title: In vitro correction of disorders of lysosomal transport by microvesicles derived from baculovirus-infected Spodoptera cells.
abstract: Infection of Spodoptera frugiperda (Sf9) cells by baculovirus (BV) is well established for transgene expression of soluble proteins, but few correctly folded transmembrane proteins have been so produced. We here report the use of the BV/Sf9 (BVES) method for the expression and transfer, via microvesicles, of the exclusive lysosomal exporters for cystine and sialic acid, human cystinosin and sialin. These proteins and their mRNA are released into the culture medium as very low-density microvesicles (~1.05 g/ml), which do not label for lysobisphosphatidic acid. The presence of the human transgene proteins in the vesicles was confirmed by western blotting and confirmed and quantified by mass spectrometry. Addition of vesicles to cultures of human fibroblast lines deficient in either cystinosin or sialin produced a progressive depletion of stored lysosomal cystine or sialic acid, respectively. The depletion effect was slow (T1/2 ~48 h), saturable (down to ~40% of initial after 4 days) and stable (>one week). Surprisingly, BV infection of Spodoptera appeared to induce expression and release into microvesicles of the insect orthologue of cystinosin, but not of sialin. We conclude that BVES is an effective method to express and transfer functional transmembrane proteins so as to study their properties in mammalian cells, and has a generic potential for transport protein replacement therapy.
authors: Department of Pediatric Genetics, University of Michigan, Ann Arbor, MI 48109-5629, USA. jthoene@umich.edu
authors: Jess
authors: J
authors: Thoene
authors: Thomas
authors: T
authors: Goss
authors: Marc
authors: M
authors: Witcher
authors: Jodi
authors: J
authors: Mullet
authors: Francisca
authors: F
authors: N'Kuli
authors: Patrick
authors: P
authors: Van Der Smissen
authors: Pierre
authors: P
authors: Courtoy
authors: Si Houn
authors: SH
authors: Hahn
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D018014:Gene Transfer Techniques; D006801:Humans; D066298:In Vitro Techniques; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D055806:Microvessels / Q000378:metabolism; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D029461:Sialic Acid Storage Disease / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D018411:Spodoptera / Q000166:cytology; D027981:Symporters / Q000235:genetics / Q000378:metabolism*
chemical_list: D026921:Amino Acid Transport Systems, Neutral; C111445:CTNS protein, human; D008565:Membrane Proteins; D027361:Organic Anion Transporters; D027981:Symporters; C421224:sialic acid transport proteins