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. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: Exp Mol Pathol. 2011 Jul 23;91(3):723–732. doi: 10.1016/j.yexmp.2011.05.011

Antibodies to cell surface proteins redirect intracellular trafficking pathways

Christine A St Pierre a, Deborah Leonard b, Silvia Corvera b, Evelyn A Kurt-Jones a, Robert W Finberg a,*
PMCID: PMC3315679  NIHMSID: NIHMS331569  PMID: 21819978

Abstract

Antibody-mediated intracellular delivery of therapeutic agents has been considered for treatment of a variety of diseases. These approaches involve targeting cell-surface receptor proteins expressed by tumors or viral proteins expressed on infected cells. We examined the intracellular trafficking of a viral cell-surface-expressed protein, rabies G, with or without binding a specific antibody, ARG1. We found that antibody binding shifts the native intracellular trafficking pathway of rabies G in an Fc-independent manner. Kinetic studies indicate that the ARG1/rabies G complex progressively co-localized with clathrin, early endosomes, late endosomes, and lysosomes after addition to cells. This pathway was different from that taken by rabies G without addition of antibody, which localized with recycling endosomes. Findings were recapitulated using a cellular receptor with a well-defined endogenous recycling pathway. We conclude that antibody binding to cell-surface proteins induces redirection of intracellular trafficking of unbound or ligand bound receptors to a specific degradation pathway. These findings have broad implications for future developments of antibody-based therapeutics.

Keywords: Endosomes, degradation, antibody, trafficking, internalization

Introduction1

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.

Materials and Methods

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 glycoprotein amino acid sequence of the rabies virus ERA strain (GenBank: AF406693) (Prehaud et al., 2003). N-terminal fluorescently tagged human Rab4a, canine Rab9a, and canine Rab11a expression plasmids were kindly provided by E. Latz (University of Massachusetts, Worcester, MA). N-terminal GFP-tagged mouse clathrin light chain A expression plasmid was provided by J.H. Keen (Kimmel Cancer Institute, Philadelphia, PA), first referenced in (Gaidarov et al., 1999). To generate dominant negative Rab5a (S34N), site-directed mutagenesis was performed on a N-terminal GFP-tagged human Rab5a expression plasmid (gift from D. Lambright, University of Massachusetts, Worcester, MA) using a Phusion site-directed mutagenesis kit (Finnzymes). C-terminal GFP-tagged human TfR expression plasmid was generated by subcloning the human TfR cDNA (kindly provided by T. E. McGraw, Weill Medical College of Cornell University, New York, NY) in frame with eGFP in the pEGFP-N1 vector (Clontech) at the XhoI and BamHI restriction sites.

Antibodies and reagents

ARG1 antibody was purified from hybridomas generated from rabies vaccine immunized mice. Mouse anti-human CD71 (αCD71) antibody was obtained from BD Biosciences. Antibodies were directly labeled with Alexa fluor 647 (for confocal studies) or Alexa fluor 568 (for TIRF studies) using Alexa fluor protein labeling kits (Molecular Probes, Invitrogen). Fluorescent human transferrin (Tf) was obtained from Molecular Probes (Invitrogen). Latrunculin-A, used at a concentration of 1.25 μM for inhibition studies, was purchased from Sigma.

Flow Cytometric Analysis

Cells were grown to subconfluency on 6-well dishes (Falcon) in complete medium. Cells were resuspended in FACS buffer (PBS containing 1% BSA [bovine serum albumin] and 0.01% sodium azide) and surface stained with indicated antibodies for 60 min at 4°C. When necessary, secondary anti-IgG-APC (Invitrogen) was added to cells for 60 min at 4°C in FACS buffer. Following staining, cells were washed with PBS and analyzed on a LSRII (BD Biosciences). Data were acquired by DIVA (BD Biosciences) and were analyzed with FlowJo 8.8.6 software (Tree Star Inc.).

Confocal Microscopy

For live imaging, cells were cultured on glass-bottom 35-mm tissue-culture dishes (MatTek) in complete medium. For fixed imaging, cells were cultured as above, followed by fixation with 4% formaldehyde for 15 min. Cells were then incubated in 1X PBS containing 1% fetal bovine serum and 0.5% TritonX-100 for permeabilization and blocking. Rabies G staining was performed with 3 μg/ml ARG1-647 for 30 min. in 1X PBS, followed by 2 washes. Images were taken on a Leica SP2 AOBS confocal laser-scanning microscope with a 63x objective, using Leica Confocal Software. Multi color images were acquired by sequential scanning with only one laser active per scan to avoid cross-excitation. Overall brightness and contrast of images were optimized using Adobe Photoshop CS3.

