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 beta-Defensin linked to the targeting sequence of prosaposin (beta D-PSAP) could be redirected from its secretory pathway to the endolysosomal compartment. To this effect, beta D-PSAP was transfected into COS-7 cells. The sub-cellular distribution of beta D-PSAP was analyzed by confocal microscopy and differential centrifugation. Confocal microscopy demonstrated that beta D-PSAP overlaid with the lysosomal marker LAMP1, indicating that the construct reached endosomes and lysosomes. Differential centrifugation also showed that beta D-PSAP was in the lysosomal fractions. In addition, our binding inhibition assay demonstrated that beta D-PSAP bound specifically to sortilin. Similarly, the delivery of beta D-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 beta D-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.
[1]McGill Univ, Dept Anat & Cell Biol, 3640 Univ St, Montreal, PQ H3A 0C7, Canada.
[2]Univ Nacl Cuyo, Fac Ciencias Exactas & Nat, IHEM CONICET, Mendoza, Argentina.
[3]Jilin Univ, China Japan Union Hosp, Dept Endocrinol, Jilin, Jilin, Peoples R China.
(8.0+极速模式)