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Magnetic Nanoparticle-Based Isolation of Endocytic Vesicles Reveals a Role of the Heat Shock Protein GRP75 in Macromolecular Delivery

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Magnetic Nanoparticle-Based Isolation of Endocytic Vesicles Reveals a Role of the Heat Shock Protein GRP75 in Macromolecular Delivery Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery Anders Wittrupa, Si-He Zhanga, Katrin J. Svenssona, Paulina Kucharzewskaa, Maria C. Johanssona, Matthias Mörgelinb, and Mattias Beltinga,1 Sections of aOncology and bClinical and Experimental Infectious Medicine, Department of Clinical Sciences, Lund University, SE-221 85, Lund, Sweden Edited by Erkki Ruoslahti, Burnham Institute for Medical Research at the University of California, Santa Barbara, CA, and approved June 18, 2010 (received for review March 2, 2010) An increased understanding of cellular uptake mechanisms of dependent pathway involved in the internalization of a diverse set macromolecules remains an important challenge in cell biology with of cargoes (7, 8), share many characteristics with cholesterol- and implications for viral infection and macromolecular drug delivery. glycosphingolipid-rich membrane rafts. By using comparative pro- Here, we report a strategy based on antibody-conjugated magnetic teomics on detergent-resistant raft membrane preparations from − − nanoparticles for the isolation of endocytic vesicles induced by cav-1 / and wild-type fibroblasts, polymerase I and transcript re- heparan sulfate proteoglycans (HSPGs), key cell-surface receptors lease factor recently was identified as a constituent involved in of macromolecular delivery. We provide evidence for a role of the caveolae formation (7). Similar approaches have been used to de- glucose-regulated protein (GRP)75/PBP74/mtHSP70/mortalin (here- fine the anatomy of other trafficking vesicles (9, 10). These findings after termed “GRP75”) in HSPG-mediated endocytosis of macromo- have contributed profoundly to our understanding of endocytic lecules. GRP75 was found to be a functional constituent of intra- mechanisms; however, they are limited by their reliance on the cellular vesicles of a nonclathrin-, noncaveolin- dependent pathway fractionation and analysis of detergent-resistant cell-membrane that was sensitive to membrane cholesterol depletion and that preparations. showed colocalization with the membrane raft marker cholera toxin Heparan sulfate proteoglycans (HSPGs), a family of proteins subunit B. We further demonstrate a functional role of the RhoA substituted with polysulfated heparan sulfate (HS) polysaccharides, GTPase family member CDC42 in this transport pathway; however, have a key role in the endocytic uptake of a diverse set of macro- the small GTPase dynamin appeared not to be involved. Interest- molecular drugs, growth factors, and morphogens (2, 5, 11, 12) with ingly, we provide evidence of a functional role of GRP75 using important implications for early developmental patterning and RNAi-mediated down-regulation of GRP75 and GRP75-blocking anti- cancer. Here, we have capitalized on the recent finding (13) that bodies, both of which inhibited macromolecular endocytosis. We magnetic nanoparticles can be endocytosed efficiently by conjuga- conclude that GRP75, a chaperone protein classically found in the tion to a specific, phage-derived anti-HS antibody (αHS) for the endoplasmic reticulum and mitochondria, is a functional constituent isolation and analysis of intact, endocytic vesicles. Our studies reveal of noncaveolar, membrane raft-associated endocytic vesicles. Our a role of the chaperone 75-kDA glucose-regulated protein (GRP75) data provide proof of principle of a strategy that should be generally as a functional constituent of a defined endocytic pathway. applicable in the molecular characterization of selected endocytic pathways involved in macromolecular uptake by mammalian cells. Results Identification of GRP75 in HSPG-Associated Endocytic Vesicles. We drug delivery | endocytosis | heparan sulfate | proteoglycan | membrane set out to develop a strategy for qualitative analysis of the HSPG- raft dependent endocytic compartment of macromolecular delivery based on the internalization of single-chain fragment anti-HS– ndocytosis is the process by which cells compartmentalize conjugated superparamagnetic nanoparticles (scFv-αHSM). Among Econstituents of the plasma membrane and the extracellular several scFv-αHS clones, only one, AO4B08, efficiently delivers fi space into intracellular vesicles for further sorting to speci c magnetic nanoparticles to intracellular vesicles (13). Magnetic – cellular locations (1 4). Endocytosis regulates signaling events separation of mechanically disrupted cells that had internalized involved in cell motility and cell fate determination and can be scFv-αHSM yielded a population of intact, scFv-αHSM–containing