TIRF Microscopy

Cells were cultured on 25-mm coverslips (Thomas Scientific, No. 1.5) in complete medium. Cells were transferred from complete medium to KRH (125 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 25 mM HEPES pH 7.4, 2 mM sodium pyruvate, and 0.5% BSA) just prior to imaging, at 35°C. Imaging hardware and software were previously described (Bellve et al., 2006). Two Coherent Innova 70C lasers were used. Argon ion and argon-krypton ion lasers were used to produce the 488 and 568 nm light, respectively. The combined beams were coupled into a single mode fiber using a KineFLEX fiber coupler manufactured by Point Source (Hamble, UK). A modified Olympus IX81 inverted microscope, a modified Olympus TIRF fiber illuminator and an Olympus Plan APO 60x objective with an NA of 1.45 were used. TIRF illumination was introduced through the edge of the objective at an angle set between 65° and 68° giving a penetration depth of 90–121 nm at 488 nm and 105–141 nm at 568 nm. Light is collimated through the objective and a layer of immersion oil onto the coverslip. The quality of the collimation was set halfway between the best for 488 nm and 568 nm. Light from the fluorophores was collected and relayed onto a 640×448 pixel CCD camera developed with Lincoln Labs (MIT). A Physik Instruments pifoc was used for fine focus control. The entire microscope was contained in a heated chamber held at 35°C.

Co-localization analysis

The total number and percent of co-localized pixels per image was calculated as described previously (Bellve et al., 2006; Leonard et al., 2008). Briefly, single fluorophore raw images were corrected by subtracting the background fluorescence outside the cell. Next, regions of potential co-localization were cropped and saved as new files for analysis. From this, the intensity of all positive-valued pixels was set to one and all other pixels to zero to generate a binary masking image. Co-localized pixels were identified from the overlap of the masked images of each fluorophore. Non-specific (background) co-localization was defined as that seen when pixel-rich regions were rotated 180° relative to each other (flipped images).

Statistical Analysis

A one-way ANOVA followed by Bonferroni’s correction for post-test comparisons was used to determining statistical significance. Values of P < 0.05 were considered significant. Statistics were performed using GraphPad (Prism v5.0a) software.

Results

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 non-ARG1-bound protein.

Clathrin-mediated internalization is most commonly associated with endocytosis of cell surface proteins and receptors. Internalization involves the binding of a ligand to a cell surface protein, clustering of membrane proteins into a coated-pit, followed by vesicle formation and budding into the cell and movement through the endosomal pathway. To examine the role of clathrin in fluorescent ARG1/rabies G complex internalization, MNAG cells were transfected with clathrin-GFP and ARG1 localization was assessed by total internal reflection fluorescence (TIRF) microscopy, an imaging technique in which fluorophores residing within approximately 100–300 nm from the plasma membrane can be selectively excited (Axelrod, 2001; Axelrod, 2003; Leonard et al., 2008). In order to determine localization, we examined the total number and the percent of ARG1 and clathrin co-localized over a 20 min. time course.

We found that internalized ARG1 quickly localizes with clathrin-GFP after addition to cells and remains co-localized through 20 min. (Fig. 1A). Total co-localized ARG1 pixels ranged from 220–480 pixels and the percent of ARG1 pixels co-localized with clathrin ranged from 33–56 percent. This was significantly higher than background localization levels, which were calculated when the images (ARG1 and clathrin) were flipped 180 degrees relative to each other (Fig. 1A, flipped images). Background levels ranged from 90–270 total and 18–34 percent co-localized pixels.

Figure 1.

Figure 1

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ARG1 localizes with clathrin-expressing vesicles at early time points following addition to cells. (A, B) MNA cells were co-transfected with 1 μg rabies G and clathrin-GFP. (A) Amount of total and percent ARG1/clathrin co-localized pixels from addition to cells (T = 0) to 20 min following addition (T = 1200). Images were taken using TIRF microscopy. Arrow indicates a wash step. (B) ARG1 (red) was incubated with cells for given time points in complete medium and imaged using confocal microscopy. Arrows indicate regions of co-localization. Data are representative of three separate experiments. Bar, 5 μm.

We also examined ARG1 localization to clathrin at later time points using confocal microscopy. Similar to TIRF results, internalized antibody localized with clathrin up to 30 min after addition to cells (Fig. 1B, arrows) with no localization by 60 min. These data indicate that the ARG1/rabies G complex internalizes in MNAG cells via a clathrin-mediated endocytosis pathway, similar to that seen with other antibody-bound viral glycoproteins (Sarmiento et al., 2007; Van de Walle et al., 2001).

