And Internalization of Heat Shock Protein 70 Role of Scavenger

The Journal of Immunology

Role of Scavenger Receptors in the Binding and Internalization of Heat Shock Protein 701

Jimmy R. The´riault,* Hideki Adachi,† and Stuart K. Calderwood2*

Extracellular heat shock protein 70 (Hsp70) exerts profound effects both in mediating tumor rejection by Hsp70-based vaccines and in autoimmunity. Further progress in this area, however, awaits the identification of the cell surface receptors for extracellular Hsp70 that mediate its immune functions. We have examined a wide range of candidate Hsp70 receptors and find significant binding through two main families of cell surface proteins, including 1) the scavenger receptor (SR) family and 2) C-type lectins of the NK family. In addition, given that the anticancer effects of Hsp70 vaccines have been shown to involve uptake of Ags by APC exposed to Hsp70-tumor Ag complexes, we have examined the ability of the receptors identified here to internalize Hsp70-peptide complexes. Our findings indicate that three members of the SR family (lectin-like oxidized low density lipoprotein receptor 1; fasciclin, epidermal growth factor-like, laminin-type epidermal growth factor-like, and link domain-containing scavenger receptor-1; and SR expressed by endothelial cells-1) are able to bind Hsp70-peptide complexes and mediate its efficient internalization. Indeed, each of the SR was able to mediate efficient uptake of Hsp70 when transfected into Chinese hamster ovary cells previously null for uptake. Curiously, Hsp70 internalization occurs independently of the intracellular domains of the SR, and Hsp70 uptake could be detected when the entire intracellular domain of lectin-like oxidized low density lipoprotein receptor 1 or SR expressed by endothelial cells-1 was truncated. The existence of a wide repertoire of cell surface Hsp70-binding structures may permit intracellular responses to extracellular Hsp70 that are cell specific and discriminate between Hsp70 family members. The Journal of Immunology, 2006, 177: 8604–8611.

eat shock protein 70 (Hsp70)3 is an intracellular molec- mammalian Hsp70 (8). However, other studies have demonstrated ular chaperone that can in certain circumstances be re- that only mycobacterial Hsp70 but not mammalian Hsp70 is able H leased into the extracellular microenvironment (1). Such to bind to CD40, casting doubt on a role of CD40 as a direct Hsp70 can be released from tumor cells in a complex with intra- receptor for mammalian Hsp70 (9). The multiligand low-density cellular polypeptides and then recognized by components of the lipoprotein (LDL) receptor-related protein-1 (LRP1)/CD91 has immune system, leading to antitumor immunity against the chap- also been suggested as a receptor for Hsp70 and other members of eroned tumor peptide Ags (1). Such Hsp70-peptide complexes the heat shock protein family including glucose-regulated protein (Hsp70.PC) interact with APCs and induce tumor immunity by 94 (Grp94)/glycoprotein 96, Hsp90, and calreticulin (10). How- by guest on September 30, 2021. Copyright 2006 Pageant Media Ltd. promoting Ag cross-presentation to T cells (2–5). However, the ever, similarly to CD40, LRP/CD91 is unlikely to participate in cell surface receptors involved in recognition and uptake of Hsp70 binding given that Chinese hamster ovary (CHO) cells, Hsp70.PC have been difficult to identify. Numerous unrelated sur- which express endogenous LRP/CD91, do not bind to Hsp70 with face receptors have been proposed bind Hsp70.PC and to partici- high affinity (11, 12). However, although LRP/CD91 appears not pate in tumor Ag cross-presentation (1, 6, 7). These include the to bind cell surface Hsp70 directly, strong evidence still supports TNFR family member CD40 which was shown to interact with a role of LRP/CD91 in Hsp-dependent tumor Ag cross-presentation (13). A functional interaction has also been shown between Hsp70 and TLR 2 and 4 (TLR2/4) in combination with the sig- https://www.jimmunol.org *Molecular and Cellular Radiation Oncology, Beth Israel Deaconess Medical Center, naling molecule CD14 (14, 15). Such interactions lead to a proin- Harvard Medical School, Boston, MA 02215; and †Laboratory of Cellular Biochem- istry, RIKEN (The Institute of Physical and Chemical Research), Saitama, Japan flammatory response triggering the induction of cytokine release Received for publication August 9, 2006. Accepted for publication September and expression of APC maturation molecules, although this inter- 19, 2006. action does not seem to involve direct Hsp70 binding to CD14 or The costs of publication of this article were defrayed in part by the payment of page TLR (14, 15). However, Hsp70 has been shown conclusively to charges. This article must therefore be hereby marked advertisement in accordance bind with high avidity to scavenger receptor (SR) family member with 18 U.S.C. Section 1734 solely to indicate this fact. Downloaded from lectin-like oxidized LDL receptor 1 (LOX-1) (5, 12). The interac- 1 This work was supported by National Institutes of Health Research Grants R01CA047407 and RO1CA094397. tion between Hsp70-PC and LOX-1 promotes Ag cross-presenta- 2 Address correspondence and reprint requests to Dr. Stuart K. Calderwood, Molec- tion on dendritic cells and the activation of an in vitro and in vivo ular and Cellular Radiation Oncology, Beth Israel Deaconess Medical Center, Har- tumor Ag-specific T cell immune response (5). We have previ- vard Medical School, 21-27 Burlington Avenue, Boston, MA 02215. E-mail address: [email protected] ously investigated Hsp70 binding to each of the candidate Hsp70 receptors mentioned above and observed binding only to LOX-1; 3 Abbreviations used in this paper: Hsp, heat shock protein; Hsp70.PC, Hsp70-peptide complex; LDL, low-density lipoprotein; oxLDL, oxidized LDL; acLDL, acetylated significant Hsp70 binding to TLR2, TLR4, CD40, or CD91/LRP1 LDL; LRP1, LDL receptor-related protein-1; SR, scavenger receptor; LOX-1, lectin- was not observed (12). We have therefore concentrated on SR like oxidized low-density lipoprotein receptor 1; FEEL-1, fasciclin, epidermal growth factor-like, laminin-type epidermal growth factor-like, and link domain-containing family members as potential Hsp70 binding structures. SR-1; SREC-1, SR expressed by endothelial cells-1; SR-PSOX, SR that binds phos- The SR have been defined as receptors for modified forms of phatidylserine and oxidized lipoprotein; CHO, Chinese hamster ovary; ID, intracellular domain. Grp94, glucose-regulated protein 94; NKG2D, killer cell lectin-like lipoproteins including oxidized and acetylated LDLs (oxLDL and receptor subfamily K, member 1. acLDL) (16). However, the spectrum of SR-binding ligands has

