Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides.

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.

Complex of Proline-Specific Peptidases in the Genome and Gut Transcriptomes of Tenebrionidae Insects and Their Role in Gliadin Hydrolysis.

A detailed analysis of the complexes of proline-specific peptidases (PSPs) in the midgut transcriptomes of the larvae of agricultural pests Tenebrio molitor and Tribolium castaneum and in the genome of T. castaneum is presented. Analysis of the T. castaneum genome revealed 13 PSP sequences from the clans of serine and metal-dependent peptidases, of which 11 sequences were also found in the gut transcriptomes of both tenebrionid species' larvae. Studies of the localization of PSPs, evaluation of the expression level of their genes in gut transcriptomes, and prediction of the presence of signal peptides determining secretory pathways made it possible to propose a set of peptidases that can directly participate in the hydrolysis of food proteins in the larvae guts. The discovered digestive PSPs of tenebrionids in combination with the post-glutamine cleaving cysteine cathepsins of these insects effectively hydrolyzed gliadins, which are the natural food substrates of the studied pests. Based on the data obtained, a hypothetical scheme for the complete hydrolysis of immunogenic gliadin peptides by T. molitor and T. castaneum digestive peptidases was proposed. These results show promise regarding the development of a drug based on tenebrionid digestive enzymes for the enzymatic therapy of celiac disease and gluten intolerance.

Effective Degradation of Gluten and Its Fragments by Gluten-Specific Peptidases: A Review on Application for the Treatment of Patients with Gluten Sensitivity.

To date, there is no effective treatment for celiac disease (CD, gluten enteropathy), an autoimmune disease caused by gluten-containing food. Celiac patients are supported by a strict gluten-free diet (GFD). However, in some cases GFD does not negate gluten-induced symptoms. Many patients with CD, despite following such a diet, retain symptoms of active disease due to high sensitivity even to traces of gluten. In addition, strict adherence to GFD reduces the quality of life of patients, as often it is difficult to maintain in a professional or social environment. Various pharmacological treatments are being developed to complement GFD. One promising treatment is enzyme therapy, involving the intake of peptidases with food to digest immunogenic gluten peptides that are resistant to hydrolysis due to a high prevalence of proline and glutamine amino acids. This narrative review considers the features of the main proline/glutamine-rich proteins of cereals and the conditions that cause the symptoms of CD. In addition, we evaluate information about peptidases from various sources that can effectively break down these proteins and their immunogenic peptides, and analyze data on their activity and preliminary clinical trials. Thus far, the data suggest that enzyme therapy alone is not sufficient for the treatment of CD but can be used as a pharmacological supplement to GFD.

The dataset of predicted trypsin serine peptidases and their inactive homologs in Tenebrio molitor transcriptomes.

Tenebrio molitor is an important coleopteran model insect and agricultural pest from the Tenebrionidae family. We used RNA-Seq transcriptome data from T. molitor to annotate trypsin-like sequences from the chymotrypsin S1 family of serine peptidases, including sequences of active serine peptidases (SerP) and their inactive homologs (SerPH) in T. molitor transcriptomes. A total of 63 S1 family tryspin-like serine peptidase sequences were de novo assembled. Among the sequences, 58 were predicted to be active trypsins and five inactive SerPH. The length of preproenzyme and mature form of the predicted enzyme, position of signal peptide and proenzyme cleavage sites, molecular mass, active site and S1 substrate binding subsite residues, and transmembrane and regulatory domains were analyzed using bioinformatic tools. The data can be used for further physiological, biochemical, and phylogenetic study of tenebrionid pests and other animal systems.

New Glutamine-Containing Substrates for the Assay of Cysteine Peptidases From the C1 Papain Family.

New substrates with glutamine in the P1-position are introduced for the assay of peptidases from the C1 papain family, with a general formula of Glp-Phe-Gln-X, where Glp is pyroglutamyl and X is pNA (p-nitroanilide) or AMC (4-amino-7-methylcoumaride). The substrates have a simple structure, and C1 cysteine peptidases of various origins cleave them with high efficiency. The main advantage of the substrates is their selectivity for cysteine peptidases of the C1 family. Peptidases of other clans, including serine trypsin-like peptidases, do not cleave glutamine-containing substrates. We demonstrate that using Glp-Phe-Gln-pNA in combination with a commercially available substrate, Z-Arg-Arg-pNA, provided differential determination of cathepsins L and B. In terms of specific activity and kinetic parameters, the proposed substrates offer improvement over the previously described alanine-containing prototypes. The efficiency and selectivity of the substrates was demonstrated by the example of chromatographic and electrophoretic analysis of a multi-enzyme digestive complex of stored product pests from the Tenebrionidae family.

Human proline specific peptidases: A comprehensive analysis.

BACKGROUND: Proline specific peptidases (PSPs) are a unique group of enzymes that specifically cleave bonds formed by a proline residue. The study of PSPs is important due to their role in the maturation and degradation of peptide hormones and neuropeptides. In addition, changes in the activity of PSPs can result in pathological conditions, including various types of cancer. SCOPE OF REVIEW: PSPs annotated from the Homo sapiens genome were compared and classified by their physicochemical and biochemical features and roles in vital processes. In addition to catalytic activity, we discuss non-enzymatic functions that may regulate cellular activity. MAJOR CONCLUSIONS: PSPs apparently have multiple functions in animals. Two functions rely on the catalytic activity of the enzyme: one involved in a regulatory pathway associated with the ability of many PSPs to hydrolyze peptide hormones and neuropeptides, and the other involved in the trophic pathway associated with the proteolysis of total cellular protein or Pro-containing dietary proteins in the digestive tract. PSPs also participate in signal transduction without proteolytic activity by forming protein-protein interactions that trigger or facilitate the performance of certain functions. GENERAL SIGNIFICANCE: PSPs are underestimated as a unique component of the normal human peptidase degradome, providing the body with free proline. A comparative analysis of PSPs can guide research to develop inhibitors that counteract the abnormalities associated with changes in PSP activity, and identify natural substrates of PSPs that will enable better understanding of the mechanisms of the action of PSPs in biological processes and disease.

Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates.

A method is described for the direct detection of unstable cysteine peptidase activity in polyacrylamide gels after native electrophoresis using new selective fluorogenic peptide substrates, pyroglutamyl-phenylalanyl-alanyl-4-amino-7-methylcoumaride (Glp-Phe-Ala-AMC) and pyroglutamyl-phenylalanyl-alanyl-4-amino-7-trifluoromethyl-coumaride (Glp-Phe-Ala-AFC). The detection limit of the model enzyme papain was 17 pmol (0.29 µg) for Glp-Phe-Ala-AMC and 43 pmol (0.74 µg) for Glp-Phe-Ala-AFC, with increased sensitivity and selectivity compared to the traditional method of protein determination with Coomassie G-250 staining or detection of activity using chromogenic substrates. Using this method, we easily identified the target digestive peptidases of Tenebrio molitor larvae by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. The method offers simplicity, high sensitivity, and selectivity compared to traditional methods for improved identification of unstable cysteine peptidases in multi-component biological samples.

Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures.

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes.