CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery.

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Divergent roles for ferric ions in the biological activity of amidated and non-amidated gastrins.

Amidated forms of the peptide hormone gastrin act via the cholecystokinin-2 receptor to stimulate gastric acid secretion, whereas non-amidated forms stimulate colonic mucosal proliferation via a novel, as yet uncharacterised, receptor. Nuclear magnetic resonance (NMR) and fluorescence spectroscopic studies have revealed that glycine-extended gastrin17 bound two ferric ions, and that ferric ion binding was essential for biological activity. We have therefore investigated the role of ferric ions in the biological activity of amidated gastrin17. As with glycine-extended gastrin17, fluorescence quenching experiments indicated that Glu7 Ala and Glu8,9 Ala mutants of amidated gastrin17 each bound only one ferric ion. The affinity of the mutant peptides for the cholecystokinin-2 receptor on transfected COS-7 cells or on Tlymphoblastoid Jurkat cells, and their potency in stimulation of proliferation in Jurkat cells and inositol phosphate production in transfected COS-7 cells, were similar to the values obtained for amidated gastrin17. In addition, the iron chelator desferrioxamine did not significantly inhibit either binding of amidated gastrin17 to the cholecystokinin-2 receptor, or stimulation of inositol phosphate production by amidated gastrin17 in transfected COS-7 cells. We conclude that, in contrast to glycine-extended gastrin17, binding of ferric ions is not essential for the biological activity of amidated gastrin17. Our results support the concept of distinct modes of action for amidated and non-amidated gastrins, and raise the possibility of developing selective antagonists of the actions of non-amidated and amidated gastrins.

Mutation of lysine residues of the 78-kDa gastrin-binding protein reduces gastrin binding.

The 78-kDa gastrin-binding protein (GBP) is a likely target for the antiproliferative effects of gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. Both the N- and C-terminal halves of the GBP bind gastrin, but the affinity of the N-terminal half for gastrin is 7.2-fold higher than the affinity of the C-terminal half. In order to define the gastrin-binding sites of the GBP in greater detail, we have constructed a truncation mutant lacking residues 221-318 of the N-terminal domain and a series of point mutants in which the lysine residues in the first 220 residues of the N-terminal domain were mutated to arginine residues. The effect of these mutations on both the extent of covalent cross-linking of iodinated gastrin2,17 and on the affinity for gastrin17 was investigated. Deletion of residues 221-318 of the GBP decreased the affinity 5.5-fold and reduced, but did not abolish, the extent of covalent cross-linking. Mutation of the 17 lysines in residues 1-220 of the GBP decreased the affinity for gastrin between 1.7- and 3.5-fold and in some cases reduced, but did not abolish, the extent of covalent cross-linking. We conclude that one or more lysine residues are involved in binding of gastrin to the GBP, but that no single lysine residue is the preferred target for covalent cross-linking of iodinated gastrin2,17 to the GBP.

Short term infusion of glycine-extended gastrin(17) stimulates both proliferation and formation of aberrant crypt foci in rat colonic mucosa.

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo and for colorectal carcinoma cell lines in vitro. The effect of short term administration of synthetic gastrins on the colonic mucosa in vivo, however, has not been reported. The aim of our study was to determine whether continuous systemic infusion of glycine-extended gastrin(17) stimulated proliferation and accelerated carcinogenesis in the colorectal mucosa. A significant increase in colonic mucosal proliferation as assessed by metaphase index was seen in the caecum (23%, p < 0.02) and distal colon (27%, p < 0.001), but not the rectum, after treatment of intact rats with glycine-extended gastrin(17) for 1 week using implanted miniosmotic pumps. Defunctioning of the rectum reduced both the proliferative index and crypt height of the rectal mucosa of untreated rats. Treatment of rectally defunctioned animals with glycine-extended gastrin(17) for either 1 or 4 weeks resulted in a significant increase in both the proliferative index (40% and 93%, respectively) and crypt height (11% and 19%, respectively) of the rectal mucosa. The total number of aberrant crypt foci in intact rats treated with the procarcinogen azoxymethane plus glycine-extended gastrin(17) was increased by 48% compared to the value in controls treated with azoxymethane only (p = 0.01). We conclude that short term administration of glycine-extended gastrin(17) to mature rats not only has a proliferative effect upon colonic mucosa, but also increases the number of aberrant crypt foci formed in the colorectal mucosa after treatment with azoxymethane. Glycine-extended gastrin(17) could thus potentially act as a promoter of carcinogenesis.

Selective, high-affinity binding of ferric ions by glycine-extended gastrin(17).