Role of actin in ARG1-mediated internalization

Studies have indicated that actin polymerization is necessary for receptor-mediated endocytosis and antibody-directed endocytosis of viral glycoproteins in mammalian cells (Lamaze et al., 1997; Van de Walle et al., 2002). To examine the role of actin in ARG1 endocytosis, internalization was analyzed in the presence of the actin-specific inhibitor, Latrunculin-A (LA), using confocal microscopy. When actin polymerization was blocked by LA, there was no internal staining at any time point when compared to untreated cells (Fig. S2). Thus, the ARG1/rabies G complex internalizes through a clathrin-associated and actin-dependent mechanism of entry.

Early endosomal localization of rabies G in the presence or absence of antibody

The endosomal internalization pathway consists of various compartments that are differentially characterized by their expression of proteins of the Rab family of small GTPases, including Rab4, Rab5, Rab9, and Rab11. Rab4 is expressed in early endosomes and recycling endosomes, and is thought to play a role in early sorting events (Schmidt and Haucke, 2007; Zerial and McBride, 2001). Rab5 is a key regulator of early endocytosis, including movement from clathrin-coated vesicles to early endosomes as well as fusion between early endosomal compartments (Bucci et al., 1992; Gorvel et al., 1991; Zerial, 1993; Zerial and McBride, 2001). Rab9 is expressed in and regulates trafficking within late endosomes (Schmidt and Haucke, 2007; Zerial and McBride, 2001). Rab11 is expressed in recycling endosomes and regulates traffic at the trans-golgi network/recycling endosome boundary (Ren et al., 1998; Schmidt and Haucke, 2007).

To examine early endosomal localization, the role of Rab5a in ARG1-mediated internalization of rabies G was assessed in cells expressing wild type GFP-Rab5a through confocal microscopy. Internalized ARG1 localized with Rab5a positive endosomes at early time points after addition to cells (Fig. 2A, solid arrows), with maximal localization at 15 min and a complete loss of localization by 60 min. To quantify the apparent co-localization, we also analyzed the total number and percent of co-localized pixels at the given time-points. We saw a significant amount of co-localized pixels at 15 and 30 minutes, with a mean (± SEM) of 132.25 (± 18.23) and 81.25 (± 23.52) total pixels, respectively. The amount of ARG1/Rab5a co-localized pixels decreased over time, with numbers similar to background (flipped images) by 60 to 180 min ranging from a mean (± SEM) of 29.88 (± 7.66) to 16 (± 4.23) co-localized pixels, respectively (Fig. 2B). The percent of ARG1 pixels co-localized to Rab5a followed a similar pattern, with the highest percentage at 15 min (Fig. 2B).

Figure 2.

Figure 2

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Internalized ARG1/rabies G complex localizes to and requires Rab5a positive endosomes. MNA cells were co-transfected with 1 μg rabies G and either WT-GFP-Rab5a (A, B) or DN-GFP-Rab5a (C) and incubated with ARG1 (red) for given time points in complete medium or stained for rabies G using ARG1 following fixation/permeabilization (B). Solid arrows indicate regions of co-localization. Open arrows indicate internalized antibody. (B) Analysis of total and percent live ARG1/Rab5a (squares) or fixed rabies G/Rab5a (circles) co-localized pixels (n ≥ 5 per time point). P-values, calculated as ARG1 versus Fix, are shown as *** ≤0.001; n.s. = not significant. Data are representative of at least two separate experiments. Bar, 5 μm.

To characterize whether rabies G localizes to Rab5a early endosomes without antibody present, MNAG cells were transfected with GFP-Rab5a and stained for rabies G using ARG1 after fixation and permeabilization. Endogenous rabies G localized to Rab5a at levels similar to that of the 15 min and 30 min ARG1-bound complex time points with a mean (± SEM) of 69.45 (± 13.77) total and 12.11 (± 1.18) percent co-localized pixels (Fig. 2B).

The requirement of functional Rab5a in ARG1-mediated internalization of rabies G was assessed in cells expressing a GFP-tagged dominant-negative (DN) Rab5a. In order to function properly, Rab5a must cycle between the active GTP-bound and inactive GDP-bound states. The DN form of Rab5a contains a S34N amino acid change that is unable to bind to GTP and, thus, remains GDP-bound. No internalization of the ARG1/rabies G complex occurred in the absence of functional Rab5a (Fig. 2C, green cells). As a control, ARG1 internalization was unaffected in cells that did not express GFP and were, therefore, not transfected with the DN-form (Figure 2C, open arrows). These results indicate that ARG1 internalization following binding to rabies G localized with and is dependent on the presence of functional Rab5a, a key regulator of endosomal trafficking.

