Analysis of the early biogenesis of CD1b: involvement of the chaperones calnexin and calreticulin, the proteasome and ß2-microglobulin
Robert Hüttinger,
Günther Staffler,
Otto Majdic and
Hannes Stockinger
Institute of Immunology, Vienna International Research Cooperation Center at NFI, University of Vienna, Brunner Strasse 59, 1235 Vienna, Austria
Correspondence to:
H. Stockinger
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Abstract
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ß2-Microglobulin (ß2m)-associated human CD1b proteins present lipid and glycolipid antigens, which are loaded on CD1b in endosomal compartments. In contrast, the related MHC class I molecules acquire antigenic peptides in the endoplasmic reticulum. Here, we investigated the biogenesis of CD1b before ß2m binding in comparison to MHC class I. In ß2m-deficient FO-1 cells, we found CD1b heavy chains (HC) complexed with the chaperones calnexin and calreticulin, while MHC class I HC associated only with calnexin. Despite this difference, both CD1b HC and MHC class I HC were degraded when the chaperone interactions were prevented by the glucosidase inhibitor castanospermine. The degradation of both molecules included the proteasome and mannosidases. Chaperone-unassociated CD1b could be rescued from degradation by supplementing FO-1 cells with ß2m. Finally, prevention of chaperone interaction significantly reduced neoexpression of CD1b upon differentiation of monocytes to dendritic cells, underlining the importance of chaperones for proper expression of CD1b under physiological conditions.
Keywords: antigen presentation, CD1, chaperones, MHC
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Introduction
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Human CD1b protein is distantly related in sequence to the MHC proteins. It is composed of a non-polymorphic heavy chain (HC) and, like MHC class I, the soluble subunit ß2-microglobulin (ß2m). The three-dimensional structure of CD1b is presumably MHC class I-like containing a hydrophobic binding groove, which is predicted from the X-ray structure of mouse CD1 (1). In contrast to the peptide-presenting MHC molecules, CD1b functions as an antigen-presenting molecule that presents lipids and glycolipids derived from pathogenic Mycobacteria like Mycobacteria tuberculosis and Mycobacteria leprae to cytotoxic T cells (2,3). CD1b contains an endosomal-targeting motif in its cytoplasmic tail, which is essential for antigen presentation (4). CD1b can therefore be found in endosomal compartments, where it co-localizes with the mannose receptor, which delivers certain antigens for presentation by CD1b (5,6). The binding of antigens to CD1b is optimal at acidic pH, consistent with a requirement for endosomal acidification (7). These findings indicate that the endosomes are the antigen loading site of CD1b. Earlier steps in CD1b processing are only partially understood. We and others demonstrated that ß2m is required for the transport of CD1b to the cell surface (8,9). In the absence of ß2m, CD1b HC are found in the endoplasmic reticulum (ER) in association with the molecular chaperone calnexin (10).
Newly synthesized human MHC class I HC bind also to calnexin, which facilitates folding and assembly (11,12). The calnexinMHC class I HC interaction is mediated via processed N-linked oligosaccharides (13). The Glc3Man9GlcNAc2 oligosaccharide is added to newly synthesized proteins as they enter the ER (14). ER glucosidases I and II remove two of the three glucose residues to generate the Glc1Man9GlcNAc2 oligosaccharide which is recognized by calnexin and calreticulin (15). Inhibition of the oligosaccharide processing by the glucosidase inhibitor castanospermine results in degradation of unassembled MHC class I HC (16). Upon binding of ß2m to the calnexinhuman MHC class I HC complex, the chaperone calreticulin, the ER-resident lumenal homologue of calnexin, presumably replaces calnexin (17). Calnexin and calreticulin recognize the same oligosaccharide (18), but they differ in their binding references depending on the location of the oligosaccharides (1921). To complete the MHC class I complex, a suitable peptide is required, which is delivered to the ER by TAP (22). Fully assembled MHC class I proteins are directly transported through the Golgi complex to the cell surface without intersecting with the endocytic route (23). Misfolded or incompletely assembled MHC class I HC are removed from the ER to the cytosol, deglycosylated and degraded by the proteasome (24).
Given the similarities (HCß2m structure; association with calnexin in the ER) and differences (nature of presented antigens; appearance of CD1b, but not of MHC class I in endosomal compartments) between CD1b and MHC class I, we compared the early processing pathways of ß2m-unassociated CD1b HC and MHC class I HC. We investigated chaperone associations and the degradation mechanism when the chaperone interactions were inhibited. Here we show the identification of calreticulin as an additional chaperone beside calnexin to be associated with CD1b HC, but not with MHC class I HC. Furthermore, we demonstrate that chaperone- and ß2m-unassociated CD1b HC and MHC class I HC are degraded by a process involving mannosidase activity and the proteasome.
