9 Review Article Page 1 of 9 Pepsin properties, structure, and its accurate measurement: a narrative review Kyle J. Stanforth1, Matthew D. Wilcox1^, Peter I. Chater1^, Iain A. Brownlee2, Maria I. Zakhour1, Katherine M. R. M. Banecki3, Jeffery P. Pearson1^ 1Newcastle University Biosciences Institute, Medical School, Framlington Place, Newcastle upon Tyne, Tyne and Wear, UK; 2Applied Sciences, Northumbria University, 2Sandyford Rd, Newcastle upon Tyne, UK; 3Department of Pharmacology, Medical Sciences Division, University of Oxford, Level 3, John Radcliffe Hospital, Oxford, UK Contributions: (I) Conception and design: All authors; (II) Administrative support: All authors; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: All authors; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Matthew D. Wilcox. Newcastle University Biosciences institute, Medical School, Framlington Place, Newcastle upon Tyne, Tyne and Wear. NE2 4HH, UK. Email: [email protected]. Abstract: Pepsin is an aspartate protease that is generated from its proenzyme, pepsinogen by autocatalysis initiated by a fall in pH below 5. Human gastric juice contains eight isoenzymes of pepsin. The peptides released on conversion of pepsinogen to pepsin of which there are potentially five, have been shown to have antimicrobial activity against a wide range of bacteria including Escherichia coli, Pseudomonas and Staphylococcus which have also been shown to have biofilm formation inhibiting properties. The stability in response to changes in pH varies between pepsin and pepsinogen. Pepsinogen is stable up to pH 10, pepsin is only stable to pH just above 7.0 and is completely denatured at pH 8.0. Many diseases of the aerodigestive tract have been linked to reflux and the presence of pepsin. Therefore, the measurement of pepsin in tissue and lavages or in saliva or sputum, could be a good screening tool for the diagnosis of reflux related disease. However, there is no current consensus as to the best methods to measure it or the best time to sample it. For an effective pepsin ELISA, the following is required; a monoclonal/monospecific polyclonal antibody with a good lowest level of detection (LLOD) and sensitivity 1–25 ng/mL (depending on dilution) and an adequate supply of purified human pepsin as a standard for antibody-based assays. If possible, an activity assay for pepsin should also be used as the presence of pepsin protein does not indicate it is capable of damaging activity. Finally, if pepsin is associated with a disease large studies are required to confirm it with multiple samples. This review deals with several studies where pepsin quantitation is attempted, and their measurement techniques assessed. Keywords: Pepsin; pepsinogen; pepsin measurement; pepsin detection Received: 01 December 2020. Accepted: 22 February 2021. doi: 10.21037/aoe-20-95 View this article at: http://dx.doi.org/10.21037/aoe-20-95 Pepsin types and structure proteins and along with acid in the protection against Pepsins are aspartate proteases active in acidic conditions ingested pathogenic organisms gaining a foothold in the and are the major proteases in human gastric juice. gastrointestinal tract. The active enzyme is derived from Pepsins are therefore important in normal digestion of its pro-enzyme pepsinogen which is secreted into the ^ ORCID of Matthew D. Wilcox: 0000-0003-0150-0106; Peter I. Chater: 0000-0001-8679-0004; Jeffery P. Pearson: 0000-0002-9816-7866. © Annals of Esophagus. All rights reserved. Ann Esophagus 2021 | http://dx.doi.org/10.21037/aoe-20-95 Page 2 of 9 Annals of Esophagus, 2021 Table 1 Antimicrobial activity of pepsinogen activation peptides Minimum inhibitory concentration (µM) Bacterial species 1–47 1–25 26–47 Escherichia coli 6.3 25 >50 Salmonella typhimurium 6.3 25 >50 Pseudomonas aeruginosa 6.3 25 25 Staphylococcus aureus 6.3 25 >50 Listeria monocytogenes >50 >50 >50 Data taken from Pane et al. (16). lumen of the gastric glands from chief cells (peptic cells). be detected, resulting in the appearance of 5 potential Pepsinogens consist of two immunological groups PGI peptides in gastric juice 1–23, 1–25, 24–47, 26–47 and 1–47 containing pepsinogens 1, 2 ,3, 4 and 5 and PGII containing (5). In order to test the antimicrobial properties of these pepsinogens 6 and 7 (1,2). Pepsinogen is a symmetrical Pane et al. expressed recombinant proteins in E. coli BL21 molecule with N and C terminal lobes with a molecular (DE3) producing the full length 47 amino acid peptide, a weight between 40–42 K (3) and is stable up to pH values 1–25 and a 26–47 amino acid peptide. The only difference of 10 (4). However, when it is exposed to pH levels below between these peptides and the original pepsinogen peptides 5, which occurs in the gastric glands, an auto-catalytic is a proline at the N-terminal which was placed in the activation occurs. This involves the removal of a section of