exploited by microbial intruders for infection. Interestingly, the vesicles (Fig. 1A). Importantly, it has been established that up- same pathways may be used for the delivery of therapeutic mac- take of nanoparticles per se (i.e., in the absence of scFv-αHS) is romolecules (e.g., DNA, anti-sense oligonucleotides, and siRNA) insignificant, and that internalized scFv-αHSM and fluorophor- in the treatment of various diseases. A more detailed un- labeled scFv-αHSF antibody complexes show strong colocaliza- derstanding of endocytic mechanisms thus is a major challenge in tion in endocytic vesicles (13). Moreover, the uptake of scFv- basic cell biology with implications for viral infection, the regu- αHSF as well as scFv-αHSM was absent in HSPG-deficient lation of signaling networks in cancer (3), and the development of cells, indicating that the magnetic nanoparticle does not affect macromolecular drugs (5). the endocytic route itself and that the uptake of scFv-αHS is High-resolution, live-cell imaging techniques have unraveled M the heterogeneity of vesicular compartments in terms of kinetic/ dynamic parameters as well as ligand specificity. In addition to the Author contributions: A.W., S.-H.Z., K.J.S., P.K., M.C.J., M.M., and M.B. designed research; classical clathrin-dependent mechanism of endocytosis, several A.W., S.-H.Z., K.J.S., P.K., M.C.J., and M.M. performed research; M.M. contributed new clathrin-independent endocytic pathways are emerging (3, 4, 6). reagents/analytic tools; A.W., S.-H.Z., K.J.S., P.K., M.C.J., M.M., and M.B. analyzed data; Collectively, published data from several groups indicate that and A.W. and M.B. wrote the paper. ligands can be taken up by multiple lipid raft-mediated pathways; The authors declare no conflict of interest. however, these pathways require further definition at the molecular This article is a PNAS Direct Submission. level. Classification schemes based on the dependence on/associa- 1To whom correspondence should be addressed. E-mail: [email protected]. tion with dynamin, caveolin-1, and the RhoA family GTPases Rac1, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. RhoA, and CDC42 have been proposed. Caveolae, a dynamin- 1073/pnas.1002622107/-/DCSupplemental. 13342–13347 | PNAS | July 27, 2010 | vol. 107 | no. 30 www.pnas.org/cgi/doi/10.1073/pnas.1002622107 Downloaded by guest on September 27, 2021 showing the enrichment of several proteins in the vesicular prepa- B Non- PNS mag Mag ration (Fig. 1 B and C). We decided to focus on one of these pro- kDa teins, the heat shock protein (HSP) family member GRP75 that A Non-magnetic Magnetic 191 consistently appeared as one of the dominating vesicular compo- 97 nents, as determined by MALDI-TOF and LC-MS/MS. Previous 64 studies have suggested that GRP75 (also known as “PBP74”) not 51 only is present in the endoplasmic reticulum (ER) but also is asso- ciated with the plasma membrane and in early endocytic vesicles in 39 immune cells, suggesting a role in antigen processing (14). Others 28 have reported on cytosolic localization of GRP75 (also known as 19 “mortalin”), where it may regulate the activity of p53 (15) and FGF (16). More recent proteomic analyses of detergent-resistant mem- brane fractions have suggested GRP75 is a ubiquitously expressed C PNS Magnetic 31pH 031pH 0 membrane lipid raft protein (17); however, the role of GRP75 in High endocytic mechanisms remains unknown. MS data were confirmed MW D by immunoblotting experiments, showing substantial enrichment of PNS Mag GRP75 in endocytic vesicles (Fig. 1D).Thesedatawerecorrobo- rated by confocal microscopy, showing the localization of GRP75 to Anti-GRP75 scFv-αHS–induced vesicular compartments in intact cells (Fig. 1E). Importantly, immunogold staining and electron microscopy showed Low F MW the enrichment of GRP75 in magnetically isolated vesicles (Fig. 1 ). ThefactthatbothscFv-αHSM and scFv-αHSF colocalized with E αHS GRP75 GRP75 provides additional evidence that the nanoparticle per se fi Detail has no signi cant impact on the endocytic route. To address further whether GRP75 is located inside oratthesurfaceofisolatedvesi- cles, trypsin-treated and untreated vesicles were analyzed for GRP75 levels. As shown in Fig. S1C, GRP75 remained virtually intact after trypsin treatment. Notably, cell-surface GRP75 was shown to be highly trypsin sensitive(seecell-surfacebiotinylation experiments below). As a positive control, we found efficient deg- radation by trypsin of the cytoskeleton proteins actin and tubulin C F Non-magnetic Magnetic that are associated with the surface of endocytic vesicles (Fig. S1 ). Together, these data indicate that GRP75 is a genuine resident of endocytic vesicles. Immunoprecipitation of cell lysates with anti-GRP75 antibody or scFv-αHS did not result in coimmunoprecipitation of HSPG or GRP75, respectively. Moreover, GRP75 showed no specific binding to
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