recently been expanded to other molecules including Gram-posi- Materials and Methods tive and Gram-negative bacteria, LPS, advanced glycation end Cell culture products, fucoidan, and polyanionic ligands (16). The SR family Wild-type CHO K1 cells and all stable CHO transfectant cells were grown can be subdivided into eight different subclasses (A–H), and most in Ham’s F-12 medium supplemented with 10% FBS. In the case of stable receptors belonging to this family are expressed on the surface of transfectant CHO human SREC-1, CHO human FEEL-1, CHO human APC (16, 17). Class A and B SR such as SR-A, macrophage re- Dectin-1, CHO human CD94-NKG2A, and CHO human CD94-NKG2C, ceptor with collagenous structure, human CD36 and LIMPII anal- clonal selection was kept in 0.4 mg/ml G418, whereas LOX-1 expression (CHO-LOX-1) was maintained in blasticidin S (10 mg/ml). Stable CHO- ogous receptor (CLA-1), and CD36 are integral parts of the im- Dectin-1 transfectant was produced through transfection of pCDNA3 Flag mune response increasing phagocytosis of bacteria and yeast (16). human Dectin-1 and populations of G418 (0.4 mg/ml)-resistant cells were class C dSR-C1 is only expressed in the fruit fly Drosophila mela- generated after 2 wk of cell culture. nogaster and has no mammalian counterpart, whereas CD48 (class Generation of LOX-1 deletion mutants D SR) shares homology to lysosome-associated membrane protein 1 and is mainly expressed in endosomal/lysosomal vesicles (17). LOX-1 deletion mutants were constructed especially for this study using a Besides its role in antitumor immunity, LOX-1, the sole member PCR-based Site-directed Mutagenesis Kit (ExSite; Stratagene). Briefly, PCRs were performed on pcDNA3 myc human LOX-1 using as template of the class E SR, serves as a tethering receptor for leukocyte two 21-bp oligonucleotides corresponding of the extremities of the deleting homing and rolling on the surface of endothelial cells (18). The site of each mutant and the ExSite PCR-Base Site Directed Mutagenesis kit class F SR expressed by endothelial cells-1 (SREC-1) interacts (Stratagene) according to the manufacturer’s suggestions. After amplifica- either with its homolog SREC-2 or with itself through extracellular tion, the reaction products were digested with DpnI (Invitrogen Life Tech- motifs and induces cell-cell homophilic and heterophilic trans- nologies), and the PCR products were ligated with T4 DNA ligase (In- vitrogen Life Technologies). interaction (19). Interestingly, phagocytosis and cell corpse engulfment can be initiated by Caenorhabditis elegans worm Caeno- Alexa 488-labeled purified Hsp70 preparation rhabditis elegans death gene 1, a surface receptor containing an Human melanoma A375-MEL cells or mouse MISA cells have been used extracellular domain that is structurally homologous to SREC-1 as starting material for mammalian Hsp70.PC preparations. Recombinant and an intracellular domain (ID) functionally related to CD91/ Hsp70 was purified from competent BL-21 Escherichia coli (Invitrogen LRP1 (20). The class G SR that binds phosphatidylserine and ox- Life Technologies) transformed with a pET-5 plasmid encoding human LDL has also been discovered as a ligand of the chemokine re- HSP70.1 cDNA (BamHI-HindIII insertion). Hsp70 purification was conducted as previously reported (34). Briefly, a 10-ml cell pellet was homog- ceptor CXCR6 (CXCL16) (21, 22). The class G SR that binds enized in 40 ml of hypotonic buffer (10 mM NaHCO3, 0.5 mM PMSF, pH phosphatidylserine and oxLDL possesses angiogenic and prolifer- 7.1) by Dounce homogenization. Bacterial rHsp70 was produced by first ative properties (23, 24). Finally, class H receptor FEEL-1 also growing the BL-21 bacteria (dilution 1/100) in 1 liter of LB with