Pepsin B Research Articles

An analysis of subdomain orientation, conformational change and disorder in relation to crystal packing of aspartic proteinases

The analysis reported here describes detailed structural studies of endothiapepsin (the aspartic proteinase from Endothia parasitica), with and without bound inhibitors, and human pepsin 3b. Comparison of multiple crystal structures of members of the aspartic proteinase family has revealed small but significant differences in domain orientation in different crystal forms. In this paper, it is shown that these differences in domain orientation do not necessarily correlate with the presence or absence of bound inhibitors, but appear to stem at least partly from crystal contacts mediated by sulfate ions. However, since the same inherent flexibility of the structure is observed for other enzymes in this family such as human pepsin, the native structure of which is also reported here, the observed domain movements may well have implications for the mechanism of catalysis.

Receptor-Mediated Uptake of Pepsin by Laryngeal Epithelial Cells

Previous data suggest a mechanistic link between exposure to pepsin and cellular changes that lead to laryngopharyngeal disorders. Initial confocal microscopy analysis of pepsin uptake by cultured hypopharyngeal epithelial cells revealed that pepsin may be taken up by a specific process. The objective of this study was to use electron microscopy to confirm the initial confocal findings and to determine whether uptake of pepsin by laryngeal epithelial cells is receptor-mediated. Cultured human hypopharyngeal FaDu cells and human laryngeal biopsy specimens, taken from the posterior larynx of "control" patients without symptoms or findings of laryngopharyngeal reflux, were exposed to purified human pepsin 3b with or without transferrin (a marker for receptor-mediated endocytosis) in vitro. Uptake of pepsin was documented by electron microscopy. Pepsin co-localized with transferrin in intracellular vesicles; this finding confirms that pepsin is taken up by laryngeal epithelial cells by receptor-mediated endocytosis. This is a novel finding that further defines the role and mechanism of pepsin-mediated injury in laryngopharyngeal reflux. The objective of ongoing research is to identify the receptor and investigate potential antagonists as a new therapeutic option for patients with reflux-attributed disease--in particular, those patients who have persistent symptoms despite acid suppression therapy.

Activity/Stability of Human Pepsin: Implications for Reflux Attributed Laryngeal Disease

Exposure of laryngeal epithelia to pepsin during extra-esophageal reflux causes depletion of laryngeal protective proteins, carbonic anhydrase isoenzyme III (CAIII), and squamous epithelial stress protein Sep70. The first objective of this study was to determine whether pepsin has to be enzymatically active to deplete these proteins. The second objective was to investigate the effect of pH on the activity and stability of human pepsin 3b under conditions that might be found in the human esophagus and larynx. Prospective translational research study. An established porcine in vitro model was used to examine the effect of active/inactive pepsin on laryngeal CAIII and Sep70 protein levels. The activity and stability of pepsin was determined by kinetic assay, measuring the rate of hydrolysis of a synthetic pepsin-specific substrate after incubation at various pH values for increasing duration. Active pepsin is required to deplete laryngeal CAIII and Sep70. Pepsin has maximum activity at pH 2.0 and is inactive at pH 6.5 or higher. Although pepsin is inactive at pH 6.5 and above, it remains stable until pH 8.0 and can be reactivated when the pH is reduced. Pepsin is stable for at least 24 hours at pH 7.0, 37 degrees C and retains 79% +/- 11% of its original activity after re-acidification at pH 3.0. Detectable levels of pepsin remain in laryngeal epithelia after a reflux event. Pepsin bound there would be enzymatically inactive because the mean pH of the laryngopharynx is pH 6.8. Significantly, pepsin could remain in a form that would be reactivated by a subsequent decrease in pH, such as would occur during an acidic reflux event or possibly after uptake into intracellular compartments of lower pH.