Uptake of dietary iron is essential for replenishment of body stores. A role for the hormone gastrin in iron uptake as a chelator of ferric ions in the gastric lumen has been proposed previously [Baldwin, G. S. (1992) Med. Hypotheses 38, 70-74]. Here, spectroscopic evidence of selective, high-affinity binding of ferric ions to progastrin-derived peptides in aqueous solution at low pH is provided. The maximum at 281 nm in the absorption spectrum of glycine-extended gastrin(17) at pH 4.0 increased (2.07 +/- 0.30)-fold in the presence of > or =2 equiv of ferric ions. Titration of glycine-extended gastrin(17) with ferric ions under stoichiometric conditions indicated that the stoichiometry of binding was 2.00 +/- 0.28 mol of Fe(3+)/mol of peptide. Fluorescence quenching experiments yielded values for the stoichiometry and apparent dissociation constant of the ferric ion-glycine-extended gastrin(17) complex at pH 4.0 of 2.39 +/- 0.17 mol of Fe(3+)/mol and 0.62 +/- 0.19 microM, respectively. No interaction was detected with Co(2+), Cu(2+), Mn(2+), or Cr(3+). Electron paramagnetic resonance spectroscopy suggested that the iron ligands were either oxygen or sulfur atoms. Fluorescence quenching experiments with peptides derived from the glycine-extended gastrin(17) sequence indicated that one or more of the five glutamic acid residues were necessary for iron binding. The binding of ferric ions by glycine-extended gastrin(17) at low pH is consistent with a role for progastrin-derived peptides in iron uptake from the lumen of the gastrointestinal tract.

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells.

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.

A kinetic analysis of substrate recognition by uracil-DNA glycosylase from herpes simplex virus type 1.

Uracil-DNA glycosylase (UDG) is responsible for the removal of uracil from DNA. It has previously been demonstrated that UDG exhibits some sequence dependence in its activity, although this has not been well characterised. This study has investigated the sequence-dependent activity of UDG from herpes simplex virus type-1 (HSV-1). A more detailed analysis has been possible by using both kinetic and binding assays with a variety of different oligonucleotide substrates. The target uracil has been placed in substrates with either A-T-rich or G-C-rich flanking sequences and analyses have been performed on both the single- and double-stranded forms of each substrate. In the latter the uracil has been placed in both a U.A base pair and a U.G mismatch. It is observed that the sequences flanking the target uracil have a greater effect on UDG activity than the partner base of the uracil. Furthermore, the sequence context effects extend to single-stranded DNA. Systematic examination of the kinetics and binding of UDG with these different substrates has enabled us to examine the origin of the sequence preferences. We conclude that the damage recognition step in the HSV-1 UDG reaction pathway is modulated by local DNA sequence.

Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells.

The various molecular forms of gastrin can act as promoters of proliferation and differentiation in different regions of the gastrointestinal tract. We report a novel stimulatory effect of glycine-extended gastrin(17) only on cell/cell dissociation and cell migration in a non-tumorigenic mouse gastric epithelial cell line (IMGE-5). In contrast, both amidated and glycine-extended gastrin(17) stimulated proliferation of IMGE-5 cells via distinct receptors. Glycine-extended gastrin(17)-induced dissociation preceded migration and was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) but did not require mitogen-activated protein (MAP) kinase activation. Furthermore, glycine-extended gastrin(17) induced a PI3-kinase-mediated tyrosine phosphorylation of the adherens junction protein beta-catenin, partial dissociation of the complex between beta-catenin and the transmembrane protein E-cadherin, and delocalization of beta-catenin into the cytoplasm. Long lasting activation of MAP kinases by glycine-extended gastrin(17) was specifically required for the migratory response, in contrast to the involvement of a rapid and transient MAP kinase activation in the proliferative response to both amidated and glycine-extended gastrin(17). Therefore, the time course of MAP kinase activation appears to be a critical determinant of the biological effects mediated by this pathway. Together with the involvement of PI3-kinase in the dissociation of adherens junctions, long term activation of MAP kinases seems responsible for the selectivity of this novel effect of G(17)-Gly on the adhesion and migration of gastric epithelial cells.

HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line.

The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.

Inhibition of the preferential binding of actin to the N-terminal hydratase domain of the 78-kDa gastrin-binding protein by non-steroidal anti-inflammatory drugs and gastrin receptor antagonists.

