Intracellular trafficking pathway of newly synthesized CD1b molecules
2002; Springer Nature; Volume: 21; Issue: 4 Linguagem: Inglês
10.1093/emboj/21.4.825
ISSN1460-2075
Autores Tópico(s)Immune Cell Function and Interaction
ResumoArticle15 February 2002free access Intracellular trafficking pathway of newly synthesized CD1b molecules V. Briken V. Briken Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461 USA Search for more papers by this author R.M. Jackman R.M. Jackman Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461 USA Search for more papers by this author S. Dasgupta S. Dasgupta Department of Biochemistry & Biophysics, University of California, San Francisco, CA, 94143-0534 USA Search for more papers by this author S. Hoening S. Hoening Georg-August University, Gosslerstrasse 12d, D-37073 Göttingen, Germany Search for more papers by this author S.A. Porcelli Corresponding Author S.A. Porcelli Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461 USA Search for more papers by this author V. Briken V. Briken Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461 USA Search for more papers by this author R.M. Jackman R.M. Jackman Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461 USA Search for more papers by this author S. Dasgupta S. Dasgupta Department of Biochemistry & Biophysics, University of California, San Francisco, CA, 94143-0534 USA Search for more papers by this author S. Hoening S. Hoening Georg-August University, Gosslerstrasse 12d, D-37073 Göttingen, Germany Search for more papers by this author S.A. Porcelli Corresponding Author S.A. Porcelli Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461 USA Search for more papers by this author Author Information V. Briken1, R.M. Jackman1, S. Dasgupta2, S. Hoening3 and S.A. Porcelli 1 1Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 10461 USA 2Department of Biochemistry & Biophysics, University of California, San Francisco, CA, 94143-0534 USA 3Georg-August University, Gosslerstrasse 12d, D-37073 Göttingen, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:825-834https://doi.org/10.1093/emboj/21.4.825 Correction(s) for this article Intracellular trafficking pathway of newly synthesized CD1b molecules15 March 2002 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The intracellular trafficking of major histocompatibility complex (MHC) class I and class II molecules has evolved to support their function in peptide antigen presentation optimally. We have analyzed the intracellular trafficking of newly synthesized human CD1b, a lipid antigen-presenting molecule, to understand how this relates to its antigen-presenting function. Nascent CD1b was transported rapidly to the cell surface after leaving the Golgi, and then entered the endocytic system by internalization via AP-2-dependent sorting at the plasma membrane. A second sorting event, possibly involving AP-3 complexes, led to prominent accumulation of CD1b in MHC class II compartments (MIICs). Functional studies demonstrated the importance of nascent CD1b for the efficient presentation of a foreign lipid antigen. Therefore, the intracellular trafficking of nascent CD1b via the cell surface to reach MIICs may allow the efficient sampling of lipid antigens present in endocytic compartments. Introduction The CD1 cell surface glycoproteins are now well established as a third distinct family of antigen-presenting molecules that have the unique capacity to bind lipid ligands and present them to T cells (Porcelli, 1995). The human group 1 CD1 proteins, consisting of CD1a, CD1b and CD1c isoforms, have been shown to present lipids and glycolipids from mycobacteria to T cells belonging to a variety of different phenotypic subsets (Burdin and Kronenberg, 1999; Porcelli and Modlin, 1999). In addition, T-cell responses to a CD1c-presented mycobacterial isoprenoid glycolipid antigen have been shown to be elevated in the blood of human subjects previously infected with Mycobacterium tuberculosis (Moody et al., 2000). These findings underscore the potential importance of antigen presentation by the CD1 system in the immune response to pathogens. CD1a, -b and -c isoforms are expressed by lymphoid and myelomonocytic cells, with the most prominent expression occurring on myeloid lineage dendritic cells (DCs) (Porcelli, 1995). Recent studies have characterized the intracellular localization of different CD1 isoforms as overlapping but not identical. Whereas CD1a and CD1c accumulate at the cell surface and in early endosomes (Sugita et al., 1999; Briken et al., 2000a), CD1b