Targeting exogenous ß-Defensin to the endolysosomal compartment via a vehicle guided system.

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 ß-Defensin linked to the targeting sequence of prosaposin (ßD-PSAP) could be redirected from its secretory pathway to the endolysosomal compartment. To this effect, ßD-PSAP was transfected into COS-7 cells. The sub-cellular distribution of ßD-PSAP was analyzed by confocal microscopy and differential centrifugation. Confocal microscopy demonstrated that ßD-PSAP overlaid with the lysosomal marker LAMP1, indicating that the construct reached endosomes and lysosomes. Differential centrifugation also showed that ßD-PSAP was in the lysosomal fractions. In addition, our binding inhibition assay demonstrated that ßD-PSAP bound specifically to sortilin. Similarly, the delivery of ßD-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 ßD-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.

The interactomics of sortilin: an ancient lysosomal receptor evolving new functions.

The delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptor (MPR). The MPR has been demonstrated to attain the early endosomes via a process that requires the interaction of its cytosolic domain with the GGA and AP-1 adaptor proteins. Additionally, the MPR can be recycled back to the trans-Golgi network (TGN) through its interaction with the retromer complex. Interestingly, in I-cell disease (ICD), in which the MPR pathway is non-functional, many soluble lysosomal proteins continue to traffic to the lysosomes. This observation led to the discovery that sortilin is responsible for the MPR-independent targeting of the sphingolipid activator proteins (SAPs) and acid sphingomyelinase (ASM). More recently, our laboratory has tested the hypothesis that sortilin is also capable of sorting a variety of cathepsins that exhibit varying degrees of MPR-independent transport. We have demonstrated that the transport of cathepsin D is partially dependent upon sortilin, that cathepsin H requires sortilin, and that cathepsins K and L attain the lysosomes in a sortilin-independent fashion. As a type-1 receptor, sortilin also has numerous cytosolic binding partners. It has been observed that like the MPR, the anterograde trafficking of sortilin and its cargo require both GGAs and AP-1. Similarly, the retrograde recycling pathway of sortilin also involves an interaction with retromer through a YXXphi site in the cytosolic tail of sortilin. In conclusion, the cytosolic domains of sortilin and MPR possess a high degree of functional homology and both receptors share a conserved trafficking mechanism.