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

Abstract

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.

Fighting Celiac Disease: Improvement of pH Stability of Cathepsin L In Vitro by Computational Design

“…Tribolium castaneum cathepsin L variants of the wild type and with the suggested mutations V277T, V277D, V277I, V277A and V277Y were obtained in the form of recombinant proenzymes in the yeast expression system of Pichia pastoris (rpTcCathL1 WT, rpTcCathL1 V277T, rpTcCathL1 V277D, rpTcCathL1 V277I, rpTcCathL1 V277A and rpTcCathL1 V277Y). To obtain active enzymes of mutant forms of cathepsin L, autocatalytic processing was carried out according to the method developed for wild-type cathepsin, which resulted in an active enzyme rTcCathL1 WT [11]. However, in practice, we were able to obtain mature forms of recombinant enzymes for only three mutant proteins: rTcCathL1 V277I, rTcCathL1 V277T and rTcCathL1 V277A.…”

“…The cloning, expression, and isolation of the wild-type T. castaneum procathepsin L (Tc-CathL1, Uniprot D6X519, NCBI NP_001164001) were performed as described in a previous study [11]. The pTcCathL1 WT gene was cloned into the pPICZalphaA vector, expressed in P. pastoris GS115-II-3 transformants, and recombinant procathepsin L (rpTcCathL1 WT) was isolated from the culture medium by ammonium sulfate precipitation.…”

“…Processing of the wild-type and mutant forms of procathepsin L was performed autocatalytically by incubation at pH 4.0 and 37 • C for 60-150 min as described in [11].…”

“…In this work, we implement the second strategy, based on conferring pH stability to an enzyme that already has sufficient glutenase activity. Earlier, we have shown that the main digestive cathepsin L from the insect pest Tribolium castaneum (TcCathL1, Uniprot D6X519, NCBI NP_001164001) has high post-glutamine cleaving activity and is able to degrade 33-, 26-, 10-and 8-mer immunogenic prolamin peptides and their fluorogenic analogs [10,11]. Successful experiments were carried out both with the native enzyme isolated from the T. castaneum midgut extract [10] and with the recombinant preparation obtained as a zymogen in the Pichia pastoris expression system followed by autoprocessing to the mature active enzyme [11].…”

“…Earlier, we have shown that the main digestive cathepsin L from the insect pest Tribolium castaneum (TcCathL1, Uniprot D6X519, NCBI NP_001164001) has high post-glutamine cleaving activity and is able to degrade 33-, 26-, 10-and 8-mer immunogenic prolamin peptides and their fluorogenic analogs [10,11]. Successful experiments were carried out both with the native enzyme isolated from the T. castaneum midgut extract [10] and with the recombinant preparation obtained as a zymogen in the Pichia pastoris expression system followed by autoprocessing to the mature active enzyme [11]. However, the disadvantage of the obtained preparations of cathepsin L was their low stability at acidic pH values (2)(3) corresponding to the physiological conditions in the human stomach [12].…”