Differential localization of rabies G to recycling and late endosomal compartments

To assess localization with recycling endosomes, internalized ARG1 was compared to GFP-tagged Rab4a and Rab11a in MNAG cells through confocal microscopy. The ARG1/rabies G complex did not localize with recycling endosomal proteins, Rab4a or Rab11a at any time point tested (Figs 3 and 4). In the absence of antibody, however, rabies G significantly localized with Rab11a, exhibiting a mean (± SEM) of 128.8 (± 17.98) total and 27.66 (± 3.74) percent co-localized pixels (Fig. 4B). This data suggests that ARG1 binding can shift the endosomal localization of rabies G within cells.

Figure 3.

Figure 3

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ARG1 does not localize with Rab4a positive endosomes. MNA cells were co-transfected with 1 μg Rabies G and GFP-Rab4a and incubated with ARG1 (red) for given time points in complete medium. Data are representative of two separate experiments. Bar, 5 μm.

Figure 4.

Figure 4

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Rabies G alone, but not the ARG1/Rabies G complex localizes to Rab11a positive endosomes. (A, B) MNA cells were co-transfected with 1 μg Rabies G and GFP-Rab11a. Cells were either (A, B) incubated with ARG1 (red) for given time points in complete medium or (B) stained for rabies G using ARG1 following fixation/permeabilization. (B) Analysis of total and percent of live ARG1/Rab11a (squares) or fixed rabies G/Rab11a (circles) co-localized pixels (n ≥ 5 per time point). P-values, calculated as ARG1 versus Fix, are shown as *** ≤0.001; ** < 0.01. Data are representative of at least two separate experiments. Bar, 5 μm.

In contrast to recycling endosomes, internalized ARG1 begins to significantly localize with the late endosomal protein, Rab9a by 60 min after addition to cells, with maximal co-localized pixels at 120 and 180 min (Fig. 5, A B) exhibiting a mean (± SEM) of 402.58 (± 45.92) and 432.09 (± 59.79) total and 28.11 (± 2.39) and 31.46 (± 2.8) percent co-localized pixels, respectively. Background co-localized pixels ranged from a mean (± SEM) of 11.25 (± 7.57) to 86.09 (± 20.66) total and 1.4 (± 0.99) to 6.65 (± 0.61) percent co-localized pixels at all time points. These results are in contrast to the endogenous endosomal pathway followed by rabies G in expressing cells, which does not localize with Rab9a (Fig. 5B), indicating that ARG1 can shift the endosomal localization of rabies G.

Figure 5.

Figure 5

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Internalized ARG1/rabies G complex, but not rabies G alone localizes to late endosomes. (A, B) MNA cells were co-transfected with 1 μg Rabies G and YFP-Rab9a. Cells were either (A, B) incubated with ARG1 (red) for given time points in complete medium or (B) stained for rabies G using ARG1 following fixation/permeabilization. Arrows indicate regions of co-localization. (B) Analysis of total and percent live ARG1/Rab9a (squares) or fixed rabies G/Rab9a (circles) co-localized pixels (n ≥ 4 per time point). P-values, calculated as ARG1 versus Fix levels, are shown as *** ≤0.001; * < 0.05; n.s. = not significant. Data are representative of three separate experiments. Bar, 5 μm.

To test for a possible role for Fc-receptors in antibody internalization or trafficking, the internalization of ARG1 F(ab′)2 fragments was assessed. Fluorescently labeled F(ab′)2 fragments of ARG1 exhibited a staining and endosomal localization pattern similar to that of full length ARG1 (Fig. S3), indicating that the Fc region of ARG1 does not play a role in internalization and endosomal localization.

ARG1 localization to lysosomes

Lysosomal localization of the ARG1/rabies G complex in MNAG cells was also assessed through confocal microscopy. Internalized ARG1 began to localize with the cell-permeant, acidotrophic dye, LysoTracker Green DND-26, at 60 min after addition to cells (Fig. 6). The total number of co-localized pixels increased over time, maximal co-localized pixels at 180 min exhibiting a mean (± SEM) of 345 (± 55.78) total and 33.54 (± 5.39) percent co-localized pixels (Fig. 6B). These results indicate that the ARG1/rabies G complex traffics from Rab5a-positive early endosomes to Rab9a-positive late endosomes and finally to a lower pH, lysosomal-like compartment within 180 min.

