title: Toward PKU enzyme replacement therapy: PEGylation with activity retention for three forms of recombinant phenylalanine hydroxylase.
abstract: Phenylketonuria (PKU) is a disease in which phenylalanine and phenylalanine-derived metabolites build up to neurotoxic levels due to mutations in the phenylalanine hydroxylase gene (PAH). Enzyme replacement therapy is a viable option to supply active PAH. However, the inherent protease sensitivity and potential immunogenicity of PAH have precluded adoption of this approach. In this report, we have used polyethylene glycol derivatization (PEGylation) to produce protected forms of PAH for potential therapeutic use. Three recombinantly produced PAH enzymes were reacted with activated PEG species, with the aim of developing a stable and active PKU enzyme replacement. Tetrameric full-length human PAH, dimeric double-truncated (DeltaN102-DeltaC428) human PAH, and monomeric Chromobacterium violaceum PAH were PEGylated with succinimidyl succinate polyethylene glycol of molecular weight 5000 or 20,000 Da. Characterization of the PEGylated species was accomplished with MALDI-TOF mass spectrometry, SDS-PAGE, and specific activity measurements using ESI mass spectrometry. All PEG-derivatized PAH species retained catalytic activity, and, at low numbers of PEG molecules attached, these PEGylated PAH proteins were found to be more active and more stable than their non-derivatized PAH counterparts.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Gámez
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Mary
authors.initials: M
authors.lastname: Straub
authors.forename: Marianne G
authors.initials: MG
authors.lastname: Patch
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D004795:Enzyme Stability; D006801:Humans; D010649:Phenylalanine / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000378:metabolism / Q000627:therapeutic use; D010661:Phenylketonurias / Q000201:enzymology / Q000628:therapy; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013388:Succinimides; D011092:Polyethylene Glycols; D010649:Phenylalanine; C050761:polyethylene glycol bis(succinimidyl succinate); D010651:Phenylalanine Hydroxylase