pepsinogen sequence to produce acid labile sequences and the pesinogen’s N-terminal (5-7). The generated pepsins this proline is unlikely to affect any anti-microbial properties consist of 1, 2, 3a, 3b, 3c, 4, 5, 6, 7 and can be separated by (16,17). Computational analysis was used to identify anion exchange high performance liquid chromatography cryptic antimicrobial peptides (AMPs) based on amino acid (HPLC) and agar gel electrophoresis (8,9). Pepsin composition, charge, and hydrophobicity of known AMPs. 7 isolated from gastric mucosa also referred to as a slow- This analysis indicated that the pepsinogen peptides would moving protease is an aspartate protease. However, it is not have antimicrobial activity. The full-length peptide was a pepsin but instead a cathepsin E which is an intracellular shown to have a minimum inhibitory concentration (MIC) enzyme (10). of ≤12.5 µM against a wide range of bacterium including E. We present the following article in accordance with the coli, Pseudomonas’, and Staphylococcus’, but greater than 50 µM Narrative Review reporting checklist (available at http:// for Listeria. dx.doi.org/10.21037/aoe-20-95). The two shorter peptides performed worse but were still effective around 50 µM (Table 1). To determine if this effect also occurred at acidic conditions pertaining to Pepsin activation and function of activation the stomach the full-length peptide was tested at pH 3.5. peptides Bacterial viability was measured as CFU/mL and Salmonella The mechanism of activation by just a change in pH is typhimurium which has resistance to low pH had ~50% well understood in human and porcine pepsin (5,11). survival after 45 minutes exposure to pH 3.5. When the The activation peptide, the N-terminal of the pepsinogen full-length peptide was added this was reduced to ~20% (12). molecule which is cleaved off when pepsin is activated, has The 1–25 peptide was also effective in preventing biofilm recently been shown to have antimicrobial properties (12). formation of Pseudomonas aeruginosa and Staphylococcus Human pepsinogen has a predicted amino acid sequence of aureus. Finally, in a mouse model of Pseudomonas aeruginosa 373 amino acids (13). In human pepsin the cleaved peptide infection the full-length peptide showed 4 orders of is 47 amino acids long and is generally released as 2 peptides magnitude reduction in CFU/mL and the two shorter generating an active enzyme with a molecular weight 34–37 peptides ~2 orders of magnitude (12). KD (5,14,15). The cleavage occurs in the region Leu23- These findings highlight that pepsin’s activation peptides Lys24-Asp25-Phe26 but the full-length peptide can also generated from secreted pepsinogen may form an important © Annals of Esophagus. All rights reserved. Ann Esophagus 2021 | http://dx.doi.org/10.21037/aoe-20-95 Annals of Esophagus, 2021 Page 3 of 9 Table 2 Disease associated with reflux and identified by the from refluxate is recognised as a biomarker of reflux as well presence of pepsin as in tissue damage. In addition to pepsin, bile acids have Diseases Reference been strongly implicated when present in the refluxate as Laryngopharyngeal reflux (LPR) (28-32) biomarkers of reflux and damaging agents (19,23). However, many of the papers dealing with bile acids are using assays Gastro-oesophageal reflux (GORD) (33,34) which do not have the required sensitivity (24-28). Otitis media with effusion (OME) (35,36) Laryngomalacia (37,38) Diseases associated with reflux identified by Vocal fold leucoplakia (associated with LPR) (39,40) the presence of pepsin Rhinitis and sinusitis (41) Over the past ten years or more the presence of pepsin has Lung transplant rejection (42) been associated with many diseases of the aerodigestive Oesophageal atresia (43) tract including gastro-oesophageal reflux disease (GORD), Laryngopharyngeal reflux (LPR), rhinitis and sinusitis, vocal fold leucoplakia (VFL), laryngomalacia and several element of the antimicrobial barrier function of gastric lung diseases (28-43) (Table 2). Reflux of gastric contents juice. This may be particularly important in the case of associated diseases have become so prevalent that in the microbial resistance to low pH. recent Incredibles 2 film a new superhero Reflux was born. Pepsinogen and pepsin stability What do we need to measure pepsin? Is pepsin being measured correctly? Pepsin or Unlike pepsinogen, pepsin is not stable at alkaline pH. pepsinogen? When purified pepsin 3 was incubated over 24 hours at 37 Pepsin is not secreted at proximal sites in the gastrointestinal with pH ranging from 2–8, when measuring at pH 2, 100% ℃ tract. Therefore, it represents a rational and objective of activity could be recovered after the pre-incubation marker of recent reflux events when detected in biological between pH 4–6.8 but no activity was recovered after pH samples from the aerodigestive tract, like saliva and sputum.