Figure 6.

Figure 6

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Internalized ARG1/rabies G complex localizes to a lower pH, lysosomal compartment. (A, B) MNAG cells were incubated with ARG1 (red) and LysoTracker Green DND-26 (50 nM) for 30 min in complete medium. Arrows indicate regions of co-localization. (B) Analysis of total and percent ARG1/Lysotracker co-localized pixels (n ≥ 4 per time point). P-values, relative to flipped levels, are shown as ***≤0.001; ** < 0.01; n.s. = not significant. Data are representative of three separate experiments. Bar, 5 μm.

Internalization and endosomal localization of the transferrin receptor/transferrin complex

In order to determine whether the antibody-mediated shift in intracellular trafficking that we observed with the rabies glycoprotein was a generalized phenomenon, we examined the internalization pathway of an endogenous cell surface protein, the transferrin receptor (TfR), bound to an antibody or bound to its ligand, transferrin (Tf). We first verified the natural internalization pathway of the TfR in HEK cells. TfR bound to Tf is known to internalize via clathrin-coated vesicles almost exclusively through a recycling pathway (Green et al., 1997; Harding et al., 1983; Sonnichsen et al., 2000).

To examine localization with clathrin, fluorescent Tf was added to HEK cells transfected with clathrin-GFP. Tf localization to clathrin was examined using TIRF microscopy. As expected, Tf localized with clathrin very rapidly after addition to cells, consistent with published findings (Bellve et al., 2006; Leonard et al., 2008) (Fig. S4A). TIRF analysis revealed a mean (± SEM) of 220.5 (± 94.7) total and 31.21 (± 11.89) percent Tf co-localized pixels, respectively. This was higher than background co-localization (flipped images), which exhibited a mean (± SEM) of 20.88 (± 23.64) total and 2.692 (± 2.4) percent co-localized pixels. Background co-localization was calculated when the images (Tf and clathrin) were flipped 180 degrees relative to each other.

To examine endosomal localization of Tf-bound TfR, HEK cells were transfected with GFP-Rab4a, GFP-Rab5a, YFP-Rab9a, or GFP-Rab11a expressing plasmids and imaged using confocal microscopy. As expected, when bound to fluorescent Tf, internalized TfR localized with the early endosomal markers GFP-Rab4a (Fig. S5A) and GFP-Rab5a (Fig. S5B) and the recycling endosomal marker GFP-Rab11a (Fig. S5C), but not with the late endosomal marker, YFP-Rab9a (Fig. S5D). These data demonstrate that the TfR/Tf complex internalizes through a recycling endosomal pathway, and confirm the results obtained by others (Green et al., 1997; Harding et al., 1983; Sonnichsen et al., 2000)

Endosomal localization of the transferrin receptor/antibody complex

We next examined whether or not the internalization of the TfR to recycling endosomes would be affected if the receptor were bound to an antibody instead of Tf. Other groups have studied the ability to use antibodies specific for the TfR for drug delivery across the blood brain barrier, reviewed in (Qian et al., 2002). To mimic an antibody-based therapeutic scenario, we used an antibody specific for the exofacial domain of the transferrin receptor protein, αCD71. As described above, HEK cells were transfected with GFP-Rab4a, GFP-Rab5a, YFP-Rab9a, or GFP-Rab11a. Surprisingly, when bound to fluorescent αCD71, the TfR/αCD71 complex localized with all endosomal markers, including the late endosomal marker, YFP-Rab9a (Figs 7, 8), which was not seen in the natural TfR/Tf pathway. As a control, fluorescent αCD71 localized to GFP-tagged TfR at all time points tested (Figure 8C). Internalized antibody also localized with clathrin, similar to that seen with both the ARG1/rabies G and TfR/Tf complexes (Fig. S4B).

Figure 7.

Figure 7

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Antibody-mediated transferrin receptor early and recycling endosomal localization is similar to that of the natural pathway. HEK cells were transfected with 1 μg GFP-Rab4a (A), GFP-Rab5a (B), or GFP-Rab11a (C) and incubated with αCD71 (red) for given time points in complete medium. Arrows indicate regions of co-localization. Data are representative of three separate experiments. Bar, 5 μm.

Figure 8.