ampicillin named stabilin-1/CLEVER-1 has been initially characterized as a (50 mg/ml) for3hat37°C. Hsp70 expression was induced by adding 1 mM isopropyl ␤-D-thiogalactoside for 4 h. A 1-liter bacterial pellet was receptor interacting with advanced glycation end products and me- washed twice with 30 ml of buffer A (50 mM HEPES, 50 mM NaCl, 10 diating trans-migration of lymphocyte and vascular cells on the mM DTT, 0.5 mM EDTA, 0.5 mM PMSF, and 1ϫ protease inhibitor endothelium (25–28). Intriguingly, members of the heat shock pro- mixture from Roche; pH 7.4) and then sonicated and homogenized tein family other than Hsp70 have been shown to interact with (Dounce homogenization) in 12.5 ml in buffer A supplemented with 2 mM MgCl2, 1.25 mg/ml lysozyme and 11.2 U/ml DNase I. The homogenate by guest on September 30, 2021. Copyright 2006 Pageant Media Ltd. some SRs. Grp94/glycoprotein 96 and calreticulin show significant was first centrifuged at 10,000 ϫ g for 30 min, and the supernatant was affinity to SR-A and are internalized by this receptor (29). Extra- recentrifuged for 60 min at 100,000 ϫ g. The sample buffer was changed cellular calreticulin uptake can also be mediated by SREC-1 but to buffer D (20 mM Tris-acetate, 20 mM NaCl, 15 mM 2-ME, 3 mM MgCl , and 0.5 mM PMSF, pH 7.5) using a PD-10 column (Amersham apparently not by LOX-1 (29, 30). 2 Biosciences). The sample was applied directly to a 5-ml ADP-agarose col- As mentioned earlier, a receptor-mediated Ag uptake mechanism umn (Sigma-Aldrich) which was equilibrated with buffer D. Hsp70 was appears to mediate Hsp70-induced cross-presentation. Receptor-li- eluted from ADP-agarose column with 3 mM ADP or 3 mM ATP in buffer gand complex internalization is mainly governed through motifs D (ADP-bound Hsp70 vs ATP-bound Hsp70). The sample buffer was within the IDs of endocytic receptors. Such motifs are sufficient to changed to FPLC buffer (20 mM sodium mono- and diphosphate, 20 mM NaCl, pH 7.0) with a PD-10 column. The supernatant was applied to a trigger ligand uptake through coated pits or lipid raft caveolin-related DEAE anion exchange column equilibrated with FPLC buffer (Amersham https://www.jimmunol.org mechanisms (31, 32). Tyrosine-based and dileucine-based motifs ac- Biosciences). Hsp70 was eluted with the FPLC buffer containing 150 mM count for the vast majority of intracellular sorting signals and usually NaCl. All proteins were quantitated with Bradford assay. Alexa 488 label- direct ligands to specific intracellular compartments such as lysosome ing on Hsp70.PC was conducted according to the manufacturer’s instructions (Molecular Probes). BSA was used as negative control. Almost no or endosome (32). After internalization of extracellular Hsp70.PC by degradation (Ͻ2%) of the Hsp70 purified preparation was observed by APC, the complex is thought to traffic into the cytoplasm or endoso- Coomassie staining, and the presence of Hsp70 in the preparation was mal vesicles before the release, processing and re-presentation of the confirmed by Western blotting using a mouse mAb specific against Hsp70 Downloaded from immunogenic peptide cargo by class I and/or II MHC receptors at the (SPA-810; Stressgen). surface of APC (2, 33). Hsp70 binding assay In this study, we show that a subgroup of the SR family includ- 5 ing LOX-1, SREC-1 and FEEL-1 can bind avidly both to Nontrypsinized cells (2 ϫ 10 ) were washed twice in PBS containing 0.5% FBS, 0.05% NaN3, and 1 mM CaCl2 and incubated with 150 nM Alexa Hsp70.PC purified from mammalian cells and to purified recom- 488-labeled BSA, mammalian Hsp70.PC, ATP-bound recombinant Hsp70 binant Hsp70. Interestingly, the nature of the adenosine phosphate and ADP-bound recombinant Hsp70 for 30 min on ice with gentle shaking.

