Esophageal pepsin and proton pump synthesis in barrett's esophagus and esophageal adenocarcinoma.

OBJECTIVES/HYPOTHESIS: Gastroesophageal reflux disease and associated metaplasia of the esophagus (Barrett's esophagus [BE]) are primary risk factors for esophageal adenocarcinoma (EAC). Widespread use of acid suppression medications has failed to stem the rise of EAC, suggesting that nonacid reflux may underlie its pathophysiology. Pepsin is a tumor promoter in the larynx and has been implicated in esophageal carcinogenesis. Herein, specimens from the esophageal cancer spectrum were tested for pepsin presence. Pepsin-induced carcinogenic changes were assayed in an esophageal cell culture model. STUDY DESIGN: Laboratory analysis. METHODS: Pepsin was assayed in reflux and cancer free esophagi, BE, EAC, and esophageal cancer lacking association with reflux (squamous cell carcinoma [SCC]). Refluxed or locally synthesized pepsin was assayed by Western blot. Local synthesis of pepsin and proton pumps was assayed via reverse transcription-polymerase chain reaction. The effect of pepsin on BE and EAC markers was investigated via enzyme-linked immunosorbent assay and quantitative polymerase chain reaction in human esophageal epithelial cells treated with pepsin or control diluent. RESULTS: Pepsinogen and proton pump mRNA were observed in BE (3/5) and EAC (4/4) samples, but not in normal adjacent specimens, SCC (0/2), or reflux and cancer-free esophagi. Chronic pepsin treatment (0.1-1 mg/mL, 4 weeks) of human esophageal cells in vitro induced BE and EAC markers interleukin 8 and KRT8 and depleted normal esophageal marker KRT10 (P < .05) expression. CONCLUSIONS: Local synthesis of pepsin and proton pumps in BE and EAC is not uncommon. Absence of these molecules in normal (noncancer) esophagi, SCC, and in vitro data support a role for pepsin in reflux-attributed carcinogenic changes in the esophagus. LEVEL OF EVIDENCE: NA Laryngoscope, 129:2687-2695, 2019.

Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively.

Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin.

Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity.

Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans.

The widespread presence of pepsin-like enzymes in eukaryotes together with their relevance in the control of multiple biological processes is reflected in the large number of studies published so far for this family of enzymes. By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from Shewanella amazonensis provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the pepsin homolog from Shewanella denitrificans, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the enzymatic properties of recombinant shewasin D are strongly reminiscent of eukaryotic pepsin homologues. We determined the specificity preferences of both shewasin D and shewasin A using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1, thereby confirming their phylogenetic proximity. Moreover, we provide first evidence of expression of active shewasin D in S. denitrificans cells, confirming its activity at acidic pH and inhibition by pepstatin. Finally, our results revealed an unprecedented localization for a family A1 member by demonstrating that native shewasin D accumulates preferentially in the cytoplasm.

Effect of insoluble fiber supplementation applied at different ages on digestive organ weight and digestive enzymes of layer-strain poultry.