The 78 kDa gastrin-binding protein (GBP) is a likely target for the antiproliferative effects of gastrin receptor antagonists and non-steroidal anti-inflammatory drugs (NSAIDs) on colorectal carcinoma cells (Baldwin GS, Murphy VJ, Yang Z, and Hashimoto T. J Pharmacol Exp Ther 1998;286:1110-14). This study tested the hypotheses that the GBP bound actin, and that the interaction could be disrupted by gastrin receptor antagonists and NSAIDs. Binding of actin to the GBP was assessed by competition with (125)I-[Nle(15)]-gastrin(2,17) in a covalent cross-linking assay, and by comparison of (125)I-actin binding to the N- and C-terminal GBP domains, which had been expressed independently in E. coli as glutathione-S-transferase (GST) fusion proteins. The ability of gastrin receptor antagonists and NSAIDs to interfere with the actin-GBP interaction was measured by release of (125)I-actin from preformed complexes with the N- and C-terminal domain-GST fusion proteins. Actin purified from skeletal muscle or from gastric mucosal cytosol competed with (125)I-[Nle(15)]-gastrin(2,17) for binding to the GBP with IC(50) values of 2.6 +/- 0.7 microM, and 2.1 +/- 0.7 microM, respectively. The amount of (125)I-actin from either source bound to the N-terminal GBP domain was 8.2 times greater than the amount bound to the C-terminal domain. Binding of actin to both domains was inhibited by the gastrin receptor antagonists proglumide and benzotript, and by NSAIDs. We conclude that the GBP may associate with the cytoskeleton via an interaction between its N-terminal domain and actin, and that the association may be disrupted either by gastrin receptor antagonists or by NSAIDs.

Binding sites for progastrin-derived peptides in colonic crypts.

BACKGROUND AND AIMS: Gastrin17gly acts as a growth factor for the colonic mucosa. Studies on the binding properties of the receptor involved in transducing the proliferative effects have generally been confined to colorectal carcinoma cell lines, and no investigation of gastrin17gly receptors on normal colonocytes has yet been reported. The aim of this study was to investigate the binding of 125I-[Met15]-gastrin17gly to normal colonic crypts. METHODS: Crypts were released from normal rat and rabbit colonic mucosa by treatment with EDTA and isolated by centrifugation. The binding of 125I-[Met15]-gastrin17gly was measured in displacement experiments with increasing concentrations of either gastrin17gly, gastrin17 or gastrin receptor antagonists. The concentrations required for 50% inhibition were determined by the use of curve fitting. RESULTS: 125I-[Met15]-Gastrin17gly bound to both rat and rabbit crypts, and displacement experiments with unlabeled gastrin17gly revealed that the IC50 values were 1.0 +/- 0.6 and 0.6 +/- 0.2 micromol/L, respectively. Binding was also competed by gastrin17, with IC50 values of 2.4 +/- 1.7 and 2.4 +/- 0.7 micromol/L, respectively. Binding was inhibited by the non-selective gastrin/CCK receptor antagonists proglumide and benzotript, but not by the cholecystokinin (CCK)-A receptor antagonist L364 718, or the gastrin/CCK-B receptor antagonist L365 260. CONCLUSION: We conclude that the gastrin17gly binding site on normal colonic crypts has properties consistent with the gastrin/CCK-C receptor.

Biologically active recombinant human progastrin(6-80) contains a tightly bound calcium ion.

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.

Quantitative measurement of mRNA coding for the receptors controlling acid secretion in the ovine fundus and antrum by using RT-PCR.

BACKGROUND AND AIMS: Gastric acid secretion is stimulated by the action of gastrin, histamine and acetylcholine on their respective receptors. To determine the regulation of synthesis of these receptors during different gastric secretory states a competitive RT-PCR method for quantitating the mRNA for these receptors was developed. METHODS: Partial cDNA clones (400-500 base pairs (bp)) for the ovine gastrin, histamine (H2) and acetylcholine (M3) receptors were isolated and sequenced. These cDNA constructs were modified by the inclusion of approximately 100 bp of unrelated sequence within the plasmids. cDNA was synthesized from a mixture of known amounts of RNA transcribed from the modified plasmids and from total RNA extracted from sheep stomach. Proportional coamplification of mixed cDNA was demonstrated using common primer sets. RESULTS: All three receptors were more highly expressed in the fundus than the antrum. The concentration of cholecystokinin-B/gastrin receptor mRNA was 75-fold higher in the fundus than in the antrum, and the concentration of both histamine and acetylcholine receptor mRNA were fivefold higher in the fundus than in the antrum. Infusion of gastrin caused a significant increase in fundic histamine mRNA receptor expression, but not in the expression of the gastrin or muscarinic receptors. CONCLUSIONS: No significant differences were observed in the levels of receptor mRNA between normal adult and fetal animals despite markedly reduced gastric secretion in the fetus, suggesting that gastric receptor gene expression is not the rate-limiting factor in determining gastric acid secretion in the neonatal animal.

Do NSAIDs contribute to acute fatty liver of pregnancy?