Figure 8

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Antibody-mediated transferrin receptor internalization includes movement though late endosomes. (A) HEK cells were transfected with 1 μg YFP-Rab9a (green) and incubated with αCD71 (red) for given time points in complete medium. Arrows indicate regions of co-localization. Bar, 5 μm. (B) Analysis of total and percent of Rab9a/Tf (black squares) versus Rab9a/αCD71 (red triangles) co-localized pixels (n ≥ 5 per time point). P-values, calculated as αCD71/Tf versus Rab9a/Tf levels, are shown as *** ≤ 0.001; ** < 0.01; * <0.05. Data are representative of three separate experiments. (C) HEK cells were transfected with 1μg TfR-GFP (green) and incubated with αCD71 (red) for given time points in complete medium. Bar, 5μm. Data are representative of two separate experiments.

To quantify the apparent antibody-mediated shift in trafficking to include late endosomes, the amount of co-localized pixels between the TfR/Tf complex or the TfR/αCD71 complex and YFP-Rab9a were compared. The number of co-localized pixels between the TfR/Tf complex and Rab9a were at levels similar to background (flipped images) at all time points tested, ranging from a mean (± SEM) of 49.33 (± 17.93) to 116 (± 21.22) total and 9.84 (± 1.48) to 13.93 (± 1.83) percent co-localized pixels. Comparatively, the number of co-localized pixels between the TfR/αCD71 complex and Rab9a were significantly higher (P ≤ 0.01) than the TfR/Tf complex at all time points, ranging from a mean (± SEM) of 200.5 (± 34.79) to 422.67 (± 99.86) total and 19.22 (± 3.22) to 24.98 (± 2.35) percent co-localized pixels (Fig. 8B). These results indicate that, when bound to an antibody, the internalization pathway of the TfR can shift from an exclusively recycling endosomal pathway to include localization with late endosomes.

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, 2010).

Studies have also indicated the benefit of using antibodies to treat viral infections. Virus-specific antibodies are known to modulate or neutralize viral infection and decrease virus-induced cell death (Sarmiento et al., 2007). It has been suggested that the ability of antibodies to neutralize virus is partially due to the fact that an antibody efficiently binds to and internalizes the envelope protein to which it is specific (Chesebro et al., 1979; Sarmiento et al., 2007). One potential mechanism for neutralization is to mediate the internalization of the viral glycoproteins on infected cells. It is possible that this internalization renders virus within a cell unable to appropriately bud from an infected cell, leading to a decrease in viral titers and an ineffective viral infection (Chesebro et al., 1979; Dowdle et al., 1974). It has been shown that antibody-mediated internalization of viral proteins can be used to target antiviral therapeutics to infected cells only. For example, Song et al. reported that an antibody against the HIV glycoprotein can specifically deliver functional siRNA only to infected cells, leading to a decrease in viral protein expression (Song et al., 2005). Others have shown that antibodies directed against the hepatitis B surface antigen can effectively deliver antiviral siRNAs to infected cells both in vitro and in vivo leading to decreased viral gene expression (Wen et al., 2007).

Using a monoclonal antibody specific for rabies G, ARG1, we show that the ARG1/rabies G complex internalizes through endosomes associated with the degradation pathway, including localization with Rab5a, Rab9a and low pH lysosomes. We have also shown that although endogenous rabies G protein expressed in MNA cells maintains localization to Rab5a early endosomes, it also exhibits localization to Rab11a recycling endosomes, which was not seen with the ARG1/rabies G complex. Conversely, endogenous rabies G did not localize to Rab9a late endosomes, while the ARG1/rabies G complex did. Although this is not a true infection model, this suggests that antibody binding can induce a shift in endosomal localization of rabies G to late endosomes and away from recycling endosomes. This finding does not appear to be virus-specific as preliminary studies have suggested that a monoclonal antibody specific to the Hepatitis C virus envelope also mediates localization of the protein to late endosomes and lysosomes in HEK cells (C. A. St. Pierre, unpublished).