moiety associated with Hsp70 affects its binding to all three SR. The cells were washed in PBS containing 0.5% FBS, 0.05% NaN3, and 1 We show that each of the SR is subsequently able to mediate mM CaCl2 twice. and Alexa 488-labeled Hsp70.PC binding was monitored efﬁcient internalization of Hsp70.PC after expression in CHO cells by ﬂow cytometry (BD Biosciences). previously null for uptake. Surprisingly, deletion mutation studies Transfection and Western blot indicate that sequences in the ID of both LOX-1 and SREC-1 are ϫ 5 dispensable for Hsp.PC uptake, ruling out conventional pathways CHO-K1 cells (2.5 10 ) were transiently transfected with 5 mg of empty vector (pCDNA3) or pCDNA3 plasmids encoding various myc-tagged hu- of internalization involving internalization motifs found in the ID man LOX-1, human LOX-1 deletion mutants, full-length human SREC-1, of many receptors. or SREC-1 D370 (deletion of the last 370 aa of the C terminus) for 48 h 8606 Hsp70 UPTAKE BY SRs

using the Superfect transfection reagent (Qiagen) according to the manufacturer’s instructions. The cells were harvested in 200 ml of SDS-PAGE sample buffer once, and 20 mg of protein extracts were loaded on SDS- PAGE, transferred, and subjected to Western blot using the mouse monoclonal Myc Ab (clone 9E10; Stratagene).

Hsp70 internalization and immunofluorescence experiments In an 8-well CultureSlide (Labtek), 2 ϫ 104 cells were rinsed twice in ice-cold medium (Ham’s F-12, 10% FBS) and incubated with 750 nM (50 mg/ml) Alexa 488-labeled BSA (negative control) or Hsp70 for 30 min with intermittent shaking at 4°C. After the incubation, the cells were washed four times with ice-cold medium and once with warm medium (37°C). Hsp70.PC internalization was induced by incubating the cells for 15 min at 37°C. Then, the cells were washed twice in Dulbecco’s PBS and fixed in 3.7% paraformaldehyde for 15 min and processed for microscope visualization. LOX-1 and SREC-1 surface expression was illustrated using 9E10 and SREC-1 (SR-4) (dilution, 1/100) Ab. Images were obtained using a fluorescent microscope NIKON Eclipse E600, at ϫ40 magnification; with a SPOT RT color camera and SPOT advanced software (Diagnostics Instruments).

Results Binding of Hsp70 to cell surface SRs We first examined the ability of Hsp70.PC from human tumor cells to bind to CHO cells transfected with expression plasmids encoding a number of the known SRs. Binding of Hsp70 to wild-type CHO was minimal, whereas significant binding was seen with cells stably transfected with the SR family members LOX-1, SREC-1, and FEEL-1 (Fig. 1). Cell surface expression of the overexpressed receptors was confirmed by immunofluorescence as shown below. Each of the three SRs was able to bind mammalian Hsp70.PC with a different relative avidity compared with Alexa-labeled BSA (negative control) which did not show significant binding to cells expressing these receptors or to wild type CHO (Fig. 1A). Binding to these SR family members is relatively selective as previous FIGURE 1. Hsp70 purified from mammalian cells or E. coli binds to studies show that, whereas LOX-1 binds Hsp70, a number of other SRs. A, CHO K1 cells or CHO cells stably overexpressing human SR LOX-1, SREC-1, or FEEL-1 were incubated with Alexa 488-labeled BSA SR family members including SR-A1, CD36, macrophage receptor