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Methods
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Cells
The human ß2m-negative melanoma cell line FO-1 and the human ß2m-positive embryonic kidney cell line 293 were cultured in RPMI 1640 medium (Gibco/BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS (PAA, Linz, Austria), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 18.75 µg/ml gentamycin sulfate.
Monocytes were isolated from peripheral blood mononuclear cells (PBMC) by plastique adherence. For differentiation, they were incubated for 24 h in RPMI 1640 medium containing 10% heat-inactivated FCS, 1:50 diluted MEM essential amino acids (Gibco/BRL), 1:100 diluted MEM non-essential amino acids (Gibco/BRL), 50 µM 2-mercaptoethanol (Gibco/BRL), 10 mM HEPES, 50 µg/ml granulocyte macrophage colony stimulating factor (GM-CSF) and 200 U/ml IL-4 (both kindly provided by Novartis) in the presence or absence of 0.5 mM castanospermine.
Antibodies
The anti-pTag mAb H902-producing hybridoma cell line is reagent no. 521 from the NIH AIDS Research and Reference Reagent Program (25). CD1b mAb 4A7.6 was kindly provided by Dr Daniel Olive (INSERM U119, Institut Paul, Marseilles, France) (26). mAb HC-10 (27) that recognizes ß2m-unassociated MHC class I heavy chains was obtained from Dr Hidde Ploegh (Massachusetts Institute of Technology, Cambridge, MA). The hybridoma cell line producing mAb W6/32 which reacts with ß2mMHC class I complexes was from the ATCC (Rockville, MD) (28). Anti-human B7-1 (CD80) was purchased from R & D (Wiesbaden, Germany). The negative control mAb (anti-insulin IN-06) was provided by Dr Vaclav Horejsi (Institute of Molecular Genetics, Prague, Czech Republic). The rabbit sera recognizing calreticulin or the C-terminus of calnexin respectively were purchased from StressGen (Victoria, BC, Canada). Biotinylation of mAb H902 and FITC labeling of anti-ß2m mAb BBM.1 (29) was done in our Institute.
Construction and expression of tagged CD1b and CD147 molecules
The fusion of the 30 amino acid pTag peptide, provided by Dr Gottfried Baier (Institute of Medical Biology and Human Genetics, Innsbruck, Austria), to CD1b has been already described previously (8). CD147pTag was constructed using the same method. Briefly, the cDNA sequences encoding CD1b and CD147 were amplified by PCR to remove the stop codon and to allow C-terminal fusion to the pTag sequence in the pTag/CMV-neo plasmid (25). Lipofection with the cationic lipid DMRIE-C (Life Technologies, Gibco/BRL, Gaithersburg, MD) was performed according to the manufacturer's instruction, and was used to introduce the plasmid expression constructs into FO-1 cells and 293 cells, followed by selection for G418 resistance and cloning.
Metabolic labeling
FO-1 cells (1x106) were starved for 60 min in methionine- and cysteine-free RPMI 1640 medium supplemented with 10% dialyzed FCS, and then labeled for 20 min with 40 µCi of EasyTag Express protein labeling mix containing [35S]methionine and [35S]cysteine (NEN, Beverly, MA). For pulsechase experiments, 10xcold cysteine and methionine were added, and the cells were incubated for the indicated time periods. When indicated, 0.3 mM castanospermine (Sigma, St Louis, MO), 10 µM lactacystin (purchased from E. J. Corey, Harvard University, Cambridge, MA) and 2 mM deoxymannojirimycin (dMNJ; Sigma) were present during starvation, pulse and chase periods.
Immunoprecipitation and Western blotting
Cells were lysed in PBS, pH 7.0, containing 1% Brij 58 or 1% NP-40 (Pierce, Rockford, IL) as detergent, and the protease inhibitors iodoacetamide (5 mM), aprotinin (5 mM), leupeptin (5 mM), PMSF (1 mM) and pepstatin (1 µM; all from Sigma). After incubation for 30 min on ice, nuclei were removed by centrifugation at 10 000 g for 10 min at 4°C. The lysates were incubated with Ni2+-nitrilo-tri-acetic acid (NTA) agarose (Qiagen, Hilden, Germany) for 2 h at 4°C. Alternatively, samples were precleared with Protein ASepharose CL-4B (Pharmacia, Uppsala, Sweden) for 1.5 h before a 2 h incubation with the indicated antibodies prebound to Protein ASepharose. Immunoprecipitates were washed 3 times with 0.1% Brij 58 or 0.1% NP-40 in PBS and eluted by boiling in reducing or non-reducing SDSPAGE sample buffer. Immunoprecipitates were resolved on 8% SDSPAGE gels (30). Gels with radioactive proteins were treated with Enlightning (NEN), dried and exposed to X-OMAT AR films (Eastman Kodak, Rochester, NY) together with BioMax TranScreen-LE intensifying screen (Kodak) at 70°C for various lengths of time. Non-radioactive samples were transferred to Immobilon-P membranes (Millipore, Bedford, MA). After a 40 min incubation in blocking buffer [Tris-buffered saline (TBS), pH 7.5, containing 4% non-fat powdered milk], blots were probed with the indicated antibodies for 40 min, washed in TBS/0.1% Tween 20 and incubated for 30 min with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (BioRad, Richmond, CA). Proteins were visualized with enhanced chemiluminescence (Boehringer Mannheim, Mannheim, Germany) on a Lumi-Imager (Boehringer Mannheim).