Several groups have suggested that anti-receptor antibodies can mediate a decrease in cell receptor expression of a variety of receptors, including the asialoglycoprotein receptor (Schwartz et al., 1986), the insulin receptor (Roth et al., 1983; Taylor and Marcus-Samuels, 1984), and the mannose-6-phosphate receptor (von Figura et al., 1984). To add to these findings and demonstrate that antibody binding can re-direct endosomal trafficking of cellular proteins as well as viral proteins using specific endosomal markers and confocal microscopy, we analyzed the antibody-mediated internalization of an endogenous cell surface protein, the TfR. The TfR is expressed on most cell types, including cells in the brain and is highly expressed in tumor cells (Qian et al., 2002). As such, groups have studied the ability to use the TfR as a therapeutic target. Some groups have looked at conjugating drugs or DNA to Tf to mediate delivery into cells, while others have examined conjugating drugs or DNA to antibodies specific for the TfR (Qian et al., 2002). It has been shown that antibody binding can mediate degradation of the TfR (Hopkins and Trowbridge, 1983; Lesley and Schulte, 1985; Lesley et al., 1989; Weissman et al., 1986). These studies used electron microscopy to demonstrate that IgG bound to the TfR mediates a loss of surface expression and internalization through a pathway different from the recycling receptor. Due to the lack of known markers, localization of IgG-bound TfR to endosomal compartments was based solely on morphology. We have further characterized this by specifically examining the endosomal localization of antibody-bound TfR using confocal microscopy and known endosomal/lysosomal markers.

We observed a significant increase in localization to Rab9a-positive late endosomes when the receptor was bound to an antibody, indicating that the internalization pathway had been shifted to include degradation-associated endosomes, in agreement with our hypothesis (Fig. 9). As expected, when bound to its natural ligand, Tf, the receptor internalized exclusively through a recycling endosomal pathway, including localization with endosomal proteins Rab4a, Rab5a, and Rab11a, but not with Rab9a. Since the TfR normally recycles, there is a possibility that antibodies that reach late endosomes are no longer associated with the TfR. In other words, the fluorescence that co-localizes with Rab9 could be due to free αCD71 antibodies. We found that fluorescent αCD71 co-localized to a GFP-tagged TfR at all time points tested, ruling out this possibility. These findings are in agreement with work by Hopkins and Trowbridge (Hopkins and Trowbridge, 1983), which determined that antibodies bound to the TfR remained associated with the receptor following internalization through Sepharose-Tf purification. These results demonstrate that there is a specific antibody-mediated internalization pathway that occurs when an antibody is bound to a cell surface protein.

Figure 9.

Figure 9

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Diagram of specific antibody-mediated internalization pathway. Antibodies specific for both viral (Rabies G = ARG1) and endogenous proteins (TfR = αCD71) mediate localization to Rab9a-positive late endosomes (LE). EE = Early endosomes, RE = recycling endosomes.

Previous studies by Lesley et al. suggest that the Fc region of antibodies may be involved in sorting cell surface receptors to different endosomal compartments since Fab′-IgG bound to the TfR did not induce a significant loss of surface expression of the receptor (Lesley et al., 1989). Although we cannot completely rule-out this possibility without further experimentation, our results with the endosomal localization of internalized F(ab′)2-ARG1 suggest that antibody-mediated trafficking to degradation endosomes is Fc-independent. It is possible that antibody valence, rather than the Fc region, mediates internalization of the receptor to a non-recycling pathway, as suggested by (Lesley et al., 1989; Lesley and Schulte, 1985). It will be of interest to further examine this possibility with Fab′ fragments of both ARG1 and αCD71.

It is thought that in order for an antibody conjugate to be functional, it must internalize and localize with early and late endosomes, and potentially lysosomes, so that the antibody/protein complex will be degraded or, in the case of a drug/siRNA-conjugate, the drug (e.g., siRNA or inhibitors) will be released into the cytoplasm (Perera et al., 2007; Sarmiento et al., 2007; Tampellini et al., 2007). Studies thus far examining the potential of an antibody to be used as a delivery therapeutic have focused on determining whether or not the cell surface protein selected will localize with late endosomes (Perera et al., 2007). Since our findings indicate that binding a cell surface protein with an antibody shifts the endosomal localization of the protein from a recycling pathway to a degradative pathway, such selection of targets should not be necessary. In other words, our findings suggest that one may not have to choose an antibody therapeutic based on the intrinsic property of a cell surface protein, since the binding of an antibody to that protein would automatically guide internalization to include the degradative compartments. Thus, we conclude that since antibodies alter the intrinsic degradation pathway of a given cell surface protein, this expands the possible targets for a monoclonal antibody to be used to treat cancer or infections. Proteins that can be successfully targeted by therapeutic monoclonal antibodies are not limited to those that are normally processed through late endosomes, but should include any cell surface protein, including recycling receptors and viral envelope proteins.

Supplementary Material

01. Figure S1.

ARG1 specifically binds to and internalizes in rabies G-expressing cells. (A) MNA cells were transfected with rabies G (red lines), or empty vector (dashed lines) and surface stained with indicated antibodies (non-specific or ARG1). Gray plots indicate isotype staining. (B) MNAG cells were incubated with ARG1 at 37°C and 4°C for given time points in complete medium. Bar, 5 μm.

