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Computational design of an α-gliadin peptidase.

Gordon SR, Stanley EJ, Wolf S, Toland A, Wu SJ, Hadidi D, Mills JH, Baker D, Pultz IS, Siegel JB - J. Am. Chem. Soc. (2012)

Bottom Line: The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics.Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease.The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States.

ABSTRACT
The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.

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Related in: MedlinePlus

Schematic depicting the role of enzyme therapeuticsin the treatmentof celiac disease. Gluten is comprised of many glycoproteins includingα-gliadin. Partial proteolysis of α-gliadin results inprotease-resistant peptides enriched in a PQ dipeptide motif thatcan lead to inflammation and disease. An enzyme that is functionalin the stomach and capable of specifically degrading the immunogenicpeptides could potentially act as a therapeutic for this disease.
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fig1: Schematic depicting the role of enzyme therapeuticsin the treatmentof celiac disease. Gluten is comprised of many glycoproteins includingα-gliadin. Partial proteolysis of α-gliadin results inprotease-resistant peptides enriched in a PQ dipeptide motif thatcan lead to inflammation and disease. An enzyme that is functionalin the stomach and capable of specifically degrading the immunogenicpeptides could potentially act as a therapeutic for this disease.

Mentions: Celiac disease is characterized byan inflammatory reaction inthe digestive tract to α-gliadin, an important component ofthe glycoprotein gluten, which is found in any food containing wheat,rye, or barely.4,5 Upon ingestion, α-gliadinis partially degraded by digestive proteases to oligopeptides thatare resistant to further proteolysis due to their unusually high prolineand glutamine content5 (Figure 1). These proteolytically resistant oligopeptidesthat are highly enriched in a proline–glutamine (PQ) motifare believed to trigger an autoimmune response, which elicits manyof the symptoms in celiac patients.6 Anideal oral enzyme therapeutic (OET) for celiac disease would havethe following traits: (1) optimal activity at the pH of the stomachafter a meal (in the range of 2–4);7 (2) resistance to common digestive proteases; (3) facile recombinantproduction in a soluble form; and (4) specificity for the common proline–glutamine(PQ) motif found in immunogenic α-gliadin oligopeptides.6,8 While there are several OETs being explored for the treatment ofceliac disease,6,9−11 none conformto all of these properties.


Computational design of an α-gliadin peptidase.

Gordon SR, Stanley EJ, Wolf S, Toland A, Wu SJ, Hadidi D, Mills JH, Baker D, Pultz IS, Siegel JB - J. Am. Chem. Soc. (2012)

Schematic depicting the role of enzyme therapeuticsin the treatmentof celiac disease. Gluten is comprised of many glycoproteins includingα-gliadin. Partial proteolysis of α-gliadin results inprotease-resistant peptides enriched in a PQ dipeptide motif thatcan lead to inflammation and disease. An enzyme that is functionalin the stomach and capable of specifically degrading the immunogenicpeptides could potentially act as a therapeutic for this disease.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526107&req=5

fig1: Schematic depicting the role of enzyme therapeuticsin the treatmentof celiac disease. Gluten is comprised of many glycoproteins includingα-gliadin. Partial proteolysis of α-gliadin results inprotease-resistant peptides enriched in a PQ dipeptide motif thatcan lead to inflammation and disease. An enzyme that is functionalin the stomach and capable of specifically degrading the immunogenicpeptides could potentially act as a therapeutic for this disease.
Mentions: Celiac disease is characterized byan inflammatory reaction inthe digestive tract to α-gliadin, an important component ofthe glycoprotein gluten, which is found in any food containing wheat,rye, or barely.4,5 Upon ingestion, α-gliadinis partially degraded by digestive proteases to oligopeptides thatare resistant to further proteolysis due to their unusually high prolineand glutamine content5 (Figure 1). These proteolytically resistant oligopeptidesthat are highly enriched in a proline–glutamine (PQ) motifare believed to trigger an autoimmune response, which elicits manyof the symptoms in celiac patients.6 Anideal oral enzyme therapeutic (OET) for celiac disease would havethe following traits: (1) optimal activity at the pH of the stomachafter a meal (in the range of 2–4);7 (2) resistance to common digestive proteases; (3) facile recombinantproduction in a soluble form; and (4) specificity for the common proline–glutamine(PQ) motif found in immunogenic α-gliadin oligopeptides.6,8 While there are several OETs being explored for the treatment ofceliac disease,6,9−11 none conformto all of these properties.

Bottom Line: The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics.Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease.The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States.

ABSTRACT
The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.

Show MeSH
Related in: MedlinePlus