by guest on September 30, 2021. Copyright 2006 Pageant Media Ltd. or mammalian Hsp70.PC at a concentration of 10 ng/ml (150 nM) on ice with collagenous structure, and CLA-1 do not (5). These experi- for 30 min with gentle shaking. Mammalian Hsp70.PC binding was mon- ments therefore show selective association of Hsp70 with a subset itored by flow cytometry. Shaded gray and thick black line histograms of the SR. We next conducted similar experiments using recom- corresponded, respectively, to BSA and mammalian Hsp70.PC binding. B, binant human Hsp70 preparation, purified from E. coli and ob- Experiments were conducted in a way similar to that of A except that the served binding to the LOX-1-, SREC-1-, and FEEL-1-expressing CHO K1 and CHO stable transfectant were incubated with human recom- cell lines (Fig. 1B). However, in the case of LOX-1 and SREC-1, binant Hsp70 produced in E. coli bound to ATP (thick black line histo- such binding was significantly reduced when compared with the grams) or ADP (dotted line histograms) (H70-ATP/ADP). Shaded gray degree of association with the preparation of Hsp70 purified from corresponded to the BSA binding to the cells. Experiments were conducted mammalian cells (Fig. 1B). Interestingly, the association of the three times with similar results. https://www.jimmunol.org recombinant Hsp70 with CHO-SREC-1 was greatly reduced and almost at the minimal level of Hsp70 binding observed in wild- type CHO cells (Fig. 1A). By contrast, in the case of FEEL-1- Some members of the SR family, including SR-A and CLA-1, expressing cells, recombinant Hsp70 binds at similar levels com- have been shown to participate in bacterial LPS uptake raising pared with mammalian purified Hsp70 (Fig. 1). The nature of the concerns about the potential involvement of such endotoxins in adenosine phosphate moiety (ATP vs ADP) bound to Hsp70 also SR-mediated Hsp70 internalization (35, 36). Indeed, the presence Downloaded from affected the degree of Hsp70 interaction with LOX-1, SREC-1, and of endotoxins in Hsp70 preparations has been recently proposed to FEEL-1 expressed in CHO cells (Fig. 1A). ADP-bound Hsp70 participate in Hsp70-mediated proinflammatory responses, sug- slightly surpassed the binding level of ATP-bound Hsp70 to all 3 gesting that LPS could also take part in Hsp70 internalization (37, SR (Fig. 1B). Differences in Alexa 488 fluorochrome incorporation 38). However, SR-mediated LPS internalization is observed only in the ADP-bound or ATP-bound Hsp70 preparations used here when LPS is preassociated to LPS binding protein, soluble CD14, were unlikely to be responsible for the differences in binding to SR or even high-density lipoprotein signifying that LPS cannot be because fluorochrome incorporation into each of the Hsp70 prep- internalized by itself and requires prior binding to a putative SR arations was similar (5.25 Alexa 488 molecules per ATP-bound binding ligand (35, 39). Although there is no indication that SR-A Hsp70 vs 5.75 Alexa 488 molecules per ADP-bound Hsp70). and CLA-1 directly interact with Hsp70, it is still possible that Overall, these experiments demonstrate that the source of the Hsp70 can act as a SR-binding ligand facilitating LPS internaliza- Hsp70 preparation used (recombinant Hsp70 compared with tion (5). Nevertheless, we conducted Hsp70 binding experiments Hsp70 from mammalian cells) and the adenosine phosphate moi- comixing LPS-FITC and Alexa 488-labeled Hsp70 and saw no ety (ATP vs ADP) bound to Hsp70 are important factors affecting additive effect of LPS on Hsp70 binding to RAW 264.7 cells (data Hsp70 binding to LOX-1, SREC-1, and FEEL-1. not shown). The Journal of Immunology 8607

FIGURE 2. Mammalian Hsp70.PC binds to speciﬁc C-type lectin receptors. CHO K1 cells or CHO cells stably overexpressing human C- type lectin receptors Dectin-1, NGK2D-Ly49 chimera, CD94-NKG2A, or CD94-NKG2C were incubated with Alexa 488-labeled BSA or mammalian Hsp70.PC at a concentration of 10 ng/ml (150 nM) on ice for 30 min with gentle shaking. Mammalian Hsp70.PC binding was monitored by ﬂow cytometry. Shaded gray and thick black line histograms corresponded, respectively, to BSA and mammalian Hsp70.PC binding. Experiments were conducted three times with similar results.

