Foldase and inhibitor functionalities of the pepsinogen prosegment are encoded within discrete segments of the 44 residue domain.

Pepsin is initially produced as the zymogen pepsinogen, containing a 44 residue prosegment (PS) domain. When folded without the PS, pepsin forms a thermodynamically stable denatured state (refolded pepsin, Rp). To guide native folding, the PS binds to Rp, stabilizes the folding transition state, and binds tightly to native pepsin (Np), thereby driving the folding equilibrium to favor the native state. It is unknown whether these functionalities of the PS are encoded within the entire sequence or within discrete segments. PS residues 1p-29p correspond to a highly conserved region in pepsin-like aspartic proteases and we hypothesized that this segment is critical to PS-catalyzed folding. This notion was tested in the present study by characterizing the ability of various truncated PS peptides to bind Rp, catalyze folding from Rp to Np, and to inhibit Np. Four PS truncations were examined, corresponding to PS residues 1p-16p (PS1-16), 1p-29p (PS1-29), 17p-44p (PS17-44) and 30p-44p (PS30-44). The three PS functionalities could be ascribed primarily to discrete regions within the highly conserved motif: 1p-16p dictated Rp binding, 17p-29p dictated Np binding/inhibition, while the entire 1p-29p dictated transition state binding/catalyzing folding. Conversely, PS30-44 played no obvious role in PS-catalyzed folding; it is hypothesized that this more variable region may serve as a linker between PS1-29 and the mature domain. The high sequence conservation of PS1-29 and its role in catalyzing pepsin folding strongly suggest that there is a conserved PS-catalyzed folding mechanism shared by pepsin-like aspartic proteases with this motif.

[Physiological and clinical implications of gastric pepsinogens].

This review deals with pepsinogen metabolism, physiological role, and clinical implications. Effects of various factors, e.g H. pylori, on pepsinogen levels are considered. It is concluded that non-invasive screening of gastric precancer conditions provides a cost-effective and efficacious approach to the prevention of this pathology.

Structural and phylogenetic comparison of three pepsinogens from Pacific bluefin tuna: molecular evolution of fish pepsinogens.

The amino acid sequences of three pepsinogens (PG1, PG2 and PG3) of Pacific bluefin tuna (Thunnus orientalis) were deduced by cloning and nucleotide sequencing of the corresponding cDNAs. The amino acid sequences of the pre-forms of PG1, PG2 and PG3 were composed of a signal peptide (16 residues each), a propeptide (41, 37 and 35 residues, respectively) and a pepsin moiety (321, 323 and 332 residues, respectively). Amino acid sequence comparison and phylogenetic analysis indicated that PG1 and PG2 belong to the pepsinogen A family and PG3 to the pepsinogen C family. Homology modeling of the three-dimensional structure suggested that the remarkably high specific activity of PG2 toward hemoglobin, which had been found previously, was partly due to a characteristic deletion of several residues in the S1'-loop region that widens the space of the active site cleft region so as to accommodate protein and larger polypeptide substrates more efficiently. Including the tuna and all other fish pepsinogen sequences available to date, the molecular phylogenetic comparison was made with reference to evolution of fish pepsinogens. It was suggested that functional divergences of pepsinogens (pepsins) occurring in fishes as well as in mammals, correlated with differences in various aspects of fish physiology.

Fast detection of phosphorylation of human pepsinogen A, human pepsinogen C and swine pepsinogen using a combination of reversed-phase high-performance liquid chromatography and capillary zone electrophoresis for peptide mapping.

A combination of reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary zone electrophoresis (CZE) was used for characterization of alpha-chymotryptic digests of human pepsinogen A, human pepsinogen C (both isolated from stomach mucosa of patients suffering from gastric cancer), swine pepsinogen and their dephosphorylated forms. Combining RP-HPLC and CZE for peptide mapping allowed to detect phosphorylations in molecules of the above mentioned gastric zymogens. We have found one phosphate group in the molecule of human pepsinogen A and two phosphate groups in the molecule of human pepsinogen C. The investigated sample was obtained from stomach mucosa of a patient suffering from gastric cancer. An increased number of phosphate groups in molecules of human pepsinogen seems to be associated with gastric cancer. The developed method represent a suitable tool for studying relationships between specific phosphorylations of proteins and cancerogenesis or potentially could serve for early diagnosis of gastric cancer.

Crystal and molecular structures of human progastricsin at 1.62 A resolution.

The crystal and molecular structures of human progastricsin (hPGC) have been determined using multiple isomorphous replacement methods and anomalous scattering in conjunction with a phased translation function. The structure has been refined to a conventional R-factor (= sigma parallel Fo magnitude of - magnitude of Fc parallel / sigma magnitude of Fo magnitude of) of 0.179 with data to 1.62 A resolution. The first 37 amino acid residues of the prosegment are similar in conformation to the equivalent residues of porcine pepsinogen (pPGN). As in pPGN, the N zeta atom of Lys37p sits between the active-site carboxylate groups of Asp32 and Asp217, thereby preventing catalysis. The side-chains of Tyr38p and Tyr9 sit in the S1' and S1 substrate-binding pockets of hPGC, respectively, in an analogous manner to what is observed in porcine pepsinogen. There are large conformational differences centered around the region containing residues Arg39p to Pro6, relative to the equivalent region in the structure of pPGN. Two surface loops in the vicinity of this segment are also displaced relative to those in pPGN and in mature aspartic proteinases (Phe71 to Thr81 (the "flap"), and Tyr125 to Thr131). In hPGC, Tyr75 O eta does not make its usual hydrogen bond to Trp39 N epsilon 1. Rather, the "flap" containing Tyr75 is excluded from the active site by the polypeptide segment Arg39p to Pro6. However, the conformation of the inhibitory segment, Lys37p to Tyr38p, is virtually identical with that observed in pPGN. Hence the structures of these two proteins indicate that aspartic proteinase zymogens keep themselves inactive at neutral pH by a very similar mechanism in human progastricsin and porcine pepsinogen. This similarity likely carries over to all members of both the pepsinogen A and C families of aspartic proteinase zymogens.