Acute fatty liver of pregnancy (AFLP) and the childhood encephalopathy known as Reye's syndrome are both characterised by microvesicular steatosis. Mothers with AFLP are frequently heterozygous for a mutation which reduces the activity of the trifunctional protein (TP) of fatty-acid oxidation. Several lines of evidence suggest that blockade of fatty-acid oxidation may also be the underlying cause of Reye's syndrome, and epidemiological studies have identified aspirin taken during a viral illness as a contributing factor to the development of the disease. The hypotheses are presented:* that children with Reye's syndrome may also be heterozygous for TP mutation, and* that inhibition of the residual long-chain fatty-acid oxidation by NSAIDs including aspirin precipitates the similar symptoms observed in patients with Reye's syndrome and AFLP. Identification of NSAIDs as candidates for the unidentified factor which precipitates AFLP suggests that avoidance of NSAIDs during pregnancy may lead to a reduction in the incidence of this life-threatening disease.

Affinity of cholecystokinin receptor antagonists for the gastrin-binding protein.

A 78 kDa gastrin-binding protein is a likely target for the anti-proliferative effects of the cholecystokinin (CCK) receptor antagonists D,L-4-benzamido-N,N-dipropylglutaramic acid (proglumide) and N-4-chlorobenzoyl-L-tryptophan (benzotript) on colorectal carcinoma cell lines [Baldwin, G.S., 1994. Antiproliferative gastrin/cholecystokinin receptor antagonists target the 78-kDa gastrin-binding protein. Proc. Natl. Acad. Sci. USA 91, 7593-7597.]. Definition of the physiological role of the gastrin-binding protein has been hampered by the very low affinity of benzotript for the gastrin-binding protein. Benzotript analogues were therefore tested for their ability to inhibit the binding of iodinated gastrin to the gastrin-binding protein. The affinity of the most potent analogue (the D-isomer of benzotript, CR 665) was similar to the value reported previously for the L-isomer. In order to isolate more potent binding inhibitors, several selective CCK receptor antagonists were also tested as inhibitors of the binding of gastrin to the gastrin-binding protein. The affinity of the most potent binding inhibitor PD 149164 (benzenebutanoic acid, 4-fluoro-!b/-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[(tricyclo-[3.3 .1. 1(3,7)]dec-2-yloxy)carbonyl]amino]propyl]amino]-, [R-(R*,S*)]-) was approximately 10-fold higher than the L-isomer of benzotript. PD 149164 may serve as the lead compound for the future development of more potent and selective gastrin-binding protein inhibitors.

Expression of gastrin/CCK-B receptors does not lead to a mitogenic response to gastrin in two colon cell lines.

To clarify the impact of the classic gastrin/CCK-B receptor on the growth of benign and malignant colonic cells, two permanent cell lines expressing this receptor have been established. The conditionally immortalized nonmalignant colonic cell line YAMC and the colonic carcinoma cell line SW 403 were stably transfected with a plasmid encoding the gastrin/CCK-B receptor (GR), or with plasmid alone (V). Expression of the gastrin/CCK-B receptor in the transfected YAMC-GR and SW 403-GR cells was demonstrated by gastrin binding experiments. The YAMC-GR cell line did not respond mitogenically to pentagastrin or gastrin(17) in vitro and was not tumorigenic. The SW 403-GR cell line was stimulated by gastrin(17) in vitro, but the growth patterns of SW 403-GR and SW 403-V were the same in nude mice with cells injected either subcutaneously or into the spleen. These results provide further evidence that the gastrin/CCK-B receptor is not responsible for gastrin-stimulated growth in colonic tumors.

Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.

Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.

Binding of pepsinogen to the 78 kDa gastrin binding protein.

An endogenous ligand of the 78 kDa gastrin-binding protein (GBP) has been purified from detergent extracts of porcine gastric mucosal membranes by ion exchange chromatography and preparative gel electrophoresis. The ligand bound to the GBP with high affinity (mean IC50 value of 0.31+/-0.09 microgram/ml, or 8 nM), as assessed by inhibition of cross-linking of iodinated gastrin2,17 to the GBP. Both the N- and C-terminal halves of the GBP, which had been expressed individually as glutathione-S-transferase fusion proteins in Escherichia coli, and purified on glutathione-agarose beads, bound the ligand. Two peptides derived from the ligand were purified by reversed-phase high-performance liquid chromatography (HPLC), and characterised by mass spectrometry and Edman sequencing. The peptides were 97% and 100% identical, respectively, to amino acids 119-157 and 199-219 of porcine pepsinogen A. Commercial samples of pepsinogen also bound to the GBP, with a mean IC50 value of 3.9+/-1. 2 micrograms/ml (100 nM). We conclude that the ligand is closely related, but not identical, to pepsinogen A.