title: Mechanisms involved in serum-dependent inactivation of the immunotoxin enhancers monensin and carrier-protein-monensin.
abstract: The immunotoxin-enhancing properties of monensin and of human-serum-albumin-monensin conjugates are severely impaired in the presence of human serum. In this study we have therefore investigated the interaction between serum proteins and monensin leading to the inactivation of monensin function as immunotoxin potentiator. We found that the binding of monensin-specific mAb to thioether-cross-linked or disulfide-cross-linked protein-monensin conjugates is negatively affected by serum, as indicated by immunoenzymic (ELISA) and radioimmunobinding analysis. Size-exclusion chromatography of serum samples indicated that the greatest blocking effect is due to protein components of 40-90 kDa eluting as a broad peak (peak 4). Analysis of the proteins contained within peak 4 by ion-exchange chromatography followed by microsequencing revealed that the major components of peak no. 4 were transferrin, human serum albumin and immunoglobulin fragments. Investigations on the nature of the interactions between serum proteins and monensin leading to monensin inactivation were conducted by affinity chromatography of serum on immobilized human-serum-albumin-monensin conjugates, size-exclusion chromatography, SDS/PAGE analysis of serum-treated human-serum-albumin-monensin conjugates, and evaluation of the stability of immobilized human-serum-albumin-bound 125I-monensin following treatment with serum. Addition of esterase inhibitors (e.g. EDTA, 4-nitrophenyl phosphate) or prior treatment of the serum at 56 degrees C partially reversed the serum effects observed. We conclude that serum proteins block the immunotoxin-enhancing effect of monensin and of human-serum-albumin-monensin conjugates by multiple mechanisms involving hydrophobic and covalent interactions and enzyme-mediated cleavage of protein-bound monensin.
authors.affiliation: Istituto di Immunologia e Malattie Infettive, Università di Verona, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Franceschi
authors.forename: F
authors.initials: F
authors.lastname: Dosio
authors.forename: C
authors.initials: C
authors.lastname: Anselmi
authors.forename: R
authors.initials: R
authors.lastname: Chignola
authors.forename: C
authors.initials: C
authors.lastname: Candiani
authors.forename: M
authors.initials: M
authors.lastname: Pasti
authors.forename: G
authors.initials: G
authors.lastname: Tridente
authors.forename: M
authors.initials: M
authors.lastname: Colombatti
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology*; D009596:Nitrophenols / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D011863:Radioimmunoassay; D012709:Serum Albumin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D000922:Immunotoxins; D046911:Macromolecular Substances; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D012709:Serum Albumin; C008644:nitrophenylphosphate; D008985:Monensin; D004492:Edetic Acid