Mohamed Qaddoumi1, Vincent H L Lee. 1. Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90089, USA.
Abstract
PURPOSE: To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. METHODS: Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. RESULTS: The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. CONCLUSION: Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.
PURPOSE: To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. METHODS: Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. RESULTS: The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. CONCLUSION: Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.
Authors: Evelyn M Covés-Datson; Steven R King; Maureen Legendre; Auroni Gupta; Susana M Chan; Emily Gitlin; Vikram V Kulkarni; Jezreel Pantaleón García; Donald F Smee; Elke Lipka; Scott E Evans; E Bart Tarbet; Akira Ono; David M Markovitz Journal: Proc Natl Acad Sci U S A Date: 2020-01-13 Impact factor: 11.205