title: Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells.
abstract: Conjugation of folate to proteins permits receptor-mediated endocytosis via the folate receptor (FR) and delivery of the conjugate into the cytoplasm of cells. Since many cancers up-regulate the FR it has enabled the targeting of toxins to tumor cells resulting in specific cell death. However, current conjugation methods rely on chemistries that can affect certain catalytic subunits, such as the A-chain of the plant toxin gelonin. As a result many folate-targeted toxins are a compromise between receptor/ligand interaction and toxin activity. We describe the first example of folate conjugated to a protein via carbohydrate residues, using a novel SH-folate intermediate. The folate-gelonin conjugate retains over 99% of toxin activity in a cell-free translational assay compared with unmodified gelonin and is able to bind the FR at the same affinity as free folic acid (10(-10) m). Additionally, the conjugate exhibits prolonged inhibition of protein synthesis in FR positive cell lines in vitro. Folate linked to gelonin via amino conjugation exhibits the same affinity for FR as free folic acid but the toxin is 225-fold less active in a cell-free translational assay. The effect of different conjugation methods on toxin activity and the implications for folate targeting of other glycoproteins are discussed.
authors.affiliation: CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA United Kingdom.
authors.forename: S F
authors.initials: SF
authors.lastname: Atkinson
authors.forename: T
authors.initials: T
authors.lastname: Bettinger
authors.forename: L W
authors.initials: LW
authors.lastname: Seymour
authors.forename: J P
authors.initials: JP
authors.lastname: Behr
authors.forename: C M
authors.initials: CM
authors.lastname: Ward
mesh_terms: D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D008956:Models, Chemical; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000737:chemistry*; D011956:Receptors, Cell Surface; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000378:metabolism*; D013440:Sulfides / Q000737:chemistry; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D008024:Ligands; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011956:Receptors, Cell Surface; D054789:Ribosome Inactivating Proteins, Type 1; D013440:Sulfides; C025674:GEL protein, Gelonium multiflorum; D005492:Folic Acid