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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

Immunogenicα9-gliadin peptide degradation by KumaMax. (A)Reaction chromatograms measuring the abundance of the M + H ion ofthe parent α9-gliadin peptide after 50 min of incubation withno enzyme (gray), SC PEP (gold), or KumaMax (purple). (B) The fractionof α9-gliadin peptide remaining in the presence of KumaMax asa function of incubation time at pH 4. The curve is a sample exponentialfitting. The R2 value was 0.97.
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fig4: Immunogenicα9-gliadin peptide degradation by KumaMax. (A)Reaction chromatograms measuring the abundance of the M + H ion ofthe parent α9-gliadin peptide after 50 min of incubation withno enzyme (gray), SC PEP (gold), or KumaMax (purple). (B) The fractionof α9-gliadin peptide remaining in the presence of KumaMax asa function of incubation time at pH 4. The curve is a sample exponentialfitting. The R2 value was 0.97.

Mentions: KumaMax was incubated at 37 °C in pH 4 sodium acetatewith 500 μM of the α9-gliadin peptide at a roughly 1:100enzyme to peptide molar ratio, which is a physiologically relevantconcentration of this peptide in the human stomach (Supporting Information). SC PEP was included in this experimentfor the sake of comparison, since this enzyme has significantly lessactivity against the FQ substrate than KumaMax at pH 4. Samples fromthe incubation were quenched every 10 min in 80% acetonitrile to haltthe proteolysis reaction. The remaining fraction of intact immunogenicpeptide was determined using ultrahigh-performance liquid chromatography–massspectrometry, in which the M + H parent ion of the α9-gliadinpeptide was monitored. KumaMax demonstrated a high level of activityagainst the immunogenic peptide in this assay, as over 95% of theimmunogenic peptide had been proteolyzed after a 50-min incubationwith KumaMax, while no significant degradation of the peptide wasobserved in the presence of SC PEP or in the absence of a peptidase(Figure 4A). The half-life of the peptide inthe presence of KumaMax was determined by plotting the fraction ofpeptide remaining against the incubation time and was calculated tobe 8.5 ± 0.7 min (Figure 4B).


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)

Immunogenicα9-gliadin peptide degradation by KumaMax. (A)Reaction chromatograms measuring the abundance of the M + H ion ofthe parent α9-gliadin peptide after 50 min of incubation withno enzyme (gray), SC PEP (gold), or KumaMax (purple). (B) The fractionof α9-gliadin peptide remaining in the presence of KumaMax asa function of incubation time at pH 4. The curve is a sample exponentialfitting. The R2 value was 0.97.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Immunogenicα9-gliadin peptide degradation by KumaMax. (A)Reaction chromatograms measuring the abundance of the M + H ion ofthe parent α9-gliadin peptide after 50 min of incubation withno enzyme (gray), SC PEP (gold), or KumaMax (purple). (B) The fractionof α9-gliadin peptide remaining in the presence of KumaMax asa function of incubation time at pH 4. The curve is a sample exponentialfitting. The R2 value was 0.97.
Mentions: KumaMax was incubated at 37 °C in pH 4 sodium acetatewith 500 μM of the α9-gliadin peptide at a roughly 1:100enzyme to peptide molar ratio, which is a physiologically relevantconcentration of this peptide in the human stomach (Supporting Information). SC PEP was included in this experimentfor the sake of comparison, since this enzyme has significantly lessactivity against the FQ substrate than KumaMax at pH 4. Samples fromthe incubation were quenched every 10 min in 80% acetonitrile to haltthe proteolysis reaction. The remaining fraction of intact immunogenicpeptide was determined using ultrahigh-performance liquid chromatography–massspectrometry, in which the M + H parent ion of the α9-gliadinpeptide was monitored. KumaMax demonstrated a high level of activityagainst the immunogenic peptide in this assay, as over 95% of theimmunogenic peptide had been proteolyzed after a 50-min incubationwith KumaMax, while no significant degradation of the peptide wasobserved in the presence of SC PEP or in the absence of a peptidase(Figure 4A). The half-life of the peptide inthe presence of KumaMax was determined by plotting the fraction ofpeptide remaining against the incubation time and was calculated tobe 8.5 ± 0.7 min (Figure 4B).

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