and murine CD1d are found mainly in late endosomes, lysosomes and major histocompatibility complex (MHC) class II compartments (MIICs) (Sugita et al., 1996; Chiu et al., 1999). These results support the hypothesis that the evolution and conservation of different CD1 isoforms may be due at least in part to their ability to survey the lipid content of different intracellular compartments efficiently (Briken et al., 2000b). Most CD1 proteins contain a tyrosine-based motif (YXXZ) in their short cytoplasmic tails (Jackman et al., 1998). These YXXZ motifs (Y is a tyrosine, X is any amino acid and Z is a bulky hydrophobic amino acid) interact with cytosolic adaptor protein (AP) complexes to induce sorting into transport vesicles (Kirchhausen et al., 1997; Bonifacino and Dell'Angelica, 1999). Knowledge of the intracellular transport of antigen-presenting molecules has provided important information about the molecular mechanisms by which they function. Thus, examination of the transport of MHC class I molecules revealed that the rate-limiting step of their cell surface appearance was the transport from the endoplasmic reticulum (ER) to the Golgi apparatus. From the Golgi, MHC class I proteins traffic rapidly to the plasma membrane (PM) where they accumulate, since internalization from the cell surface is inefficient. These results were consistent with experiments showing loading of the MHC class I proteins with antigenic peptides in the ER (Germain and Margulies, 1993; Heemels and Ploegh, 1995; Pamer and Cresswell, 1998). In contrast, the intracellular transport of MHC class II molecules is characterized by their sorting at the trans-Golgi network (TGN) for transport to the endocytic pathway where MHC class II molecules reside for 2–3 h, to allow degradation of their associated invariant chain (Ii chain) and binding to antigenic peptides, before reaching the cell surface (Mellman et al., 1995; Pieters, 1997). Internalization and recycling of MHC class II from the cell surface is inefficient but nevertheless represents a minor alternate pathway for MHC class II peptide loading (Pinet et al., 1995). CD1b molecules mainly accumulate in lysosomes and MIICs in immature dendritic cells (iDCs), which is very similar to the intracellular distribution of MHC class II molecules (Sugita et al., 1996; Briken et al., 2000a). However, the transport pathway that newly synthesized CD1b molecules employ to reach MIICs remains to be elucidated. In this study, we analyzed the trafficking of newly synthesized CD1b molecules to determine the route that these take to reach lysosomal compartments where they are most efficiently loaded with lipid antigens. Our results showed that CD1b molecules have evolved an unusual intracellular trafficking pathway that is unlike those previously found for MHC class I or class II antigen-presenting molecules, and suggest that this is likely to be essential to the unique function of CD1b in the binding and presentation of lipid antigens throughout the endocytic pathway. Results Direct transport of CD1b from the Golgi apparatus to the cell surface To study the intracellular transport of CD1b, the human B-lymphoma cell line C1R stably transfected with CD1b was used. To validate the use of these cells as a model for intracellular trafficking of CD1b, the steady-state distribution of CD1b in iDCs and C1R cells was compared by confocal immunofluorescence microscopy using Lamp-1 as a marker of late endosomes and lysosomes (Figure 1A). In addition, the distribution of CD1b in fractions of total cellular membranes separated on Percoll gradients was compared for iDCs and C1R cells (Figure 1B) (Briken et al., 2000a). With both methods, the steady-state distribution of CD1b molecules was very similar in iDCs and C1R cells, indicating similar intracellular trafficking of CD1b in these cells. Figure 1.Similar steady-state distribution of CD1b in iDCs and CD1b-transfected B lymphoblastoid cells. (A) The distribution of CD1b in either iDCs (top row) or CD1b-transfected C1R cells (bottom row) was analyzed by immunofluorescence and confocal microscopy. Single channel images show CD1b staining (BCD1b3.1) in red (left column) and Lamp-1 staining (rabbit anti-Lamp1 serum) in green (middle column). Co-localization of the two proteins is revealed as yellow staining in the overlay of the two scanned images (right column). Scale bar: 2 μm. (B) Lysosomal compartments of C1R cells or iDCs were separated from endosomes and the plasma membrane using Percoll gradients. The distribution of CD1b in the fractions