Antibodies to cell surface proteins redirect intracellular trafficking pathways
Highlights
► Comparison of rabies virus glycoprotein internalization alone or bound to antibody. ► Internalization of the transferrin receptor bound to ligand or antibody. ► Bound antibodies mediate localization to degradation endosomes. ► Antibodies can redirect endogenous internalization pathways of receptor proteins.
Introduction
The use of antibodies to target specific cells or cell types has become an increasingly desirable method of treatment for a variety of infections and diseases. Several publications have indicated the benefit to using monoclonal antibodies to specifically deliver drugs, including the targeted delivery of cytokines and siRNAs to tumor cells (Kamizuru et al., 2001, Kaspar et al., 2007, Marecos et al., 1998, Peer et al., 2007, Polson et al., 2007, Trachsel et al., 2007). It has also been shown that glycoprotein-specific antibodies can mediate internalization of viral glycoproteins (Favoreel et al., 1999, Favoreel et al., 2004, Sarmiento et al., 2007, Van de Walle et al., 2001).
Rabies virus infections occur in over 100 countries and territories and are fatal once symptoms develop (WHO, 2007). To prevent development of the disease, treatment of exposed individuals includes administration of the rabies vaccine and human rabies immune globulin, which helps to neutralize the virus. There are several ways that rabies G-specific antibodies have been shown to mediate inhibition of the virus. Neutralizing antibodies can bind to the virion-expressed glycoprotein to either block infection of target cells or to inhibit escape of the virus from endosomal compartments following entry (Dietzschold et al., 1987), an important step in viral uncoating. Antibodies can also bind to rabies G expressed on the surface of infected cells to inhibit cell-to-cell spread (Lodmell and Ewalt, 1987). Virus-specific antibodies have also been exploited to target virus-infected cells and deliver antiviral agents, while sparing uninfected cells, by targeting cell surface-expressed viral proteins (Song et al., 2005, Wen et al., 2007). Using mouse neuroblastoma (MNA) cells that express rabies G on the cell surface to mimic an infected cell state (Wiktor and Koprowski, 1978), we examined the ability of a rabies G specific antibody to induce internalization and localization of the rabies G protein to degradative endosomal compartments as a model of antibody conjugates.
Due to the broad-range of cell surface proteins that have been successfully utilized for antibody-based internalization or delivery, we hypothesized that the internalization pathway seen when an antibody is bound to a cell surface protein may remain the same, regardless of the natural internalization pathway of the membrane protein to which the antibody is specific. In other words, we theorized that there might be a common antibody-mediated internalization pathway that exists when an antibody is bound to a cell surface protein. In order to examine this, we studied the internalization of a surface expressed viral protein, with and without antibody. We also analyzed the internalization of an endogenous cell surface protein, the transferrin receptor, when bound to an antibody (αCD71) and compared this to the well-known, recycling pathway observed when the receptor is bound to its ligand, transferrin.
Section snippets
Cells and cell culture
Mouse neuroblastoma (MNA) cells and HEK293T (ATCC) cells were grown in complete medium (Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin, 1% l-glutamine, and 1% sodium pyruvate) at 37 °C with 10% CO2. For localization experiments, 60–80% confluent cells were transfected using GeneJuice reagent (Novagen) according to the manufacturer's protocol.
Plasmids
The codon-optimized rabies G glycoprotein expression plasmid was generated from the
Initial binding and internalization of the ARG1/rabies G complex
We examined the internalization and fate of a viral cell surface glycoprotein, rabies G, by using a fluorescent rabies-specific antibody, ARG1, which specifically binds to and internalizes in mouse neuroblastoma cells expressing rabies G (MNAG) on the surface (Fig. S1). The endosomal localization of rabies G has not been studied. We therefore performed an in-depth analysis of the internalization and endocytic pathway of ARG1-bound rabies G, and compared this to the endosomal pathway of
Discussion
Studies have shown that the use of antibodies to target cellular proteins can be beneficial for the treatment of various diseases and cancers. The presence of antibodies specific for the β-amyloid peptide on the surface of neuronal cells, which can mediate internalization and degradation of the protein (Tampellini et al., 2007), have been shown to slow cognitive deterioration and reduce plaque burden in mice and Alzheimer's patients (Hock et al., 2003, Masliah et al., 2005, Solomon and Frenkel,
Conflict of interest
The authors express no conflicts of interest for this work.
Acknowledgments
We thank the following individuals at UMass: E. Latz for the XFP-Rab expression plasmids, D. Lambright for the GFP-Rab5a expression plasmid, and the Biomedical Imaging Group, especially Clive Standley and Karl Bellve, for assistance with TIRF image collection and analysis. This work was supported by NIH grants R01AI64349 to RWF and P01AI083215-01 to RWF and EKJ, NIH/NIAID grant U54AI057159 to RWF, and JDRF grant to RWF. Core resources supported by the Diabetes Endocrinology Research Center
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