK binding were calculated; CCK-8 was equal to human CCK-33, 3-fold stronger than natural porcine CCK-33 and 39, and 700-fold stronger than the unsulphated form of synthetic human CCK-33. Our data suggest that CCK-33, one of the longer molecular forms of CCK, is as important as CCK-8 in the mechanism of physiological actions of CCK.     

The molecular nature of vascularly released cholecystokinin from the isolated perfused porcine duodenum

Using sequence-specific radioimmunoassays, the quantities and molecular nature of cholecystokinin (CCK) have been determined in extracts of porcine duodenal mucosa and in the vascular perfusate from the isolated porcine duodenum. The basal concentration of CCK in the perfusate was 84 pM equiv. CCK-8 (mean; range: 32–173 pM, n = 5). After intraluminal stimulation with amino acids, acidified fat emulsions and hydrochloric acid, the concentrations increased 2–5-fold. Both in the basal and stimulated state the concentrations of the related hormone, gastrin, were below 5 pM equiv. gastrin-17. CCK in the perfusate was concentrated by affinitychromatography using antibodies directed against the bioactive C-terminus. Subsequent gel chromatography revealed a form with a size like or slightly larger than the C-terminal dodecapeptide (CCK-12), a predominant form resembling the C-terminal octapeptide (CCK-8), and a form resembling the C-terminal tetrapeptide (CCK-4). The duodenal mucosa contained in addition CCK-33, -39 and CCK-peptides with further N-terminal extensions. The results suggest that small CCK peptides are the principal circulating forms, while CCK-33 and larger forms are biosynthetic precursors.     

Isolation and characterization of sulphated and nonsulphated forms of cholecystokinin-58 and their action on gallbladder contraction.

Cholecystokinin (CCK) exists in multiple molecular forms with different polypeptide lengths and the absence or presence of sulphation. We have isolated sulphated and nonsulphated forms of CCK-58 from porcine intestine and have determined their bioactivities in a guinea-pig gallbladder contraction assay. Both forms co-eluted in cation-exchange chromatography and in several rounds of reverse-phase (RP)-HPLC, but separated upon RP-HPLC using a water/acetonitrile system with heptafluorobutyric acid as counter ion. Nonsulphated CCK-58 was the form detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry because of desulphation in that process. The biological activity of CCK-58 and CCK-33 is equipotent, although the kinetics of the response differ. Sulphated CCK-58 was found to be 35 times more potent than nonsulphated CCK-58. In contrast, sulphated CCK-8 is 150 times more potent than nonsulphated CCK-8, and for sulphated and nonsulphated CCK-33, the activities differ by a factor of 100. This type of correlation indicates that the N-terminal end of CCK-58 partially compensates for the decrease in activity arising from the lack of sulphated tyrosine. Given its fairly high bioactivity, nonsulphated CCK-58 may have a physiological significance.     

Characterization of canine intestinal cholecystokinin-58 lacking its carboxyl-terminal nonapeptide. Evidence for similar post-translational processing in brain and gut.

An antibody raised against a synthetic cholecystokinin (CCK) analog, (1-27)-(CCK)-33, corresponding to the midregion of CCK-58, detected immunoreactivity in intestinal extracts which eluted between the positions of CCK-33/39 and CCK-58 on high performance liquid chromatography. This peak, lacking carboxyl-terminal cholecystokinin immunoreactivity, was purified by reverse phase and cation-exchange chromatographies. Amino acid, mass spectral, and microsequence analysis established that it was the amino-terminal desnonapeptide fragment of cholecystokinin-58, (1-49)-CCK-58. It was demonstrated further that CCK-58 has less biological activity than CCK-8, suggesting that the amino terminus either sterically hindered the ability of CCK-58 to exert its biological activity or that its amino terminus acted at another site to inhibit release of amylase from rat pancreatic acini. The desnonapeptide of CCK-58 by itself had no biological activity, nor did it affect CCK-8-stimulated amylase release from isolated rat pancreatic acini, suggesting that the amino terminus shields the carboxyl terminus from expressing its biological activity. Its presence in intestine suggests that it is released into the circulation where it could be detected by midregion antibodies. The presence of high proportions of (1-49)-CCK-58 indicates that most CCK-8 is directly derived from CCK-58. Its occurrence in brain and intestine indicates similar processing for procholecystokinin in both tissues.     

Distribution and heterogeneity of immunoreactive cholecystokinin (CCK) in the mucosa of the porcine gastrointestinal tract

The concentration and molecular nature of cholecystokinin-like immunoreactivity (CCK-LI) in extracts of porcine intestinal mucosa were determined using sequence-specific radioimmunoassays. Highest CCK concentrations were measured in duodenal mucosa (258 +/- 60 pmol/g in the distal duodenum) followed by jejunal mucosa (204 +/- 36 pmol/g in the proximal jejunum) and pylorus (51 +/- 9 pmol/g). All other gastrointestinal regions proximal to the pylorus and distal to the jejunum contained less than 20 pmol/g. Pancreas contained less than 1 pmol/g. Gel chromatography in 6 M urea revealed four immunoreactive forms and this was confirmed by reverse-phase high-pressure liquid chromatography (HPLC). The predominant molecular form in acid extracts of duodenal mucosa resembled CCK-33 although high concentrations of the larger CCK form ('CCK-58') and of the form intermediate in size between CCK-33 and CCK-8 were measured. A molecular form resembling CCK-8 was the principal form in neutral extracts of the duodenum.     

Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.     

Cholecystokinin-58 is the major molecular form in man, dog and cat but not in pig, beef and rat intestine

Cholecystokinin (CCK)-58 was found to be the most abundant form in upper small intestinal mucosa of man, dog and cat. However, in pig, beef and rat upper small intestinal mucosa CCK-33/39 and smaller CCK-forms were dominant. The differences in the distribution of the molecular forms of cholecystokinin between these species presumably reflects altered posttranslational processing of procholecystokinin. This may be caused by the different feeding habits of the investigated species. The different forms of cholecystokinin were distributed over the entire length of the mucosa in canine small intestine. The total amount of CCK decreased from the duodenal mucosa towards the colon. In the canine duodenal mucosa, CCK-58 accounted for 85% of the total CCK-like immunoreactivity. The relative amounts of small forms of CCK increased towards the distal jejunum.     

Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.