HSP70 binding to C-type lectin receptors Alexa 488-labeled Hsp70 to bind to the cell surface at 4°C and Besides its functional relationship to other SR family members, then incubating for an additional 15 min at 37°C to trigger energy- LOX-1 shares structural homology to type V C-type lectin family dependent Hsp70 uptake before fixation. Hsp70 internalization members such as Dectin-1, killer cell lectin-like receptor subfam- was assessed by visualization of intracellular punctuate staining ily K, member 1 (NKG2D), and CD94 (40). These C-type lectin (vesicle staining) using fluorescence microscopy. Fluorescence receptor family members usually exist as homo- or heterodimers images are in each case accompanied by matched phase-contrast im- (40). Previous studies have demonstrated that another type V C- ages of the same field. As seen in Fig. 3, CHO cell lines overexpress- type lectin CD94 binds to Hsp70 but not to type II C-type lectin ing, respectively, each of the SR, LOX-1, SREC-1, or FEEL-1 were DC-SIGN (12, 41). From these experiments, it has been suggested able to internalize mammalian Hsp70.PC, whereas uptake was not that the C-type lectin domain, a conserved ligand interaction do- seen in wild-type CHO control cells or cells transfected with empty main in these receptors is responsible for the association with expression plasmid. Control experiments indicate minimal uptake of Hsp70. To determine whether the C-type lectin domain can func- Alexa-labeled BSA by the SR transfectants (Fig. 3). tion as an Hsp70 binding site, we examined Hsp70 binding to CHO Recombinant Hsp70 uptake was also mediated by each of the cells overexpressing a number of members of the V V-type lectin three SR, although with contrasting efficiency (Fig. 4). Indeed, receptor family. Although Dectin-1 has been shown to possess the SREC-1-mediated HSP70 uptake was relatively ineffective, result- by guest on September 30, 2021. Copyright 2006 Pageant Media Ltd. greatest structural homology to LOX-1 of the C-type lectins, no ing in a fairly low percentage of CHO-SREC-1-expressing cells Ͻ Hsp70 binding was not detected in CHO overexpressing Dec- internalizing Hsp70 ( 5% of cells scored) as well as a lower flu- tin-1 (Fig. 2). However, a significant affinity was seen between orescence intensity (Fig. 4B). The greatest degree of internaliza- Hsp70 and NKG2D expressing cells and to a lesser extent in tion was observed in CHO-FEEL-1 cells and magnitude of hsp70 cells expressing the heterodimer CD94/NKG2A but not the internalization was positively correlated with Hsp70 binding (Fig. CD94/NKG2C heterodimer (Fig. 2). Binding to these members 1A). No obvious differences were observed between ATP- and of the C-type lectins showed selectivity in that a number of ADP-bound Hsp70 internalization (Fig. 4). In conclusion there- other family members overexpressed in CHO cells, including fore, the expression of LOX-1, SREC-1, or FEEL-1 at the cell surface was sufficient to mediate the endocytosis of extracellular https://www.jimmunol.org DC-SIGN, CLEC-1 and CLEC-2 showed minimal Hsp70 association (J. The´riault and S. K. Calderwood, unpublished obser- Hsp70.PC. vations). Thus, these findings suggest that a number of C-type The cytoplasmic portion of LOX-1 and SREC-1 is dispensable lectins can serve as receptors for Hsp70 although the C-type for Hsp70 internalization lectin domain was not the sole determinant required for Hsp70 interaction. Receptor-mediated uptake of many ligands is initiated by the association of adaptor molecules with consensus endocytic motifs Downloaded from such YXXQ and NPXY on the ID of the receptor (31, 32). How- SR-induced Hsp70 endocytosis ever, even though LOX-1 and SREC-1 are able to internalize ex- Because Hsp70-induced Ag cross-presentation requires Hsp70.PC tracellular Hsp70, no such consensus internalization motif is uptake after binding to receptor(s) on APC, we next examined present in the ID of either receptor. Therefore, to evaluate the role whether the SR mediate could such Hsp70.PC internalization (1). of the ID of LOX-1 and SREC-1 in Hsp70 uptake, constructs of We chose not to examine CD94/NKG2A and NKG2D internaliza- LOX-1 and SREC-1 with the ID truncated were generated by de- tion because these C-type lectin receptors function mainly in NK letion mutagenesis, stably transfected into CHO cells and sub- cell signaling. Moreover, NK cells are not known to efficiently jected to Hsp70 internalization assay (Figs. 5 and 6). The relative participate in extracellular Ag endocytosis. We have instead con- levels of expression of the deletion mutants in CHO transfectant centrated our effort on LOX-1, SREC-1, and FEEL-1 which are cells were examined by Western blot and/or immunofluorescence most likely be involved in Hsp70 uptake. These receptors have using an anti-myc Ab to detect expression of a LOX-1-myc tag been shown previously to mediate ligand internalization and fusion protein and an anti-SREC-1 Ab (SR-4) for SREC-1 (Figs. LOX-1, SREC-1, and FEEL-1 can mediate oxLDL and/or acLDL 5B and 6). LOX-1 glycosylation (presence suggested by a band uptake (42–44). Hsp70 internalization was initiated by permitting doublet) was observed with the full length LOX-1 and other 8608 Hsp70 UPTAKE BY SRs

FIGURE 3. Mammalian Hsp70-PC was internalized in CHO cells overexpressing LOX-1, SREC-1, and FEEL-1. Fluorescence microscopic analysis of wild-type CHO K1 (a–d), CHO LOX-1 (e–h), SREC-1 (i–l), and FEEL-1 (m–p) incubated with Alexa 488-labeled mammalian Hsp70 or Al- exa 488-labeled BSA. Hsp70 uptake was induced by preincubating the cells with 750 nM Alexa 488-labeled Hsp70-PC or BSA for 30 min at 4°C followed by an additional 15 min of incubation at 37°C. Phase contrast images of the cells were taken at the same time. Experiments were conducted three times with similar results. wt, Wild type.

LOX-1 deletion mutants except for LOX-1 D2–35 (Fig. 5B). A well as full-length LOX-1 was able to mediate the internalization LOX-1 cytoplasmic arginine-rich motif (KKAK) could potentially of mammalian Hsp70.PC (Fig. 5C). Because there was no indica- serve as a positive motif required for the expression of LOX-1 at tion that the ID of LOX-1 is involved in Hsp70 uptake, this led us the cells surface. However, the full ID deletion mutant LOX-1 to investigate the albeit unlikely possibility that adjacent extracel- D2–35 was expressed at the cell surface indicating that this motif lular regions could be involved in Hsp70 internalization. Intrigu- may be nonfunctional (Fig. 5C). To rule out the possibility of a ingly, a putative extracellular dileucine motif (DXXXLL) was deleterious effect of this large truncation on LOX-1 receptor struc- present in the extracellular portion of LOX-1 closely adjacent to ture, function or stability, we also prepared another, smaller dele- the transmembrane domain (Fig. 5A). We tested the possibility that tion mutant removing only the arginine-rich region in the ID of this motif was required for Hsp70 internalization. However dele- LOX-1 (LOX-1 D21–27) and evaluated its cell surface receptor tion of this 62–67 region (sequence containing the dileucine motif) expression. LOX-1 D21–27 was still expressed at the cell surface likewise did not affect Hsp70 uptake (Fig. 5C). A similar series of

by guest on September 30, 2021. Copyright 2006 Pageant Media Ltd. conﬁrming that the KKAK motif does not play a signiﬁcant role in experiments was executed with deletion of the ID of SREC-1, LOX-1 surface sorting (Fig. 5C). Each LOX-1 deletion mutant, as generating deletion mutant (SREC-1 CD370, which truncates the https://www.jimmunol.org Downloaded from