Two experiments were conducted to study effects of dietary insoluble fiber (IF) on digestive enzyme function in layer poultry. In Experiment 1, 8 wk old pullets were fed a control diet (Group C) or a diet (Group IF) supplemented with 1% IF (Arbocel RC). After 5 wk, 6 pullets per group were killed and organ samples collected. The remaining pullets in Group C were divided into two groups: half were fed the control diet (Group C) and half were given the IF diet (Group C-IF). Similarly, half the pullets in Group IF continued on the IF diet (Group IF) and half on the control diet (Group IF-C). At 10 wk, organ samples were collected. BW at wk 5 (IF, 1364.8 g; C, 1342.9 g) and 10 wk (IF, 1678.1 g; IF-C, 1630.5 g; C-IF, 1617.1 g; C, 1580.4 g) were not different. At wk 5, the relative proventricular weight (0.41 g/100 g BW) and activities of pepsin (75.3 pepsin units/g proventriculus/min) and pancreatic general proteolytic activity (GP) (122.9 µmol tyrosine produced/g tissue) were greater (P < 0.05) than those of Group C (proventricular relative weight, 0.36; pepsin activity, 70.6; GP activity, 94.3). At wk 10, relative weights of liver and gizzard of Group IF were heavier (P < 0.05) than other treatments; activities of pepsin, GP, trypsin and chymotrypsin of IF pullets were significantly greater than other treatments as was mRNA expression for pepsinogens A (25.9 vs. 22.9) and C (13.1 vs. 10.8). In Experiment 2, 19 wk old hens were fed a control diet or a diet containing 0.8% IF (Arbocel RC) for 12 wk. Final BW after 12 wk was not different (IF, 1919.4 g; C, 1902.1 g). Pancreatic GP activity was greater (P < 0.05) in Group IF hens than Group C at wk 12 (122.2 vs. 97.0 µmol tyrosine released/min/g tissue)) as was relative gizzard weight (1.32 vs 1.10 g/100 g BW). The significantly improved digestive organ weights and enzyme activities in IF pullets may contribute to an improvement in feed utilization.

Structure, molecular evolution, and hydrolytic specificities of largemouth bass pepsins.

The nucleotide sequences of largemouth bass pepsinogens (PG1, 2 and 3) were determined after molecular cloning of the respective cDNAs. Encoded PG1, 2 and 3 were classified as fish pepsinogens A1, A2 and C, respectively. Molecular evolutionary analyses show that vertebrate pepsinogens are classified into seven monophyletic groups, i.e. pepsinogens A, F, Y (prochymosins), C, B, and fish pepsinogens A and C. Regarding the primary structures, extensive deletion was obvious in S'1 loop residues in fish pepsin A as well as tetrapod pepsin Y. This deletion resulted in a decrease in hydrophobic residues in the S'1 site. Hydrolytic specificities of bass pepsins A1 and A2 were investigated with a pepsin substrate and its variants. Bass pepsins preferred both hydrophobic/aromatic residues and charged residues at the P'1 sites of substrates, showing the dual character of S'1 sites. Thermodynamic analyses of bass pepsin A2 showed that its activation Gibbs energy change (∆G(‡)) was lower than that of porcine pepsin A. Several sites of bass pepsin A2 moiety were found to be under positive selection, and most of them are located on the surface of the molecule, where they are involved in conformational flexibility. The broad S'1 specificity and flexible structure of bass pepsin A2 are thought to cause its high proteolytic activity.

Linear models of ovine IgG1 and IgG2 subclasses and predicted pepsin cleavage sites.

Highly purified specific Fab antibody fragments derived from sheep have a long history of therapeutic use as safe and effective emergency medicines. In more recent years simple low-cost methods have been developed, which take advantage of the ability of pepsin under optimally controlled conditions to preferentially digest ovine IgG within the Fc region to produce F(ab')2 and easy to remove low MW Fc sub-fragments. Despite these developments no information is currently available on the pepsin digestion of ovine IgG at the amino acid level hindering the development of improved F(ab')2 processing methods. To gain knowledge of the fragments properties we have constructed linear models of ovine IgG1 and IgG2 subclasses, starting from the gamma-1 and gamma-2 chain amino acid sequences, which also incorporate the inter- and intra-chain disulphide bonds. Any potential pepsin cleavage site was initially predicted in silico, then high probability points identified for each of the molecules and mapped onto the individual models. A theoretical order of digestion was subsequently constructed, which appeared to agree with the experimental data, suggesting an accurate prediction model for ovine IgG1 and IgG2 subclasses. These findings lay the foundations for a more detailed analysis of pepsin cleavage fragments in the future. Additionally, the F(ab')2 generated following pepsin digestion were predicted to contain subclass unique C-terminal octapeptide neoepitopes, despite the high 89% sequence identity of the intact gamma-1 and gamma-2 chain constant regions. These neoepitopes have the potential to be utilised for identification purposes once confirmed experimentally.

Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster.

Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.

Structure-Based Design of Mucor pusillus Pepsin for the Improved Ratio of Clotting Activity/Proteolytic Activity in Cheese Manufacture.

Previous theoretical studies have determined the intermolecular interactions between Mucor pusillus pepsin (MPP) and the key domain of κ-casein, with the aim to understand the mechanism of milk clotting in the specific hydrolysis of κ-casein by MPP for cheese making. Here, we combined the docking model with site-directed mutagenesis to further investigate the functional roles of amino acid residues in the active site of MPP. T218S replacement caused a low thermostability and moderate increase in the clotting activity. Mutations of three amino acid residues, T218A and T218S in S2 region and L287G in S4 region, led to a significant decrease in proteolytic activity. For T218S and L287G, an increase in the ratio of clotting activity to proteolytic activity (C/P) was observed, in particular 3.34-fold increase was found for T218S mutants. Structural analysis of the binding mode of MPP and chymosin splitting domain (CSD) of κ-casein indicated that T218S plays a critical role in forming a hydrogen bond with the hydroxyl group of Ser(104) around the MPP-sensitive Phe(105)-Met(106) peptide bond of κ- casein and L287G is partially responsible for CSD accommodation in a suitable hydrophobic environment. These data suggested that T218S mutant could serve as a promising milk coagulant that contributes to an optimal flavor development in mature cheese.

Conserved prosegment residues stabilize a late-stage folding transition state of pepsin independently of ground states.

The native folding of certain zymogen-derived enzymes is completely dependent upon a prosegment domain to stabilize the folding transition state, thereby catalyzing the folding reaction. Generally little is known about how the prosegment accomplishes this task. It was previously shown that the prosegment catalyzes a late-stage folding transition between a stable misfolded state and the native state of pepsin. In this study, the contributions of specific prosegment residues to catalyzing pepsin folding were investigated by introducing individual Ala substitutions and measuring the effects on the bimolecular folding reaction between the prosegment peptide and pepsin. The effects of mutations on the free energies of the individual misfolded and native ground states and the transition state were compared using measurements of prosegment-pepsin binding and folding kinetics. Five out of the seven prosegment residues examined yielded relatively large kinetic effects and minimal ground state perturbations upon mutation, findings which indicate that these residues form strengthened and/or non-native contacts in the transition state. These five residues are semi- to strictly conserved, while only a non-conserved residue had no kinetic effect. One conserved residue was shown to form native structure in the transition state. These results indicated that the prosegment, which is only 44 residues long, has evolved a high density of contacts that preferentially stabilize the folding transition state over the ground states. It is postulated that the prosegment forms extensive non-native contacts during the process of catalyzing correct inter- and intra-domain contacts during the final stages of folding. These results have implications for understanding the folding of multi-domain proteins and for the evolution of prosegment-catalyzed folding.

Understanding the mechanism of prosegment-catalyzed folding by solution NMR spectroscopy.

Multidomain protein folding is often more complex than a two-state process, which leads to the spontaneous folding of the native state. Pepsin, a zymogen-derived enzyme, without its prosegment (PS), is irreversibly denatured and folds to a thermodynamically stable, non-native conformation, termed refolded pepsin, which is separated from native pepsin by a large activation barrier. While it is known that PS binds refolded pepsin and catalyzes its conversion to the native form, little structural details are known regarding this conversion. In this study, solution NMR was used to elucidate the PS-catalyzed folding mechanism by examining the key equilibrium states, e.g. native and refolded pepsin, both in the free and PS-bound states, and pepsinogen, the zymogen form of pepsin. Refolded pepsin was found to be partially structured and lacked the correct domain-domain structure and active-site cleft formed in the native state. Analysis of chemical shift data revealed that upon PS binding refolded pepsin folds into a state more similar to that of pepsinogen than to native pepsin. Comparison of pepsin folding by wild-type and mutant PSs, including a double mutant PS, indicated that hydrophobic interactions between residues of prosegment and refolded pepsin lower the folding activation barrier. A mechanism is proposed for the binding of PS to refolded pepsin and how the formation of the native structure is mediated.