of each gradient is shown as a percentage of the total signal of CD1b in each gradient as described in Materials and methods. The quality of the separation was similar for each gradient, as controlled by the distribution of the lysosomal and plasma membrane marker enzymes β-hexosaminidase (B-Hex) and alkaline phosphodiesterase (APDE), respectively (Briken et al., 2000a). Download figure Download PowerPoint To determine whether the transport of newly synthesized CD1b molecules followed the pathway of secretory molecules or instead a pathway resembling that of MHC class II molecules, pulse–chase experiments were performed using C1R cells transfected with CD1b or HLA-A3. First, the cell surface appearance of MHC class II molecules was analyzed, serving as an example of a molecule that enters the endocytic pathway after passing through the Golgi apparatus but before reaching the cell surface. The signal of the total MHC class II α-chain precipitated remained constant from 1 to 6 h of chase. The cell surface signal of the MHC class II α-chain was weak after 1 and 2 h of chase, but strongly increased after a 4 h chase, with a further slight increase at 6 h of chase (Figures 2A and 3). This delay in surface expression is known from previous studies to be due to the transport of MHC class II into the endocytic pathway, where the catabolism of the Ii chain and loading with antigenic peptides occurs, before the appearance of newly synthesized MHC class II complexes at the cell surface (Mellman et al., 1995). Figure 2.Rapid trafficking of newly synthesized CD1b from the Golgi apparatus to the cell surface. The cell surface arrival of newly synthesized MHC class II (A), MHC class I (B), TfR (C) and CD1b (D) was compared by pulse labeling of C1R cells, followed by the indicated chase periods. The ‘total’ and ‘surface’ fractions of newly synthesized proteins were determined as described in Materials and methods. For (B–D), the proteins were subjected to EndoH digestion to differentiate between proteins in the ER or the Golgi and post-Golgi compartments. The sizes of EndoH-resistant and EndoH-sensitive proteins are indicated on the right by an ‘R’ or ‘S’, respectively. For (A), the sizes of the MHC class II α- and β-chains and the associated invariant chain (p33) are indicated. The experiment shown is representative of four independent experiments. Download figure Download PowerPoint Figure 3.Quantification of Golgi to cell surface transport of CD1b, TfR and MHC class I and II. The appearance of newly synthesized CD1b (triangles), TfR (circles), MHC class I (diamonds) and MHC class II (squares) as analyzed in Figure 2 was quantified using either a Phosphoimager (two experiments) or densitometry (two experiments). For MHC class II, the signal of the α-chain in the ‘total’ immunoprecipitation (IP) was compared with the signal in the ‘surface’ IP. For all the other molecules, only the fraction of EndoH-resistant signal in the ‘total’ was compared with the ‘surface’ IP in order to eliminate any influence of the kinetics of the ER to Golgi transport in determining the kinetics of cell surface appearance. The maximal amount of cell surface signal for each molecule was normalized and set to 100% to facilitate comparison of the kinetics of cell surface arrival between all molecules. Download figure Download PowerPoint As examples of proteins that follow the secretory pathway and that are directly transported to the cell surface after leaving the Golgi, the intracellular transport of transferrin receptor (TfR) and MHC class I was analyzed (Neefjes et al., 1990). For this analysis, the immunoprecipitated proteins were treated with endoglycosidase H (EndoH) before separation by SDS–PAGE, in order to differentiate between proteins in the ER and the Golgi apparatus. This allowed measurement of the kinetics of the Golgi to cell surface transport without any influence of the ER to Golgi transport, which may otherwise also account for a delay in the cell surface appearance of a protein. This distinction was not necessary for the analysis of MHC class II molecules described above, since the rate-limiting step of the cell surface transport for these molecules has been shown previously to be their exit from the endocytic pathway (Neefjes et al., 1990). After 1 h, ∼30% of the total HLA-A3 molecules were resistant to EndoH digestion and this signal remained constant thereafter, although the EndoH-sensitive signal decreased, most probably due to maturation and/or degradation (Figure 2B). After 1 h of chase, only a small fraction