Effect of Barley Endoprotease EP-B2 on Gluten Digestion in the Intact Rat

Abstract

Celiac Sprue is a multifactorial disease characterized by an intestinal inflammatory response to ingested gluten. Proteolytically resistant gluten peptides from wheat, rye, and barley persist in the intestinal lumen and elicit an immune response in genetically susceptible individuals. Here, we demonstrate the in vivo ability of a gluten-digesting protease ("glutenase") to accelerate the breakdown of a gluten-rich solid meal. The proenzyme form of endoprotease B, isoform 2 from Hordeum vulgare (EP-B2), was orally administered to adult rats with a solid meal containing 1 g of gluten. Gluten digestion in the stomach and small intestine was monitored as a function of enzyme dose and time by high-performance liquid chromatography and mass spectrometry. In the absence of supplementary EP-B2, gluten was solubilized and proteolyzed to a limited extent in the stomach and was hydrolyzed and assimilated mostly in the small intestine. In contrast, EP-B2 was remarkably effective at digesting gluten in the rat stomach in a dose-and time-dependent fashion. At a 1:25 EP-B2/gluten dose, the gastric concentration of the highly immunogenic 33-mer gliadin peptide was reduced by more than 50-fold within 90 min with no overt signs of toxicity. Evaluation of EP-B2 as an adjunct to diet control is therefore warranted in celiac patients.

Noninflammatory Gluten Peptide Analogs as Biomarkers for Celiac Sprue

“…Microbial prolyl endopeptidases (PEP) detoxify immunodominant gluten in vitro (Shan et al, 2002; Stepniak et al, 2006) and are especially effective when complemented with a barley endoprotease, EP-B2, that preferentially cleaves gluten C-terminal to glutamine residues (Gass et al, 2007; Siegel et al, 2006). The therapeutic promise of these oral proteases is further underscored by their ability to digest gluten in the rat stomach (Gass et al, 2006, 2007) and by the ability of EP-B2 to protect a gluten-sensitive rhesus macaque against gluten-induced clinical relapse (Bethune et al, 2008b). To bring therapeutic glutenases to bear on the human condition of celiac sprue, however, safe and effective tools for assessing enzyme efficacy in human celiac patients are needed.…”

Therapeutic approaches for celiac disease

“…A Gln-specific cysteine endoprotease B, isoform 2 (EP-B2) from germinating barley seeds is able to rapidly proteolyze gliadin peptides into short polypeptides [36]. Like the fungal and bacterial PEPs, EP-B2 maintains its structure and activity under gastrointestinal conditions [37]. Early proof-of-concept studies demonstrated EP-B2 effectively digested gluten both in the rat stomach and in gluten-sensitive rhesus macaques in a dose- and time-dependent manner [38].…”

A scaleable manufacturing process for pro‐EP‐B2, a cysteine protease from barley indicated for celiac sprue

“…One candidate for protease therapy is EP-B2 (Endoprotease B, Isoform 2), a papain-like cysteine protease that facilitates gluten breakdown and assimilation in germinating seeds of Hordeum vulgare (barley). In vitro and in vivo studies have shown that the zymogen (pro-EP-B2) form of this enzyme rapidly self-activates under gastric conditions, where mature EP-B2 can effectively detoxify gluten at pharmacologically reasonable doses (<5% w/w) Gass et al, 2006). The stage is therefore set for controlled clinical studies to evaluate the utility of this enzyme as supportive therapy for Celiac Sprue patients.…”