Confirmation of covalently-linked structure and cell-death inducing activity in site-specific chemical conjugates of human Fas ligand extracellular domain

Abstract

Objective: In this study, we aimed to identify the structural components and to clarify the biological activity in the site-specific conjugates of human Fas ligand extracellular domain (hFasLECD) with either fluorescein moiety (FL) or chicken egg-white avidin (Avi). The conjugates were characterized by molecular-weight measurement using MALDI-TOF mass-spectrometric analysis and by cell-death inducing activity measurement against a human colorectal cancer cell line, HT-29, using MTT cell-viability assay. Pretreatment effect with human interferon-γ (IFN-γ) on the cell-death inducing activity was evaluated. Results: The mass-spectrometric analysis of the hFasLECD-Avi conjugate showed that it was possible to detect the signal peak of molecular-weight to electric charge (m/z) derived from the component involved in the covalent linking as the sum of the molecular-weight of unconjugated hFasLECD- and Avi-derivative subunits, in addition to the signals from each corresponding subunit component irrelevant to the covalent linking. The cell-viability assay revealed that both conjugates possessed a remarkable death-inducing activity against HT-29 cells in synergy with the pretreatment using human IFN-γ. Following 24 h pretreatment with 100 IU/ml of human IFN-γ, almost no viable cells existed after 72 h treatment with either 100 or 1000 ng/ml of FL-hFasLECD and hFasLECD-Avi conjugates.