Biosynthesis and processing of pro CCK: recent progress and future challenges

Life Sciences 72 (2003) 747 – 757 www.elsevier.com/locate/lifescie

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Biosynthesis and processing of pro CCK: recent progress and future challenges Margery C. Beinfeld * Department of Pharmacology and Experimental Therapeutics, School of Medicine, Tufts University, 136 Harrison Ave., Boston, MA 02111, USA Received 22 July 2002; accepted 11 September 2002

Abstract Pro Cholecystokinin (CCK) like other prohormones that pass through the regulated secretory pathway, undergoes a number of post-translational modifications during its biosynthesis including tyrosine sulfation, endoproteolytic cleavages, trimming by carboxypeptidase and c-terminal amidation. This minireview summarizes what is known about this process, what specific enzymes are involved in endocrine and neuronal tumor cells and in mutant and knockout mouse strains. It also points out the major challenges that remain for future research. D 2003 Elsevier Science Inc. All rights reserved. Keywords: CCK; PC1; PC2; PC5; Post-translational modifications of proteins; Tyrosine sulfation; Amidation; Carboxypeptidase E

CCK is an important gastrointestinal hormone and neuropeptide neurotransmitter The discovery of CCK as a gastrointestinal hormonal substance regulating pancreatic enzyme secretion and gall bladder contraction coincided with the beginning of endocrinology, in the early 20th century. CCK 33 was isolated and sequenced from the porcine intestine in the late 1960’s [1]. The first observation that CCK was present in the brain was not until 1975 [2], followed a few years later by the isolation and sequencing of CCK 8 from sheep brain [3]. Since then, a large literature has emerged describing the distribution of CCK neurons, their projections, the two CCK receptor subtypes (A and B), regulation of CCK neurotransmission and the physiological and behavioral effects of CCK.

* Tel.: +1-617-636-0346; fax: +1-617-636-6738. E-mail address: [email protected] (M.C. Beinfeld). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 3 3 0 - 5

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CCK is one of the most abundant neuropeptides in mammalian brain [4]. CCK is present in high concentrations in cerebral cortex, hippocampus, thalamus and caudate-putamen, regions that are important for higher brain function. Neurochemical, physiological and behavioral studies have demonstrated that CCK is an excitatory neurotransmitter and is an important component in the neurochemical balance of the brain. CCK appears to play an important role in anxiety [5], satiety [6], dopamine-mediated reward [7], psychostimulant-induced locomotion and sensitization [8], analgesia [9], opiate tolerance [10], learning and memory [11], and neuroprotection [12].

CCK as an important example of tissue-specific differences in post-translational processing CCK displays major tissue and species-specific differences in post-translational processing. The major tissue-specific difference is between the brain and the intestine. The brain makes mainly CCK 8 [3,13] while the intestine makes larger forms like CCK 12, 22, 33, 58 and 83 amide [14]. The molecular forms in plasma mirror the forms in intestine, as this is their origin. There are also species-differences in regards which of these larger forms are more or less abundant, particularly in the intestine and in circulation. The reason for these tissue and species specific differences are not entirely clear but may be caused by differences in processing enzymes and other accessory proteins. These differences may have physiological significance. Having CCK 8 as the major form in brain may allow it to be degraded rapidly after release. The larger forms might be more difficult to degrade by the kidney and liver and allow for a longer biological half-life in circulation. The processing of pro CCK is somewhat unusual because most of the cleavage sites occur at single Arg or Lys residues (Fig. 1). This is not true for pro gastrin. There are many similarities between the sequence and structure of the prohormones of CCK and gastrin (particularly the carboxyl terminal). This is not surprising as these two prohormones are thought to have evolved from a common ancestor during evolution.

Fig. 1. Rat pro CCK sequence written in the single amino acid code with cleavage sites indicated by spaces and vertical arrows. CCK peptides are numbered backwards from the carboxyl terminal of CCK 8. The amino terminal end of the major forms of amidated CCK peptides is indicated. Stars mark location of the sulfated tyrosine residues while the potential casein kinase II serine phosphorylation site is marked by a vertical arrow below the line.