FIGURE 4. Effect of nucleotide (ATP or ADP) bound to Hsp70 internalization. Fluorescence microscopic analysis of wild-type CHO K1 (a–f), CHO LOX-1 (g–l), SREC-1 (m–r), and FEEL-1 (s–x) after exposure to Alexa 488-labeled rATP-bound Hsp70 (H70-ATP), ADP-bound Hsp70 (H70-ADP), or Alexa 488-labeled BSA. Hsp70 uptake was induced by preincubating the cells with 750 nM Alexa 488-labeled Hsp70-PC or BSA for 30 min at 4°C followed by an additional incubation of 15 min at 37°C. Phase contrast images of the cells were taken at the same time. Experiments were performed multiple times with reproducible results. The Journal of Immunology 8609

FIGURE 6. The ID of SREC-1 is dispensable for SREC-1-mediated Hsp70.PC uptake. Hsp70.PC internalization was examined in CHO cells expressing human SREC-1 or SREC-1 D370 (A´ 370) deletion mutants. Internal- ization of mammalian Alexa 488-labeled Hsp70.PC or Alexa 488-labeled BSA was examined in transiently transfected full-length SREC-1 and the SREC-1 CD370 deletion mutant as described in Materials and Methods. Ex- periments were performed three times with reproducible results. WT, Wild type.

uptake and suggest alternative mechanisms for the endocytic functions mediated by the SR.

Discussion One of the keys to understanding the role of extracellular molecular chaperones in cell regulation is to discern the receptors/ac- ceptor proteins on the cell surface that mediate extracellular activity. This has proved an exceedingly protracted process, and many candidate receptors with more or less validity have been proposed. Here we have begun to screen for high avidity Hsp70 receptors and show that three members of the SR when expressed on the surface of CHO-K1 cells (previously null for binding) have ability to bind Hsp70 (Fig. 1). Three receptors from the SR family and two a members of the NK family of C-type lectins have been uncovered as Hsp70 receptors in this study, although the signifi- by guest on September 30, 2021. Copyright 2006 Pageant Media Ltd. cance of these findings of multiple Hsp70 receptors is not yet en- tirely clear. However, the different receptors may mediate cell- specific effects through expression on different cell types or may recognize different members of the Hsp70 family. This is likely to FIGURE 5. The cytoplasmic tail of LOX-1 is dispensable for LOX- 1-mediated Hsp70.PC uptake. A, Schematic illustration of the structure be true of the NK family receptors NKG2A and NKG2D which are of LOX-1 and a series of deletion mutants. B, Western analysis of expressed on NK cells, and in the case of NKG2D, NK cells as LOX-1 deletion mutants after expression in CHO cells. Expression of well as some classes of T lymphocytes (45). These receptors may myc-tagged LOX-1 was detected using monoclonal anti-Myc (9E10) Ab play more significant roles in the lysis of tumor cells expressing and monoclonal anti-actin Ab (clone AC-40; Sigma-Aldrich; loading Hsp70 bound to the cell surface than in tumor Ag cross-presenta- https://www.jimmunol.org control) on CHO-K1 cells transiently transfected with pCDNA3, empty tion (45–47). Our experiments also suggest some selectivity for vector (EV), pcDNA3-myc-LOX-1 full length (L1), pCDNA3-myc- the SR in recognizing different Hsp70 isoforms. There are at least LOX-1 D2–35 (A´ 2–35), pcDNA3-myc-LOX-1 D21–27 (A´ 21–27), and 12 members of the human Hsp70 family many of which are ex- ´ pCDNA3-myc-LOX-1 D62–67 (A62–67). C, Hsp70.PC internalization pressed in tumor cells constitutively (48). Our experiments show by CHO cells expressing the LOX-1 deletion mutants. Internalization of that that human Hsp70 affinity purified from mammalian cells as Hsp70.PC or BSA was conducted on transiently transfected Myc-tagged well as recombinant Hsp70 which is the product of the Hsp70.1