Role of pepsin and pepsinogen: linking laryngopharyngeal reflux with otitis media with effusion in children.

OBJECTIVES/HYPOTHESIS: To analyze the relationship between laryngopharyngeal reflux (LPR) represented by pepsin and pepsinogen, and pathogenesis of otitis media with effusion (OME). STUDY DESIGN: Prospective case-control study. METHODS: Children with OME who required adenoidectomy and tympanostomy/tympanostomy tubes placement were enrolled in OME group, whereas children with adenoid hypertrophy (AH) who required adenoidectomy and individuals who required cochlear implantation (CI) were enrolled in AH and CI groups, respectively. Pepsinogen mRNA and protein levels were assessed by real-time fluorescence-based quantitative polymerase chain reaction and immunohistochemistry in adenoid specimens from the OME and AH groups. Pepsin and pepsinogen concentrations were evaluated by enzyme-linked immunosorbent assay in middle ear fluid and plasma from the OME and CI groups. RESULTS: The levels of pepsinogen protein expressed in cytoplasm of epithelial cells and clearance under epithelial cells in adenoid specimens from the OME group were significantly higher than those in the AH group. Furthermore, the concentrations of pepsin and pepsinogen in the OME group were 51.93±11.58 ng/mL and 728±342.6 ng/mL, respectively, which were significantly higher than those in the CI group (P<.001). In addition, the concentrations of pepsin in dry ears were significantly lower than those in serous and mucus ears in the OME group (F=22.77, P<.001).Finally, the concentration of pepsinogen in middle ear effusion was positively correlated with the expression intensity of pepsinogen protein in cytoplasm of epithelial cells (r=0.73, P<.05) in the OME group. CONCLUSIONS: Pepsin and pepsinogen in middle ear effusion are probably caused by LPR and may be involved in the pathogenesis of OME. LEVEL OF EVIDENCE: 3b.

Pepsin-like aspartic protease (Sc-ASP155) cloning, molecular characterization and gene expression analysis in developmental stages of nematode Steinernema carpocapsae.

Steinernema carpocapsae is an insect parasitic nematode associated with the bacterium Xenorhabdus nematophila. These symbiotic complexes are virulent against the insect host. Many protease genes were shown previously to be induced during parasitism, including one predicted to encode an aspartic protease, which was cloned and analyzed in this study. A cDNA encoding Sc-ASP155 was cloned based on the EST fragment. The full-length cDNA of Sc-ASP155 consists of 955 nucleotides with multiple domains, including a signal peptide (aa1-15), a pro-peptide region (aa16-45), and a typical catalytic aspartic domain (aa71-230). The putative 230 amino acid residues have a calculated molecular mass of 23,812Da and a theoretical pI of 5.01. Sc-ASP155 blastp analysis showed 40-62% amino acid sequence identity to aspartic proteases from parasitic and free-living nematodes. Expression analysis showed that the sc-asp155 gene was up-regulated during the initial parasitic stage, especially in L3 gut and 6h induced nematodes. Sequence comparison revealed that Sc-ASP155 was a member of an aspartic protease family and phylogenetic analysis indicated that Sc-ASP155 was clustered with Sc-ASP113. In situ hybridization showed that sc-asp155 was expressed in subventral cells. Additionally, we determined that sc-asp155 is a single-copy gene in S. carpocapsae. Homology modeling showed that Sc-ASP155 adopts a typical aspartic protease structure. The up-regulated Sc-ASP155 expression revealed that this protease could play a role in the parasitic process. In this study, we have cloned the gene and determined the expression of the pepsin-like aspartic protease Sc-ASP155 in S. carpocapsae.

