title: Structure-based epitope and PEGylation sites mapping of phenylalanine ammonia-lyase for enzyme substitution treatment of phenylketonuria.
abstract: Protein and peptide therapeutics are of growing importance as medical treatments but can frequently induce an immune response. This work describes the combination of complementary approaches to map the potential immunogenic regions of the yeast Rhodosporidium toruloides phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and to engineer the protein as a human therapeutic agent for the treatment of phenylketonuria (PKU), an inherited metabolic disorder. The identification of B and T cell epitopes on the PAL protein was performed by computational predictions based on the antigenicity and hydrophilicity of proteins, as well as by experimental epitope mapping using a PepSpots peptide array (Jerini AG). Human T cell epitope mapping was performed by applying the computational EpiMatrix algorithm (EpiVax, Inc.) for MHC Class I and Class II associated T cell epitopes on PAL, which predicts which sequences are associated with binding to several different HLA alleles, a requirement for antigen presentation and subsequent primary immune response. By chemical modification through PEGylation of surface lysine residues, it is possible to cover the immunogenic regions of a protein. To evaluate this strategy, we used mass spectrometry to determine which of the immunogenic epitopes are covered by the covalent PEGylation modification strategy. This approach has allowed us to determine whether additional lysines are needed in specific residue locations, or whether certain lysine residues can be removed in order to accomplish complete molecular coverage of the therapeutic enzyme.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Gámez
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Christineh N
authors.initials: CN
authors.lastname: Sarkissian
authors.forename: Dan
authors.initials: D
authors.lastname: Wendt
authors.forename: Paul
authors.initials: P
authors.lastname: Fitzpatrick
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Lemontt
authors.forename: Charles R
authors.initials: CR
authors.lastname: Scriver
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D005656:Fungal Proteins / Q000378:metabolism / Q000627:therapeutic use; D006801:Humans; D008958:Models, Molecular; D010446:Peptide Fragments / Q000276:immunology; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use*; D010661:Phenylketonurias / Q000188:drug therapy* / Q000201:enzymology; D011092:Polyethylene Glycols; D011487:Protein Conformation; D012248:Rhodotorula / Q000201:enzymology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000939:Epitopes; D005656:Fungal Proteins; D010446:Peptide Fragments; D011092:Polyethylene Glycols; D010650:Phenylalanine Ammonia-Lyase