Downloaded from LOX-1 and LOX-1 deletion mutants as described in Materials and Methods. Surface expression (Receptor Exp.) of each of the LOX-1 (HSPA1A) gene binds to LOX-1 and FEEL-1 (Fig. 1). However, deletion mutants was illustrated using mouse monoclonal Myc Ab SREC-1 only showed significant avidity to the mammalian Hsp70 (9E10). Experiments were performed three times with reproducible preparation and bound poorly to the HSPA1A product (Fig. 1). results. The Hsp70 preparation purified from mammalian cells is in fact a mixture of the intracellular Hsp70 family gene products, including the HSPA1A product, but also heat shock 70 KDa protein 8, to a 370 intracellular amino acids from human SREC-1). As with lesser extent, Grp78 and likely other Hsp70 family proteins (data LOX-1, we observed that the ID of SREC-1 is not essential for the not shown). We concentrated on this Hsp70 preparation as it forms internalization of mammalian Hsp70.PC (Fig. 6). Interestingly, the the basic building block of Hsp70-based vaccines and likely con- full ablation of the ID of the SREC-1 is similarly ineffective in tains peptides loaded under physiological conditions and the cor- blocking SREC-1-mediated acLDL internalization, showing rect form of physiological modification. The absence of interaction convergence between requirements for uptake of modified LDL between SREC-1 and the recombinant HSPA1A preparation sug- molecules and Hsp70 (19). These experiments demonstrate that the gests selectivity in Hsp70 family member binding to the same ID regions of LOX-1 and SREC-1 are not required for Hsp70 receptor (Fig. 1). Many of the previous studies investigating the 8610 Hsp70 UPTAKE BY SRs

properties of extracellular Hsp70 receptors used exclusively com- based motifs located in the ID of NKG2A (56). In addition, mercial preparations of the recombinant HSPA1A purified from E. NKG2D does not possess a cytoplasmic tail but can be efficiently coli, and use of this single protein may thus not identify effects internalized (57). Thus, mechanisms for endocytosis that function mediated by other Hsp70 family members and their receptors (5, independently of internalization motifs in the ID have been shown 10, 14). In addition, posttranslational modifications such as phosphor- previously in functionally related receptors, suggesting a precedent ylation or glycosylation of Hsp70 purified from mammalian cells for our findings in the present study. However, there is still much could also be modulating factors affecting binding to SR. Future stud- to learn regarding the mechanisms of Hsp70.PC uptake by the SR. ies of recombinant Hsp70 will use baculovirus expression, a condition Our experiments therefore indicate that Hsp70 can be recog- that more closely reproduces the milieu of the mammalian cell. ADP- nized by a number of cell surface molecules of the interrelated SR bound Hsp70 interacted with slightly more avidity with LOX-1, and C-type lectin families. Indeed, it is not clear whether further SREC-1, and FEEL-1 than ATP-bound Hsp70 although the differ- Hsp70 receptors may remain to be uncovered. This plethora of ences were of marginal significance. The affinities for Hsp70-SR receptors may, however, play a functional role in permitting se- binding are currently not known, although binding with Hsp70 con- lectivity in responding to different Hsp70 family members in dif- centrations as low as 15 nM can be readily detected. Attempts to study ferent cellular contexts. Future studies will determine the signifi- affinity using surface plasmon resonance with soluble (extracellular cance of these interactions in mediating the extracellular functions domains) of LOX-1 and SREC-1 were unsuccessful perhaps due to of Hsp70. incorrect folding of these forms of the SR. Because the receptors that bind Hsp70 are divergent in se- Acknowledgments quence, it is also currently unclear which motifs are involved in We thank Dr. Wayne Yokohama, Dr. David Raulet, and Dr. Tatsuya Hsp70 recognition by such receptors. It has been shown that that Sawamura for providing CHO-NKG2D/Ly49, CHO-CD94/NKG2A, oxLDL and Hsp70 can compete for binding to cell surface LOX-1; CHO-CD94/NKG2C, and CHO-LOX-1, respectively. We also acknowl- it has thus been assumed that both molecules possess overlapping edge Dr. Sachiko Machida, Dr. David Underhill, and Dr. Michael Sherman binding sites on LOX-1 (49). The oxLDL binding site is located in (Dr. Nava Zaarur) for generously providing pCDNA3 myc-LOX-1, the LOX-1 C-type lectin domain, and negative charges on the sur- pcDNA3 hemagglutinin-Dectin-1, and bacterial pET-5 vector expressing face of the oxLDL particle appear to interact specifically with ba- human HSP70.1, respectively. We also thank Kishiko Ogawa, Salamatu Mambula, and Mohammed Abdul Khaleque for technical assistance and sic residues disposed at the surface of LOX-1 homodimer (50, 51). scientific discussions. The elucidation of the crystal structure of LOX-1 has revealed that that these basic residues are in fact positioned diagonally on the Disclosures surface of a heart-shaped dimer (52, 53). In addition, the analysis The authors have no financial conflict of interest. of the NKG2D crystal structure demonstrates that a fairly similar pattern of basic residues on the surface of the protein are also responsible for its interaction with one of its own ligand MHC References 1. Calderwood, S. K., J. R. Theriault, and J. Gong. 2005. Message in a bottle: role class I polypeptide-related sequence A (53). Moreover, the shared of the 70-kDa heat shock protein family in anti-tumor immunity. Eur. J. Immunol. ability of LOX-1, SREC-1 and FEEL-1 to bind Hsp70 preparations 35: 2518–2527. suggests that LOX-1 shares functional homology with these SR. 2. Noessner, E., R. Gastpar, V. Milani, A. Brandl, P. J. Hutzler, M. C. Kuppner,

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