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Experimental models to study pro CCK processing Significant progress has been made on the elucidation of the temporal order and enzymology of pro CCK processing using endocrine and neuronal tumor cells in culture. Mouse strains that lack specific processing enzymes either as a result of spontaneous mutation or gene deletion, have yielded important information. In vitro cleavage assays with recombinant enzymes and synthetic and recombinant substrates have provided some insights. Endocrine and neuronal tumor cells CCK mRNA is expressed in a number of tumors including insulinomas, thyroid C cell carcinomas, intestinal tumors, neuroblastomas, small cell carcinomas, gastric carcinomas, acoustic neuromas and neuroepitheliomas. Most of these tumors and cells lines derived from them are unable to completely process pro CCK to amidated products probably because they lack some or all of the processing enzymes. Three cells lines derived from endocrine tumors express CCK mRNA and secrete CCK 8 amide as well as variable amounts of larger forms like CCK 22: 1. RIN5F (rat insulinoma) [15] 2. STC-1 (mouse intestinal carcinoma) [16] and 3. WE (rat thryoid c-cell carcinoma) [17]. A number of other cell lines (rat pituitary GH3, mouse pituitary At-T20, human neuroblastoma and human teratocarcinoma (NT2) cells have been useful. Although they don’t express CCK mRNA, they express specific prohormone convertase (PC) enzymes either singly or in combination. When transfected with the CCK cDNA or infected with CCK adenovirus, they process pro CCK either partially to glycineextended products or completely to amidated products. The mouse anterior pituitary tumor corticotrophic cell line At-T20 expresses PC1 but not PC2 or PC5. When it is transfected with the CCK cDNA, it makes and secretes large amounts of pro CCK, CCK 8 and lesser amounts of CCK 22 [15,18]. This cell line has been particularly useful for site-directed mutagenesis studies (described below) that have provided vital information about the importance of tyrosine sulfation, serine phosphorylation and individual cleavage sites for the processing of pro CCK [19]. Mutant and knockout mouse models The Cpe fat/fat mouse has a spontaneous mutation in the carobxypeptidase E gene [20] that has eliminated its enzyme activity. Examination of this strain has allowed us to determine the role of this enzyme in CCK processing in the rodent brain and intestine. Examination of pro CCK cleavage products from these mice has provided additional evidence for the temporal order of cleavages. Characterization of pro CCK products from PC2 and 7B2 knockout mice has further clarified the role of these enzymes in the endoproteolytic cleavage of pro CCK in rodent brain. It has also provided important evidence for the existence of enzyme(s) other than PC2 in rodent brain that insures production of active CCK in the absence of PC2. In vitro cleavage assays with recombinant PC enzymes and synthetic and recombinant pro CCK Production of recombinant PC1 [21–23], PC2 [24] and PC5 [25] has been achieved. The cleavage of synthetic CCK peptides and recombinant pro CCK by PC2 has been examined [26].

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The cellular context of CCK biosynthesis and processing The biosynthesis of CCK resembles all other secretory proteins in that it takes place in the regulated secretory pathway. Drugs that disrupt the Golgi (Brefeldin) or prevent the acidification of secretory granules (chloroquine and ammonium chloride) greatly decrease the cellular content of processed CCK (Beinfeld, unpublished observations). Similar results have been observed for gastrin. The signal sequence of pre-pro CCK allows it to be inserted into the ER. This sequence is removed immediately and pro CCK is correctly folded. It is probably not further modified until the trans-golgi network (TGN). There is no evidence that pro CCK is glycosylated, and it contains no consensus sequence for this modification. In the TGN, three out of four of its tyrosine residues are sulfated. This enzymatic reaction is preformed by protein tyrosine sulfotransferase activity [27–29]. These tyrosines have adjacent acidic residues that constitute the consensus sequence for sulfation. Sulfation of the tyrosine residue in CCK 8 is essential for its biological activity at the CCK A or CCK 1 receptor. This sulfation appears to be important for some aspect of CCK biosynthesis because treatment of a CCK expressing endocrine tumor cell with sodium chlorate causes a large decrease in cellular content and secretion of amidated CCK [30]. This effect has been replicated with site directed mutagenesis studies in which these tyrosine residues in pro CCK have been changed to phenylalanine residues prior to expression in pituitary At-T20 cells [19]. It is conceivable that although the sulfated tyrosines are not required for sorting or processing, they may play some role in the solubility or stability of these peptides in the cell. A serine the carboxyl terminal extension of pro gastrin is known to be phosphorylated by casein kinase II [31]. The carboxyl terminal extension of pro CCK contains a similar serine (Fig. 1) which may also be phosphorylated, although this has not been studied directly. Mutation of this serine to alanine in pro CCK followed by expression in pituitary At-T20 cells had no effect on its processing [19]. In the TGN, sulfated pro CCK is sorted into regulated secretory granules with other proteins destined for secretion, including the enzymes responsible for its processing. The nature of the molecular recognition of pro CCK as material for these granules is still a mystery. Whether there are specific sorting receptors that recognize pro CCK or whether some structural feature is recognized is unknown. It is not known whether pro CCK is cleaved entirely within secretory granules or whether the cleavage starts earlier in the ER or Golgi. Inside the secretory granule, there is a concentration step, the internal calcium concentration increases while the pH drops.

