Title : A serine peptidase from burkholderia gladioli with hydrolytic activity toward celiac disease- eliciting pro-immunogenic peptides and potency enhancement through modification of its active site pocket
Abstract:
Celiac disease is an autoimmune disease triggered by oral ingestion of gluten found inside wheat, barley, and rye grains. Gluten is a water-insoluble protein group composed of ethanol-soluble prolamins and ethanol-insoluble glutelins. The most common prolamins are the ones in wheat grains called gliadins, which possess high proline and glutamine content. When consumed orally, specific residues of gliadin are resistant to enzymes secreted by digestive tracts, such as 33-mer peptides and 26-mer peptides. Inside the duodenum, these remaining peptides incite immunogenic responses, including the production of autoantibodies and long-term inflammation, eventually causing irreversible damage. The major pathological features of celiac disease are intestinal villous atrophy and crypt hyperplasia. The prevalence of celiac disease in Caucasians is about 1% population. No permanent cure has been found, and patients will continuously suffer without following strict gluten-free diets. Here we found a gram-negative bacterium, Burkholderia gladioli, producing a serine peptidase with gliadin-degrading activity, named Bga1903. Heterologous expression of Bga1903 by E. coli can be done in an extracellular secretory manner, then purified by immobilized metal affinity chromatography. Bga1903 can hydrolyze pro-immunogenic peptides with a preference for glutamine in the P1 position. The cleavage pattern of Bga1903 indicated its ability to diminish the toxicity of immunogenic peptides completely. The crystal structure of Bga1903 was determined by x-ray crystallography, featuring an α/β/α-folded core with a twisted six-stranded parallel β-sheet sandwiched between two layers of α-helices. Adjacent to the active site pocket, a hood-like loop protruded from the core and together formed a negatively charged cleft. The single-substitution mutations at the active site were predicted by FoldX software, based on the calculation of energy change in protein stability. The method is to enhance the peptidase’s affinity toward tetrapeptide PQPQ, mimicking the toxic gluten-derived peptides. Wild-type Bga1903 and four mutations were evaluated by enzyme kinetic assays utilizing four chromogenic substrates, Z-HPK-pNA, Z-HPQ-pNA, Z-HPL-pNA, and Z-QPQ-pNA. One of the mutants, S387L showed a 17-fold increase in specificity constant toward Z-QPQ-pNA compared to the wild type, and QPQ motifs were repetitively present inside immunogenic peptides. Mutant S387L also improved the hydrolysis effectiveness toward 33-mer peptides and 26-mer peptides. These results uncover the characteristics of Bga1903 and its potential as an oral therapy enzyme for celiac disease future.
Audience Take Away:
- Understand the basic information and the risks of celiac disease.
- A potential oral enzyme therapy to ease the symptoms of celiac disease.
- Provide the modification methods to enhance the effectiveness of future enzymes to treat celiac disease