of HLA-A3 was found at the cell surface, but after 2 h most of the molecules (∼80%) had reached the cell surface with only a minor further increase at subsequent time points (Figures 2B and 3). Parallel analysis of the TfR also showed that its transport to the cell surface from the Golgi was very rapid, since after 1 h of chase the maximal amount of TfR molecules was found at the cell surface and thereafter slowly decreased until it equilibrated at ∼70% of the maximal level (Figures 2C and 3). The significantly lower plateau of cell surface signal of the TfR (∼70%) compared with MHC class I (∼100%) most probably reflected the higher internalization and recycling rates of the TfR. Using the same approach, the cell surface transport of newly synthesized CD1b molecules was also analyzed (Figures 2D and 3). It was apparent from the relative levels of EndoH-sensitive and -resistant CD1b during the chase period that the transit of CD1b molecules from the ER to the Golgi was significantly slower than that of HLA-A3 and TfR. Thus, even after 2 h of chase, the amount of EndoH-resistant CD1b still strongly increased, and after 4 h of chase there was still a small increase of signal (Figure 2D). However, the increase in EndoH-resistant material was paralleled by a simultaneous increase in cell surface expression at each time point. For example, the strongest increase of EndoH-resistant signal in the total immunoprecipitate was observed between 1 and 2 h of chase, and this was also the interval in which the greatest increase in the cell surface signal of CD1b occurred (Figures 2D and 3). Thus, unlike MHC class II molecules, CD1b molecules that had reached the Golgi apparatus were rapidly transported to the cell surface with kinetics intermediate between those of the TfR and MHC class I (Figure 3). This indicated that the majority of newly synthesized CD1b molecules were not delayed in their movement to the cell surface by transit through endosomes and lysosomes as observed for MHC class II, but instead were transported directly to the cell surface after leaving the Golgi via the secretory pathway. Clathrin-mediated endocytosis of CD1b molecules from the cell surface Since newly synthesized CD1b molecules accumulate in late endosomes and MIICs, but appeared not to enter these compartments directly upon leaving the Golgi, we hypothesized that this resulted from efficient internalization of CD1b molecules from the cell surface. The internalization of CD1b, TfR and MHC class I was determined as described in Materials and methods. As expected, the internalization of TfR was very rapid and efficient, showing ∼50% internalization after 10 min. In contrast, at this time point, only ∼5% of the MHC class I molecules and 10% of CD1b molecules had been internalized. The internalization of CD1b molecules was less than that of TfR even at later time points, but was more efficient when compared with MHC class I, since it increased to ∼25% after 40 min and 30% after 60 min, whereas the amount of internalized MHC class I remained stable at ∼10% at these time points (Figure 4A, left). Internalization of CD1b from the PM was dependent on its cytoplasmic tail, since the rate of internalization of CD1b molecules from which the cytoplasmic tail had been deleted fell to the extremely low level observed for MHC class I (Figure 4A, right) Figure 4.Efficient internalization of CD1b from the cell surface was mediated by AP-2. (A) The internalization kinetics (left panel) of TfR (circles), MHC class I (diamonds) and CD1b (triangles) were analyzed in CD1b-transfected C1R cells using a cell surface biotinylation-based method as described in Materials and methods. The dependence on the cytoplasmic tail of CD1b for internalization was determined using C1R cells transfected either with the wild-type CD1b (CD1b.wt) or with a tail-deleted mutant (CD1b.td). End point internalization of CD1b.wt or CD1b.td after 75 min at 37°C was compared with TfR and MHC class I using an antibody-based internalization assay as described in Materials and methods (right panel). Results shown are means of three independent experiments; the standard deviation is indicated (left panel) or was A, TfR.Y,F>A and Lamp-1. Y>A tails (mutant tails) or empty sensor chip (control) with purified AP-2 (left panel) or AP-3 (right panel) as measured by SPR. Note: the sensorgrams for the three mutant tails are overlapping with the sensorgram of the control for AP-2 and AP-3 measurements. (C) Affinities of the tail peptides for AP-2 (left) or AP-3 (right) are directly