title: Targeting exogenous β-Defensin to the endolysosomal compartment via a vehicle guided system.
abstract: A number of pathogens for which there are no effective treatments infect the cells via endocytosis. Once in the endosomes, the pathogens complete their life cycle by overriding normal lysosomal functions. Recently, our laboratory identified the lysosomal targeting signal of prosaposin, which is recognized by the sorting receptor "sortilin". Based on this evidence, we tested whether the antimicrobial peptide β-Defensin linked to the targeting sequence of prosaposin (βD-PSAP) could be redirected from its secretory pathway to the endolysosomal compartment. To this effect, βD-PSAP was transfected into COS-7 cells. The sub-cellular distribution of βD-PSAP was analyzed by confocal microscopy and differential centrifugation. Confocal microscopy demonstrated that βD-PSAP overlaid with the lysosomal marker LAMP1, indicating that the construct reached endosomes and lysosomes. Differential centrifugation also showed that βD-PSAP was in the lysosomal fractions. In addition, our binding inhibition assay demonstrated that βD-PSAP bound specifically to sortilin. Similarly, the delivery of βD-PSAP was abolished after overexpressing a truncated sortilin. These results indicate that the prosaposin C-terminus and D/C-domain (prosaposin targeting sequence) was an effective "guidance system" to redirect βD-PSAP to the endolysosomal compartment. In the future, this and other fusion proteins with antimicrobial properties will be assembled to our "biotic vehicle" to target pathogens growing within these compartments.
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.|IHEM-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, Mendoza, Argentina.
authors.forename: Lorena
authors.initials: L
authors.lastname: Carvelli
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Libin
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
authors.forename: Sherry
authors.initials: S
authors.lastname: Esfandnia
authors.affiliation: Department of Endocrinology, China-Japan Union Hospital of Jilin University, Jilin, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
authors.forename: John F
authors.initials: JF
authors.lastname: Presley
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada. carlos.morales@mcgill.ca.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Morales
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000818:Animals; D000890:Anti-Infective Agents / Q000008:administration & dosage / Q000494:pharmacology*; D019556:COS Cells; D002522:Chlorocebus aethiops; D016503:Drug Delivery Systems / Q000379:methods*; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects*; D014677:Pharmaceutical Vehicles; D011485:Protein Binding / Q000187:drug effects; D049231:Saposins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D023083:beta-Defensins / Q000008:administration & dosage / Q000494:pharmacology*; D000099183:Sortilin
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D000890:Anti-Infective Agents; D052118:Lysosomal-Associated Membrane Protein 1; D014677:Pharmaceutical Vehicles; D049231:Saposins; D023083:beta-Defensins; D000099183:Sortilin; C483345:PSAP protein, human