The presence of pepsin in the lung and its relationship to pathologic gastro-esophageal reflux.

BACKGROUND: Pepsin has been proposed as a biomarker of reflux-related lung disease. The goal of this study was to determine (i) if there is a higher reflux burden as measured by pH-MII in patients that are pepsin positive in the lung, and (ii) the sensitivity of pepsin in predicting pathologic reflux by pH, MII, and EGD. METHODS: We recruited children between the ages of 1-21 with chronic cough or asthma undergoing bronchoscopy, esophagogastroduodenoscopy (EGD), and multichannel intraluminal impedance (pH-MII) probe placement. The reflux profiles were compared between those patients who were pepsin positive and negative; proportions were compared using Chi-squared analyses and means were compared using t-testing. KEY RESULTS: Only the mean number of non-acid reflux events was associated with pepsin positivity (0.04). The sensitivity and specificity of pepsin in predicting pathologic reflux by pH-MII or EGD was 57% and 65%, respectively. The positive predictive value of pepsin in predicting pathologic reflux by pH, MII or EGD was 50% (11/22), and the negative predictive value was 71% (20/28). There was a significantly higher mean LLMI in patients who were pepsin positive compared with pepsin negative patients (81 ± 54 vs 47 ± 26, P = 0.001). CONCLUSIONS & INFERENCES: Lung pepsin cannot predict pathologic reflux in the esophagus, but its correlation with lung inflammation suggests that pepsin may be an important biomarker for reflux-related lung disease.

Expression and activity of trypsin and pepsin during larval development of the spotted rose snapper Lutjanus guttatus.

The present study aimed to describe and understand the development of the digestive system in spotted rose snapper (Lutjanus guttatus) larvae from hatching to 40 days post-hatch (dph). The mouth opened between 2 and 3 dph, at that moment the digestive tract was barely differentiated into the anterior and posterior intestine, although the liver and pancreas were already present. Gastric glands were observed until 20 dph, followed by the differentiation of the stomach between 20 and 25 dph. Trypsinogen expression and trypsin activity were detected at hatching, increasing concomitantly to larval development and the change in the type of food. Maximum levels of trypsinogen expression were observed at 25 dph, when animals were fed with Artemia nauplii, and maximum trypsin activity was detected at 35 dph, when larvae were fed with an artificial diet. On the other hand, pepsinogen gene expression was detected at 18 dph, two days before pepsin enzymatic activity and appearance of gastric glands. Maximum pepsin activity was also observed at 35 dph. These results suggest that in this species weaning could be initiated at an earlier age than is currently practiced (between 28 and 30 dph), since larvae of spotted rose snapper develop a functional stomach between days 20 and 25 post-hatch.

Shewasin A, an active pepsin homolog from the bacterium Shewanella amazonensis.

The view has been widely held that pepsin-like aspartic proteinases are found only in eukaryotes, and not in bacteria. However, a recent bioinformatics search [Rawlings ND & Bateman A (2009) BMC Genomics10, 437] revealed that, in seven of ∼ 1000 completely sequenced bacterial genomes, genes were present encoding polypeptides that displayed the requisite hallmark sequence motifs of pepsin-like aspartic proteinases. The implications of this theoretical observation prompted us to generate biochemical data to validate this finding experimentally. The aspartic proteinase gene from one of the seven identified bacterial species, Shewanella amazonensis, was expressed in Escherichia coli. The recombinant protein, termed shewasin A, was produced in soluble form, purified to homogeneity, and shown to display properties remarkably similar to those of pepsin-like aspartic proteinases. Shewasin A was maximally active at acidic pH values, cleaving a substrate that has been widely used for assessment of the proteolytic activity of other aspartic proteinases, and displayed a clear preference for cleaving peptide bonds between hydrophobic residues in the P1*P1' positions of the substrate. It was completely inhibited by the general inhibitor of aspartic proteinases, pepstatin, and mutation of one of the catalytic Asp residues (in the Asp-Thr-Gly motif of the N-terminal domain) resulted in complete loss of enzymatic activity. It can thus be concluded unequivocally that this Shewanella gene encodes an active pepsin-like aspartic proteinase. It is now beyond doubt that pepsin-like aspartic proteinases are not confined to eukaryotes, but are encoded within some species of bacteria. The distinctions between the bacterial and eukaryotic polypeptides are discussed and their evolutionary relationships are outlined.