Review article: human pepsins – their multiplicity, function and role in reflux disease

Human gastric juice contains a multiplicity of proteinases. These are classified as aspartic proteinases because of enzymic activity dependent on two oppositely placed aspartic acids in the active site region. At least seven zones of activity can be visualized by agar gel electrophoresis and a similar number of separate proteins resolved by high performance ion exchange chromatography. The major enzyme secreted (up to 70% of the total) pepsin 3b is sensitive to the selective inhibitor pepstatin whereas gastricsin or pepsin 5 (20% of the total) is not. Minor enzymes including pepsin 1, which has an associated proteincarbohydrate complex attached is variable and can be < 5% in normal and up to 20% of the total as in peptic ulcer patients. The activity of these enzymes depends on the substrate and pH with significant digestion occurring up to pH 4.5. It has also been shown that these enzymes can bind to substrates like collagen up to pH 5.5. In gastric secretion studies of patients with reflux oesophagitis the amount of pepsin and the profile of the enzymes in basal secretions, and that after pentagastrin stimulation, was found to be not different from healthy non-refluxers. Thus the problem with reflux is that gastric juice appears in the oesophagus, an area without any natural protection from proteolytic damage. The ability to reduce gastric secretion is therefore important in effective treatment. However, being able also to inhibit enzymic activity or protect substrates from damage using alginates offers considerable scope for future therapies.

Five human gastric aspartic proteinases: N-terminal amino acid sequences and amino acid composition

Human pepsin A consists of 4 or more isoenzymes (designated 1, 3a, 3b and 3c) one of which, pepsin 1, contains up to 50% carbohydrate moieties. The amino-acid composition and N-terminal sequence of pepsin 1 and the other isoforms have been determined and compared with data obtained for pepsin 3b and gastricsin (pepsin C or pepsin 5). Pepsins were isolated from penta-gastrin stimulated gastric juice using repetitive chromatography on DEAE-cellulose, or high performance ion-exchange chromatography. Sequencing was performed using automated solid-phase Edman degradation with a microsequence facility. The amino-acid compositions were similar for pepsins 1, 3a, b and c and the N-terminal sequences of pepsins 1, 3a and c, reported for the first time, were shown to be identical with that for pepsin 3b (the main component of pepsin A) although residue 28 was unassigned in pepsin 1. Residue 30 in all four isoenzymes is valine and we cannot confirm reports of major pepsins with leucine in this position. For gastricsin the sequence differed from the pepsin isoenzymes and in position 24 we find pro rather than ala as was first described. These observations suggest that pepsin 1 is identical to 3b or a mixture of 3a, 3b and 3c but not gastricsin. This data supports the hypotheses that the four pepsin isoenzymes are products of the same gene(s) but have undergone varying levels of post translational modification.

Human pepsin 3b peptide map sequence analysis, genotype and hydrophobic nature

Peptides from a Staphylococcus aureus (V8) proteinase digest of human pepsin 3b have been identified by amino acid sequence analysis. Only 137 out of 326 expected residues were detected from the C and N terminal regions of the molecule. Comparison with amino acid sequences derived from nucleotide analysis of three different pepsinogen A genes, identified 2 out of 4 possible substitutions. The presence of valine at position 30 and leucine at 291 indicates that the major pepsin component of gastric juice, pepsin 3b, corresponds to pepsinogen genotype PGA-3. Reversed-phase chromatography of native human pepsin 3b on C 4 (300 Å), C 18 (300 Å) or polymer (1000 Å) columns was optimal on the C 4 column and gradient elution with 2-propanol rather than acetonitrile. Denaturation of the protein in guanidinium hydrochloride, urea or high pH resulted in irreversible column retention. The marked hydrophobicity of denatured pepsin 3b may thus explain why the central segment of the protein was not revealed by peptide map analysis.

Peptide maps of five human pepsin isoenzymes and other aspartic proteinases

Peptide maps of five individual human pepsins were developed using reversed-phase high-performance liquid chromatography after protein digestion with either Staphylococcus aureus proteinase (V8) or α-chymotrypsin, Human pepsins, 3a, 3b and 3c produced almost identical peptide maps suggestive of proteins with very similar amino acid sequences. The map for human pepsin 1 was similar to pepsin 3b (the most predominant human pepsin) but less than half the expected amount of each equivalent peptide fragment was generated, indicating that the actual mass of digested protein used was less than the dry weight measurement would suggest, probably as a result of carbohydrate attached to pepsin 1. Comparison of human pepsin 3b maps with other aspartic proteinases confirmed a significant homology with swine pepsine A but not with endothiapepsin. The α-chymotrypsin digests compared with V8 gave more complex peptide maps as a result of its broader bond cleavage specificities.