Targeting exogenous β-Defensin to the endolysosomal compartment via a vehicle guided system
Lorena Carvelli1,2*, Yuan Libin1*, Sherry Esfandnia1, Yan Zhang3, John F. Presley1 and Carlos R. Morales1
1Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, 2IHEM-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo, Mendoza, Argentina and 3Department of Endocrinology, China-Japan Union Hospital of Jilin University, Jilin, China
*These authors contributed equally to this work
Offprint requests to: Carlos R. Morales, Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montreal, Quebec, H3A 0C7, Canada. e-mail: carlos.morales@mcgill.ca
Summary. 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. Histol Histopathol 32, 1017-1027 (2017)
Key words: Antimicrobial proteins, β-Defensin, Prosaposin, Sortilin, Endosomes, Lysosomes, Lysosomal trafficking
DOI: 10.14670/HH-11-862