Proposed mechanism and temporal order of Pro CCK processing A strict temporal order of cleavages has been demonstrated in the biosynthesis of a number of prohormones. Mechanistically, it is thought to exist because the first cleavage is the site that is most accessible or favored for cleavage. After cleavage of this site, the remaining prohormone changes structure making additional sites available for cleavage. A model of pro CCK processing to CCK 22 and CCK 8 is depicted in Fig. 2. This model takes into account the differences in pro CCK processing in brain and gut and should be a valid model for both tissues. It is based in part on products identified in normal and fat/fat mouse brain and in neuronal and endocrine tumor cells. A site-directed mutagenesis study [32] in which individual cleavage sites were

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Fig. 2. Model of pro CCK processing: Amidated CCK 22 and CCK 8 are produced by a branched pathway from their respective carboxyl-terminal extended peptides by steps which include endoproteolytic processing, carboxypeptidase and amidation enzyme action.

mutated also provided some convincing evidence about which cleavages are required for subsequent processing. Signal peptide cleavage The signal sequence of pre-pro CCK interacts with the signal recognition particle and the translocation machinery of the ER and results in co-translational insertion of pre-pro CCK into the lumen of the ER. The signal sequence is removed by the signalase enzyme on the lumanal surface of the ER membrane. The pre-pro CCK signal sequence is recognized by mammalian cells but also insect cells (sf9 and High 5 cells). As a result, these cells when infected with recombinant baculovirus expressing pre-pro CCK, pro CCK is efficiently secreted into the media [33]. Tyrosine sulfation Three tyrosine residues are sulfated by protein tyrosine sulfotransferase in the TGN. Endoproteolytic cleavage on the carboxyl-terminal of GRR Conversion of the GRR site on the carboxyl-terminal of CCK 8 to GAA by mutagenesis results in production and secretion of normal amounts of pro CCK in AtT20 cells but no amidated CCK is detected. Surprisingly, the GAA site in this mutant is actually cleaved on its carboxyl-terminal to liberate the S9S peptide, which can be detected by RIA after removal of the sulfated tyrosine residues by arylsulfatase.

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This GAA cleavage is performed by an enzyme that has not been identified. No glycine extended CCK was detected in media samples, either before or after carboxypeptidase Y treatment. This enzyme would remove the two alanine residues without removing the glycine and generate products that are immunoreactive in the CCK Gly RIA. Thus it appears that the lack of cleavage at GRR and/or the presence of those two alanines on the carboxyl-terminal of pro CCK prevents further processing. Removal of the S9S peptide (by placing a stop codon in front of it) results in a normal pattern of processing, production of amidated CCK 8 and CCK 22. The presence of arginines on the carboxy terminal that cannot be removed (as in the case of fat/fat mice that lack carboxypeptidase E) doesn’t inhibit subsequent processing, and results in production of CCK 8 Gly Arg Arg [34]. Cleavage of RA at CCK 58 (removal of the pro-peptide) Mutation of the RA cleavage site to AA completely prevents subsequent production of amidated CCK 22 or CCK 8. Some amidated CCK 33 is produced instead, a product not normally made by At-T20 cells. This result shows that RA is the second cleavage site required for production of CCK 22 and 8. In the absence of the ability to make these smaller forms, a larger form (CCK 33) is made, though rather inefficiently. In normal processing in the brain, this peptide is not made because CCK 58 GRR is probably more efficiently cleaved to make 22GRR and 8GRR. Cleavage at CCK 22 KN and CCK 8 RD to produce CCK 22 GRR and CCK 8 GRR Removal of these cleavage sites by mutation prevents their cleavage, but otherwise has a minor impact on any other aspect of the processing. For example, mutation of the CCK 22 KN cleavage site to AN results in production of CCK 8 only, while mutation of the CCK 8 cleavage site RD to RA, results in production of CCK 22 and CCK 12 only. Production of larger forms of CCK like CCK 58 and CCK 33 by the intestine may involve a different complement of endoproteases that do not completely convert CCK 58 to smaller forms as is seen in tumor cells and in the brain. Removal of carboxyl-terminal aginine residues by carboxypeptidase E Conversion of CCK 22 G and CCK 8 G to CCK 22 amide and CCK 8 amide by the amidating enzyme The last step in CCK biosynthesis is the amidation of glycine extended CCK peptides by peptidyglycine-a-amidating monooxygenase [35].

