Targeting of Acid Sphingomyelinase, a Lysosomal Membrane Protein, Using Apolipoprotein E3-containing Nanodiscs

We developed a nanodisc (ND)-based platform with recombinant acid sphingomyelinase (ASM) embedded in a bilayer of phospholipids that are circumscribed by apolipoprotein E3 (apoE3) for targeted delivery to lysosomes. We designed a fusion protein comprising essential catalytic segments of ASM with a transmembrane helix at one end (TM-ASM_cat). Purified TM-ASM_cat was reconstituted with phospholipids and the N-terminal (NT) domain of apoE3 to promote the formation of NDs (TM-ASM_cat-ND). Physicochemical characterization revealed the presence of both TM-ASM_cat (35 kDa) and apoE3NT (24 kDa) in the preparations, and formation of large complexes (400-600 kDa). Transmission electron microscopy of TM-ASM_cat-ND revealed discoidal complexes (major/minor axes of 30.9 ± 11.9/8.6 ± 2.0 nm, respectively), while atomic force microscopy showed NDs ~ 14 nm high. The TM-ASM_cat-ND displayed a significant ability to hydrolyze sphingomyelin at pH 5. LC-MS/MS of glioblastoma cells treated with TM-ASM_cat-ND revealed a significant decrease (22.3%) in total sphingomyelin content compared to cells treated with PBS. Sphingolipidomic analysis showed a trend of decrease in specific sphingomyelin species compared to empty ND-treated cells. Immunofluorescence analysis revealed perinuclear vesicles showing colocalization of ASM with apoE3 and Lysotracker, indicative of successful targeting of TM-ASM_cat-ND to the lysosomes. Taken together, our data indicate that TM-ASM_cat has been embedded in ND, and that it retains catalytic activity in solution and inside the cells. Our findings bear the potential to treat ASM deficiency in Niemann-Pick disease Type A and B, which are lysosomal storage disorders characterized by the accumulation of sphingomyelin.