proportional to the inverse of the equilibrium dissociation constant (KD) shown in μM−1. Asterisks indicate that binding of the TfR tail peptides was below the level of detection for the assay. Results shown are representative of three independent measurements, giving a standard deviation of between 3 and 7% of the KD value. Download figure Download PowerPoint Next the association of AP-2 and AP-3 with the tail of the lysosomal protein Lamp-1 was analyzed and compared with interactions with the tail of TfR, which accumulates only in early endosomes. Both molecules contain a tyrosine-based motif in their cytoplasmic tails (Kirchhausen et al., 1997). The TfR tail peptide had the highest affinity for AP-2 as expressed in the equilibrium dissociation constant (KD = 0.04 μM), followed by the Lamp-1 tail (KD = 0.12 μM) and finally the CD1b and CD1c tails which had similar affinities (KD = 0.35 and 0.29 μM, respectively) (Figure 5C and Table I). The specificity of the interaction was controlled using peptides with alanine mutations of the tyrosine residue of the YXXZ motif (Figure 5A), which all had significantly reduced affinity for AP-2 binding (KD = 2.3–4.5 μM). In contrast, the affinity of the TfR tail peptide for AP-3 binding was not detectable, and instead the CD1b and Lamp-1 tail peptides both bound AP-3 with similar affinities (KD = 3.5 and 3.7 μM, respectively). The CD1c tail bound AP-3 only at the background level of the tyrosine-mutated peptides of CD1b or Lamp1 (KD = 14.0 μM) (Figure 5C and Table I). In conclusion, this analysis of direct binding by the biosensor system revealed a specific interaction of the cytoplasmic tail of the lysosomal proteins CD1b and Lamp-1 with the AP-3, but not of the early endosomal proteins CD1c and TfR, suggesting that AP-3 interaction may control the accumulation of CD1b in lysosomes. Table 1. Overview of cytoplasmic tail peptide interactions with AP-2 or AP-3 Tail AP-2 AP-3 ka (1/M×s) kd (1/s) KD (μM) ka (1/M×s) kd (1/s) KD (μM) CD1c wt 1.9 × 104 5.6 × 10−3 0.29 3.4 × 102 4.7 × 10−3 14.0 CDc1b wt 1.4 × 104 4.9 × 10−3 0.35 1.2 × 103 4.2 × 10−3 3.5 CDc1 Y>A 1.2 × 103 5.3 × 10−3 4.4 0.3 × 103 4.0 × 10−3 13.3 Lamp-1 wt 2.6 × 104 3.1 × 10−3 0.12 9.2 × 102 3.4 × 10−3 3.7 Lamp-1 Y>A 8.7 × 103 3.9 × 10−3 4.5 2.7 × 102 3.9 × 10−3 14.4 TfR wt 1.2 × 105 4.6 × 10−3 0.04 nda nd nd TfR Y,F>A 2.3 × 103 5.2 × 10−3 2.3 nd nd nd The interaction of purified AP-2 and AP-3 complexes with cytoplasmic tail peptides was analyzed by SPR using a BIAcore 3000 as described in Materials and methods. The rate constants for association (ka) and dissociation (kd) were determined at three different concentrations of AP-2 and AP-3 for each peptide. Shown are the values measured with AP-2 and AP-3 at 500 nM. The equilibrium dissociation constant (KD = kd/ka) was calculated for each adaptor concentration. Values obtained from three independent sets of measurements varied by 3–7%. a nd: not detectable. Requirement for de novo protein synthesis for efficient presentation of a microbial lipid by CD1b Studies on MHC class II molecules have demonstrated an alternative antigen presentation pathway for a small subset of peptide antigens that become associated with MHC class II through a recycling pathway (Pinet et al., 1995). These studies have shown that using the irreversible protein synthesis inhibitor emetine, most MHC class II antigen presentation is prevented since traffic to the MIIC for antigen loading is dependent on de novo protein synthesis of both Ii and MHC class II α- and β-chains. However, the alternative pathway is not blocked by emetine since it relies on the recycling of pre-existing MHC class II molecules from the cell surface and their loading with new peptide antigens that are presumably encountered during recycling through endosomes. Since CD1b may also have the potential to recycle from the PM, we used this experimental approach to assess if de novo CD1b synthesis was required for antigen presentation, or if a similar alternative pathway of antigen acquisition during recycling might also exist for CD1b. Emetine treatment of the iDCs before addition of tetanus toxoid completely prevented T-cell recognition of this MHC class II-presented antigen, whereas treatment of iDCs after first pulsing them with tetanus toxoid had very little effect on subsequent presentation using the HLA-DR-restricted T-cell lines (SP-F3 and SP-F14) (Figure 6A). This was consistent with the ability of the iDCs to present this antigen only through d
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