Enzymology of Pro CCK processing Tyrosyl protein sulfotranferase Two resident Golgi enzymes (TSP1 and TSP2) have been identified that are thought to be responsible for the sulfation of the three tyrosine residues in pro CCK [27–29]. These enzymes are highly homologous and widely distributed and both isoforms share a similar distribution. Which one of them is responsible for sulfation of pro CCK is unknown.

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Endoproteases The enzyme(s) responsible for the endoproteolytic cleavages in CCK during its processing have not been completely established. The most likely candidates are the prohormone convertases PC1 [36], PC2 [37] and PC5 [38,39]. These enzymes are widely distributed in the brain and are present in a number of endocrine and neuronal tumor cell lines, including both cell lines that naturally express CCK mRNA and others that don’t. In terms of catalytic activity, all three of these enzymes are good candidates for pro CCK endoproteolysis. Cell lines that express these enzymes either singly or in combination are capable of cleaving pro CCK into appropriate products. To definitely establish which enzyme is responsible for a specific cleavage in these cells lines is technically difficult because no specific (in terms of differentially inhibiting one PC vs. the others), non toxic prohormone convertase inhibitors have been developed that work on cells in culture. The At-T20 cell is the most commonly used cell line for peptide processing studies. It expresses only PC1 and is capable of correctly processing pro CCK to amidated CCK 8 and CCK 22. PC1 expressing L cells engineered to express CCK mRNA cleave pro CCK to generate the pro peptide (cleavage at CCK 58 RA) and glycine and arginine extended CCK 8 [40] (cleavage at GRR and RD) while wild type L cells not expressing PC1 did not cleave pro CCK. Further evidence for the role PC1 and PC2 in pro CCK processing has come from antisense studies. In two cell lines that normally express CCK mRNA, stable expression of the PC1 or PC2 antisense cDNA in RIN5F (rat insulinoma cells) or STC-1 (mouse intestinal cells) caused a significant inhibition of the expression of these enzymes. This inhibition caused a decrease in the amount of processed CCK and caused a shift in the ratio of CCK 22 to CCK 8, implying that PC1 was more associated with CCK 8 production [41,42] while PC2 was more associated with CCK 22 production in these cell lines[43]. These studies provided further evidence that there is no conversion of amidated CCK 22 or larger amidated forms to CCK 8. Pituitary GH3 cells, human neuroblastoma cells and human teratocarcinoma cells express these enzymes either alone or in combination and are all capable of making appropriate endoproteolytic cleavages of pro CCK when the mRNA was introduced by DNA transfection or infection with a CCKexpressing adenovirus. The neuroblastoma cells express only PC5 and generated glycine-extended CCK 22 and CCK 12, but not CCK 8 [44]. They did not make amidated CCK, this is probably due to the fact that they lack the amidating enzyme. GH3 cells express PC2 and PC5 and make mainly amidated CCK 22 and CCK 12, with trace amounts of CCK 33 [45]. Human teratocarcinoma cells express PC1, PC2 and PC5 and when infected with CCK-expressing adenovirus, make glycine extended CCK 33, CCK 22 and CCK 12. When they are differentiated with retinoic acid and mitotic inhibitors, they express only PC5 and make mainly glycine extended CCK 8 with small amounts of glycine-extended CCK 12 and CCK 22 [45]. In this example, the state of differentiation influences both PC enzyme expression and how they process pro CCK. One way to clarify the importance of these enzymes in pro CCK processing in rodent brain is to examine enzyme knockout mice. Examination of the PC2 [46] and 7B2 [47] knockout mouse brains has demonstrated the importance of PC2 for pro CCK processing [48]. 7B2 is a protein that is a chaperone and an endogenous inhibitor of PC2 [24,49,50]. Production of active PC2 requires 7B2 expression, so the 7B2 knockout mouse also lacks PC2 [46]. The levels of amidated CCK in the PC2 and 7B2 knockout mouse brains are reduced relative to wild type mice, but they are not zero. This is strong evidence that another enzyme or enzymes are able to take