Purification and characterization of pepsinogens and pepsins from the stomach of rice field eel (Monopterus albus Zuiew).

Three pepsinogens (PG1, PG2, and PG3) were highly purified from the stomach of freshwater fish rice field eel (Monopterus albus Zuiew) by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephacryl S-200 HR. The molecular masses of the three purified PGs were all estimated as 36 kDa using SDS-PAGE. Two-dimensional gel electrophoresis (2D-PAGE) showed that pI values of the three PGs were 5.1, 4.8, and 4.6, respectively. All the PGs converted into corresponding pepsins quickly at pH 2.0, and their activities could be specifically inhibited by aspartic proteinase inhibitor pepstatin A. Optimum pH and temperature of the enzymes for hydrolyzing hemoglobin were 3.0-3.5 and 40-45 °C. The K (m) values of them were 1.2 × 10⁻4 M, 8.7 × 10⁻5 M, and 6.9 × 10⁻5 M, respectively. The turnover numbers (k(cat)) of them were 23.2, 24.0, and 42.6 s⁻¹. Purified pepsins were effective in the degradation of fish muscular proteins, suggesting their digestive functions physiologically.

Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition.

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.

Pepsin degradation of Cry1A(b) protein purified from genetically modified maize (Zea mays).

The aim of this work was to study the in vitro digestion of Cry1A(b) protein by pepsin. To perform this work, a protein fraction purified from transgenic maize by immunoadsorption was employed. The undigested fraction showed several bands of molecular weight ranging between 14 and 70 kDa when assayed by SDS-PAGE. These bands were identified as corresponding to Cry1A(b) protein by immunochemical techniques and mass spectrometry. The rate of degradation of the purified fraction by pepsin estimated by ELISA was found to be about 75% within 30 min, and the protein concentration remained constant up to 4 h. In all treated samples, the full-length protein and fragments present in Cry1A(b) fraction were absent and peptides of less than 8.5 kDa were mainly found by SDS-PAGE and mass spectrometry. These peptides did not react with antiserum against Cry1A(b) protein by Western blotting. These results suggest that Cry1A(b) fraction purified from transgenic maize is rapidly and extensively degraded by pepsin, giving peptides of low molecular mass.

Functional chimera of porcine pepsin prosegment and Plasmodium falciparum plasmepsin II.

Proplasmepsin II (zPMII) represents a unique member of the aspartic proteinase family, with a prosegment-enzyme interaction that is thus far unique among the pepsin-like proteases. The role of the prosegment in aspartic proteinase structure and function was investigated by generating two chimeric proteins, one with the pepsinogen prosegment fused to the mature region of PMII (pepproPMII) and a second with the prosegment of PMII fused to pepsin (PMIIpropep). Both chimeras were expressed using Escherichia coli; however, PMIIpropep was extremely unstable suggesting protein misfolding. Alternatively, pepproPMII was capable of both autoactivation and hydrolysis of a synthetic substrate. Similarly, when the PMII enzyme was expressed without a prosegment, it too exhibited activity against the synthetic enzyme. CD measurements indicated that pepproPMII had reduced thermal stability when compared with zPMII. This reduction of temperature stability may have resulted from the inability of the pepsinogen prosegment to stabilize the C-terminal domain of the PMII enzyme. The ability of PMII to fold in the presence of a completely non-homologous prosegment and in its absence suggests that prosegment is not critical to obtaining a functional enzyme in all pepsin-like enzymes but likely plays a role in protein stabilization.