02. Figure S2.

ARG1 internalization requires actin polymerization. MNAG cells were treated with 1.25 μM of Latrunculin-A (LA treated) or DMSO carrier alone (untreated) for 30 min. prior to addition of ARG1 antibody (red) in complete medium for given time points. Bar, 5 μm.

03. Figure S3.

F(ab′)2-ARG1 internalization and endosomal localization is similar to full length ARG1. MNA cells were co-transfected with rabies G and WT-GFP-Rab5a (A) or YFP-Rab9a (B) and incubated with F(ab′)2-ARG1 (red) for given time points in complete medium. Data are representative of two separate experiments. Arrows indicate regions of co-localization. Bar, 5 μm.

04. Figure S4.

Ligand and antibody-mediated internalization of the TfR localizes with clathrin. (A, B) HEK cells were transfected with 1 μg clathrin-GFP followed by incubation with either transferrin (Tf) or antibody (αCD71). Graphs show the amount of Tf/clathrin (A) or αCD71/clathrin (B) co-localized pixels from addition to cells (T = 0 or 400) to 20 min. following addition (T = 1200). Images for analysis were taken using TIRF microscopy. Data are representative of three separate experiments.

05. Figure S5.

Ligand-mediated transferrin receptor internalization localizes with early and recycling endosomes, but not late endosomes. HEK cells were transfected with 1 μg GFP-Rab4a (A) GFP-Rab5a (B), GFP-Rab11a (C) or YFP-Rab9a (D) and incubated with Tf (red) for given time points in complete medium. Arrows indicate regions of co-localization. Data are representative of three separate experiments. Bar, 5 μm.

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.

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 grant DK32520 were also used (to RWF, EKJ, and SC).

Footnotes

1

Abbreviations: G = glycoprotein; LA = latrunculin-A; MNA = mouse neuroblastoma cells; MNAG = MNA cells expressing rabies G; TIRF = total internal reflection fluorescence; Tf = transferrin; TfR = transferrin receptor

Conflict of Interest

The authors express no conflicts of interest for this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Figure S1.

ARG1 specifically binds to and internalizes in rabies G-expressing cells. (A) MNA cells were transfected with rabies G (red lines), or empty vector (dashed lines) and surface stained with indicated antibodies (non-specific or ARG1). Gray plots indicate isotype staining. (B) MNAG cells were incubated with ARG1 at 37°C and 4°C for given time points in complete medium. Bar, 5 μm.

NIHMS331569-supplement-01.tif (735.3KB, tif)
02. Figure S2.

ARG1 internalization requires actin polymerization. MNAG cells were treated with 1.25 μM of Latrunculin-A (LA treated) or DMSO carrier alone (untreated) for 30 min. prior to addition of ARG1 antibody (red) in complete medium for given time points. Bar, 5 μm.

NIHMS331569-supplement-02.tif (525KB, tif)
03. Figure S3.

F(ab′)2-ARG1 internalization and endosomal localization is similar to full length ARG1. MNA cells were co-transfected with rabies G and WT-GFP-Rab5a (A) or YFP-Rab9a (B) and incubated with F(ab′)2-ARG1 (red) for given time points in complete medium. Data are representative of two separate experiments. Arrows indicate regions of co-localization. Bar, 5 μm.

NIHMS331569-supplement-03.tif (6.5MB, tif)
04. Figure S4.

Ligand and antibody-mediated internalization of the TfR localizes with clathrin. (A, B) HEK cells were transfected with 1 μg clathrin-GFP followed by incubation with either transferrin (Tf) or antibody (αCD71). Graphs show the amount of Tf/clathrin (A) or αCD71/clathrin (B) co-localized pixels from addition to cells (T = 0 or 400) to 20 min. following addition (T = 1200). Images for analysis were taken using TIRF microscopy. Data are representative of three separate experiments.

NIHMS331569-supplement-04.tif (827.7KB, tif)
05. Figure S5.

Ligand-mediated transferrin receptor internalization localizes with early and recycling endosomes, but not late endosomes. HEK cells were transfected with 1 μg GFP-Rab4a (A) GFP-Rab5a (B), GFP-Rab11a (C) or YFP-Rab9a (D) and incubated with Tf (red) for given time points in complete medium. Arrows indicate regions of co-localization. Data are representative of three separate experiments. Bar, 5 μm.

NIHMS331569-supplement-05.tif (11.5MB, tif)

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