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over for PC2 in its absence and provide some processed CCK. The extent to which CCK levels are reduced varies in different brain regions. CCK levels in the cerebral cortex and forebrain of PC2 mice are more reduced relative to wild type than the hypothalamus or the intestine which are identical to wild type brains. In order to further understand what enzymes may be acting to cleave pro CCK in rodent brain, we performed a series of dual label in situ hybridization experiments examining the co-localization of CCK with PC1, PC2 and PC5 in specific regions where CCK mRNA positive cell bodies are abundant, like cerebral cortex, hippocampus, thalamus and mesencephalon. This work shows that many CCK mRNA positive cell bodies also express PC2, but in addition there are significant populations of CCK mRNA positive cell bodies that also express PC1 and PC5 mRNA. It is likely that some CCK positive cells contain all three enzymes (Cain, et al. unpublished observations). The distribution of these enzymes is much broader than that of CCK, supporting a role for these enzymes in the processing of other substrates. This suggests that PC1 and PC5 may be able (in a regionally-specific manner) to cleave pro CCK in the absence of PC2 and insure production of significant amounts of amidated CCK in these animals. PC2 appears to not participate significantly in the endoproteolysis of pro CCK in the hypothalamus or the intestine. Carboxypeptidases In adult fat/fat mice that lack carboxypeptidase E, the levels of amidated CCK in brain are decreased by about 85% compared to wild type, showing that this enzyme is required for normal CCK processing in this tissue. Amidated CCK levels in duodenum were only decreased by 36% in duodenum of fat/fat mouse. This opens up the possibility that another carboxypeptidase is largely responsible for this conversion in rodent duodenum. In the fat/fat mouse, there is a large accumulation of glycine and arginine extended CCK 8 [34,51]. It is clear that CCK 8 Gly Arg Arg is the immediate precursor of CCK 8, so the amino terminal cleavage preceeds carboxypeptidase E action. It also means that CCK 33 or CCK 22 amide cannot be the immediate precursor of CCK 8 in this tissue. Amidating enzyme Although c-terminal amidation is a widespread modification of peptides, it appears to be less common than carboxypeptidase activity. In contrast to the arginine and glycine extended CCK peptides seen only in fat/fat mouse brain, glycine extended CCK appears to be more abundant in brain and intestine as well as endocrine cells. This enzyme is dependent upon copper and ascorbate and at least in some cell lines, the quantity of glycine extended CCK is decreased by adding ascorbate [52].

Future challenges Research with endocrine and neuronal tumor cells and recombinant prohormone convertases has provided many useful insights into the mechanism and enzymology of CCK processing. Mutant and knockout mice have provided additional important information. As PC1 and PC5 knockout mice become available, the precise role of the prohormone convertases in pro CCK processing should be understood in detail.

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The most difficult questions remain to be answered: What enzymes are responsible for CCK processing in brain and intestine? Are the prohormone convertases PC1, PC2 and PC5 the only endoproteases that are required? What structural features of the prohormone determine where it will be processed and how it will be recognized as secretory material by the sorting machinery? What is the mechanism of sorting? How is tissue-specific processing determined and regulated? What is the ‘‘peptidergic phenotype’’? What other proteins (other than the prohormones and processing enzymes) are required for prohormone processing? Does alteration of the expression of prohormone genes influence the expression of the processing enzymes and vice versa? What is the role of chaperones and endogenous inhibitors in regulation of prohormone processing? What is the physiological and clinical significance of the regulation of tissue specific processing? Acknowledgements This work was supported in part by NIH grants NS 18667 and NS 31602.

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