COBRE P3: HUMANIZING ALGINATE DEPOLYMERASE

Dartmouth College, Hanover NH

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Premature mortality in cystic fibrosis typically results from chronic P. aeruginosa infection of the patient's airways. The mucoid strains of P. aeruginosa associated with cystic fibrosis are characterized by their production of the exopolysaccharide alginate, a known pathogenic factor. Alginate lyases are enzymes that degrade the alginate polymer, and have shown great promise for symptomatic treatment of cystic fibrosis. However, the microbial origin of these biocatalysts predisposes them towards high level immunogenicity, and brings into question their utility in a clinical setting. Unfortunately, as is the case with numerous microbial biotherapeutic candidates, alginate lyases have no exact human counterpart. However, the human genome encodes numerous enzymes with similar catalytic functions, i.e. degradation of carbohydrate-based biopolymers. The existence of human enzymes exhibiting similar catalytic functions but different substrate selectivities suggests that the alginate lyase therapeutic activity could be deimmunized by engineering a human carbohydrate hydrolase to act on the alginate substrate. Specifically, directed evolution of a human enzyme with altered substrate selectivity could yield a biocatalyst that efficiently degrades the alginate therapeutic target, but because of its human origin would be less likely to illicit a deleterious immune response in human patients. Directed evolution will be employed to alter the substrate selectivity of human lysozyme to generate a humanized alginate depolymerase. The activity of these enzymes will be compared to an authentic bacterial alginate lyase, and the immunogenicity of the engineered enzymes will be assessed relative to the wild type human lysozyme template.