NPC1 constructs.

NPC1 domain C sequences (residues 373 to 620) flanked by sequences that form antiparallel coiled coils as previously described (35) were cloned into the pcDNA3.1(+) vector. Constructs made included glycosylation mutants in HsNPC1 domain C (L414N+D416T, K498N+G500S, and N598A), while those in DrNPC1 domain C were N414A, N498A, and R600T. DrNPC1 domain C chimeras were made by substituting these residues for human residues 373 to 475 (chimera 1), 476 to 536 (chimera 2), 537 to 620 (chimera 3), 493 to 502 (chimera 4), 502 to 512 (chimera 5), and 513 to 522 (chimera 6), and the point mutations made were E502D, Y503F, I505V, H506Y, F509Y, and S511T. The constructs were then transiently transfected into HEK 293T cells, and the supernatant with secreted protein was harvested after 72 h and used in ELISAs. Purified proteins were made by transfecting FreeStyle 293-F cells in suspension, harvesting cells 72 h posttransfection, and purifying them by incubation with His-60 nickel resin. The proteins were eluted at 500 mM imidazole and pH 7.6 and dialyzed into 50 mM 2-(N-morpholino)ethanesulfonic acid (MES), 150 mM NaCl, pH 5.5. Domain C chimeras in the full-length NPC1 were generated by seamlessly replacing the domain C sequences in HsNPC1. The constructs were subcloned into the pBABE-puro retroviral vector and stably transfected into CHO-M12 cells by retroviral transduction, as previously described (22). All constructs possessed N-terminal Flag tags.