Rearranging the domains of pepsinogen.

Most eukaryotic aspartic protease zymogens are synthesized as a single polypeptide chain that contains two distinct homologous lobes and a pro peptide, which is removed upon activation. In pepsinogen, the pro peptide precedes the N-terminal lobe (designated pep) and the C-terminal lobe (designated sin). Based on the three-dimensional structure of pepsinogen, we have designed a pepsinogen polypeptide with the internal rearrangement of domains from pro-pep-sin (native pepsinogen) to sin-pro-pep. The domain-rearranged zymogen also contains a 10-residue linker designed to connect sin and pro domains. Recombinant sin-pro-pep was synthesized in Escherichia coli, refolded from 8 M urea, and purified. Upon acidification, sin-pro-pep autoactivates to a two-chain enzyme. However, the emergence of activity is much slower than the conversion of the single-chain zymogen to a two-chain intermediate. In the activation of native pepsinogen and sin-pro-pep, the pro region is cleaved at two sites between residues 16P and 17P and 44P and 1 successively, and complete activation of sin-pro-pep requires an additional cleavage at a third site between residues 1P and 2P. In pepsinogen activation, the cleavage of the first site is rate limiting because the second site is cleaved more rapidly to generate activity. In the activation of sin-pro-pep, however, the second site is cleaved slower than the first, and cleavage of the third site is the rate limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)

Human procathepsin D: three-dimensional model and isolation.

Human procathepsin D was isolated from medium of human breast cancer cell line ZR-75-1 potentiated with estrogen. The isolation involved both immunoaffinity chromatography and ion-exchange chromatography. The affinity chromatography employed polyclonal antibodies raised against a synthetic activation peptide of human cathepsin D. We have started preliminary crystallization trials using the isolated material. A model of human procathepsin D was also built using coordinates of human cathepsin D and pig pepsinogen. The model aids understanding of multiple roles played by activation peptides of aspartic proteinases and will be used as a starting model for molecular replacement.

Multiply charged negative ions by electrospray ionization of polypeptides and proteins.

Multiply deprotonated polypeptide and protein molecules, (M - nH)n-, produced from pH approximately 11 aqueous solutions, are analyzed by electrospray ionization-mass spectrometry (ESI-MS). Aqueous ammonium hydroxide solutions of the analyte are shown to be preferable to sodium hydroxide solutions for negative-ion ESI due to the production of multiply sodiated protein species from the latter system. Proteins with Mr to 66,000 and having up to 57 negative charges have been detected. Multiply charged negative ions can be produced from ESI of the highly acidic protein pepsin (Mr approximately 34,600) because of its relatively large number of acidic residues, 42. In contrast, the small number of basic amino acid residues for pepsin (4) does not allow formation of highly protonated species essential for positive-ion detection, for mass spectrometers of limited m/z range. Similarly, negative-ion ESI-MS is extended to large oligosaccharide analysis. Preliminary tandem mass spectrometry experiments of multiply charged polypeptide anions demonstrate the utility and potential of negative-ion ESI-MS for structural elucidation.

Development-dependent expression of isozymogens of monkey pepsinogens and structural differences between them.

The developmental changes in the expression of monkey pepsinogens and structural differences between the polypeptides were investigated. Monkey pepsinogens included five different components, namely, pepsinogens A-(1-4) and progastricsin. Their respective relative levels and specific activities changed significantly during development. The sequential expression of genes for type-A pepsinogens was particularly noteworthy. Pepsinogen A-3 was the major zymogen at the newborn stage, accounting for nearly half of the total pepsinogens at this stage. Pepsinogen A-2 became predominant at the 4-month stage, and pepsinogen A-1 predominated at the juvenile and adult stages. Enzymatic properties of pepsinogens A-1, A-2 and A-3 were similar but not identical to those of pepsinogen A-4 and progastricsin, in particular with respect to the activation processes. Each pepsin digested various protein substrates but some differences in specificity were evident. cDNA clones for five pepsinogens were isolated, and the nucleotide sequences were determined. Each cDNA contained leader, pro, and pepsin regions that encoded 15, 47, and 326 amino acid residues, respectively, with the exception of the cDNA for progastricsin in which the pro and pepsin regions encoded 43 and 329 amino acid residues, respectively. Type-A pepsinogens exhibited a high degree of similarity, with over 96% of bases in the nucleotide sequences of the protein-coding regions being identical. Northern analysis revealed that the level of expression of genes for type-A pepsinogens and for progastricsin was significant at the fetal stage and increased with development.

Protein modeling of human prorenin using the molecular dynamics method.

To study the activation-inactivation mechanism of the renin zymogen, prorenin, a tertiary structural model of human prorenin was constructed using computer graphics and molecular dynamics calculations, based on the pepsinogen structure. This prorenin model shows that the folded prosegment polypeptide can fit into the substrate binding cleft of the renin moiety. The three positively charged residues, Arg10, Arg15, and Arg20, in the prosegment make salt bridges with Asp225, Glu331, and Asp60, respectively, in renin. Arg43, which is in the processing site, forms salt bridges with the catalytic residues of Asp81 and Asp269. These ionic interactions between the prosegment and the renin may contribute to keeping the prorenin structure as an inactive form.