Specificity Enhancement of Glutenase Bga1903 toward Celiac Disease-Eliciting Pro-Immunogenic Peptides via Active-Site Modification.

Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicates its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q/S387L exhibits an approximately 34-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. The increased specificity of E380Q/S387L not only enhances its ability to break down pro-immunogenic peptides but also positions this enzyme variant as a promising candidate for oral therapy for celiac disease.

Crystal structure of a Burkholderia peptidase and modification of the substrate-binding site for enhanced hydrolytic activity toward gluten-derived pro-immunogenic peptides.

Celiac disease (CD) is a human autoimmune disease triggered by toxic gluten peptides. Recently, oral enzyme therapy has been proposed to ameliorate the health condition of CD patients based on the concept of removing pepsin-insensitive gluten-derived pro-immunogenic peptides. A Burkholderia peptidase, Bga1903, with promising gluten-degrading activity was characterized previously. Here, we report the crystal structure of Bga1903, in which the core has a α/ß/α fold featured with a twisted six-stranded parallel ß-sheet sandwiched between two layers of α-helices. The mutations at the substrate-binding pocket that might enhance the peptidase's affinity toward tetrapeptide PQPQ were predicted by FoldX. Accordingly, four single-substitution mutants, G351A, E380L, S386F, and S387L, were created. The specificity constant (kcat/KM) of wild type toward chromogenic peptidyl substrates Z-HPK-pNA, Z-HPQ-pNA, Z-HPL-pNA, and Z-QPQ-pNA are 30.2, 7.9, 3.3, and 0.79 s-1·mM-1, respectively, indicating that the QPQ motif, which frequently occurs in pro-immunogenic peptides, is not favorable. Among the mutants, E380L loses the hydrolytic activity toward Z-HPK-pNA, suggesting a critical role of E380 in preferring a lysine residue at the P1 position. S387L shows a 17-fold increase in the specificity constant toward Z-QPQ-pNA and hydrolyzes the pro-immunogenic peptides more efficiently than the wild-type peptidase.

Efficient Hydrolysis of Gluten-Derived Celiac Disease-Triggering Immunogenic Peptides by a Bacterial Serine Protease from Burkholderia gladioli.

Celiac disease is an autoimmune disorder triggered by toxic peptides derived from incompletely digested glutens in the stomach. Peptidases that can digest the toxic peptides may formulate an oral enzyme therapy to improve the patients' health condition. Bga1903 is a serine endopeptidase secreted by Burkholderia gladioli. The preproprotein of Bga1903 consists of an N-terminal signal peptide, a propeptide region, and an enzymatic domain that belongs to the S8 subfamily. Bga1903 could be secreted into the culture medium when it was expressed in E. coli. The purified Bga1903 is capable of hydrolyzing the gluten-derived toxic peptides, such as the 33- and 26-mer peptides, with the preference for the peptide bonds at the carbonyl site of glutamine (P1 position). The kinetic assay of Bga1903 toward the chromogenic substrate Z-HPQ-pNA at 37 °C, pH 7.0, suggests that the values of Km and kcat are 0.44 ± 0.1 mM and 17.8 ± 0.4 s-1, respectively. The addition of Bga1903 in the wort during the fermentation step of beer could help in making gluten-free beer. In summary, Bga1903 is usable to reduce the gluten content in processed foods and represents a good candidate for protein engineering/modification aimed to efficiently digest the gluten at the gastric condition.