Immunofluorescence
For cytoplasmic immunofluorescence analysis of FO-1 transfectants, cells (8x105) were permeabilized with 0.3% saponin for 10 min at room temperature and fixed with 1% paraformaldehyde in PBS for 15 min. The cells were then incubated for 20 min with biotinylated anti-pTag mAb H902, followed by a 20 min incubation with streptavidinPerCP and FITC-conjugated ß2m mAb BBM.1. Irrelevant biotinylated and FITC-conjugated mAb were used as negative control. Fluorescence analysis was performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Cytokine-stimulated monocytes were surface stained using the indirect immunofluorescence technique. Briefly, 50 µl of cells (1x105) were incubated with 50 µl of mAb (20 µg/ml) for 20 min on ice. After washing, the samples were incubated with fluoresceinated sheep F(ab')2 anti-mouse Ig antibodies (An der Grub, Kaumberg, Austria) as second-step reagent. Immunofluorescence was analyzed on a FACScan flow cytometer.
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Results
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Construction of tagged CD1b
Western blotting analysis revealed that all available CD1b mAb, including those from the Fourth and Fifth Workshop on Human Leukocyte Differentiation Antigens, did not recognize CD1b HC in the absence of ß2m (8). To detect and study ß2m-unassociated CD1b HC, we constructed a tagged form of CD1b by fusion of pTag to the C-terminal end of the CD1b cDNA. The pTag consists of a Ni2+ ion binding site and an HIV-derived peptide that is recognized by the H902 mAb (25). CD1bpTag and CD147pTag, which was chosen as pTag control due to its independence from ß2m, were stably expressed in the human ß2m-negative melanoma cell line FO-1. To prove the quality of the constructs, we performed an immunoprecipitation analysis with Ni2+-NTA agarose and mAb H902 (Fig. 1
). With both isolation methods, a protein of ~45 kDa which corresponds to the mol. wt of CD1b, was isolated from CD1bpTag transfectants. In CD147pTag transfectants, two dominant CD147 protein species were found: a 5055 and a 40 kDa polypeptide. The 5055 kDa form corresponds in size to the mature form of CD147 precipitated from hemopoietic cell lines; the 40 kDa form represents presumably an immature partially glycosylated form (31). This assumption is supported by the short-term metabolic labeling procedure described below, which revealed that newly synthesized CD147 existed for at least 2 h only in the 40 kDa form (see Figs 2 and 4
). These data show that the recombinant CD1bpTag and CD147pTag constructs are correctly expressed by FO-1 transfectants.

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Fig. 1. Quality control of tagged CD1b. FO-1 transfectants expressing CD1bpTag or the control protein CD147pTag were lysed in NP-40 lysis buffer and the recombinant molecules were isolated by using Ni2+-NTA agarose or the anti-pTag mAb H902. Precipitated proteins were separated by non-reducing SDSPAGE, transferred to a membrane, and visualized by mAb H902 and chemiluminescence. Mol. wt markers are indicated in kDa.
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Fig. 2. Co-immunoprecipitation analysis of CD1b HC from metabolically labeled FO-1 cells. ß2m-negative FO-1 transfectants expressing either CD1bpTag or CD147pTag, or untransfected (untransf.) FO-1 cells were radiolabeled with [35S]methionine and [35S]cysteine for 20 min. The cells were lysed in NP-40 lysis buffer, and the tagged CD1b and CD147 molecules were isolated by using H902 mAb. Isolation of MHC class I HC was performed with HC-10 mAb. Anti-insulin mAb IN-06 was used as control. Proteins were analyzed by reducing SDSPAGE and visualized by autofluorography. The positions of CD1bpTag, CD147pTag, MHC class I HC (MHC I) and the co-isolated 90 kDa protein are shown. Mol. wt markers are indicated in kDa. The data of one out of three independent experiments are shown.
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Fig. 4. Effect of castanospermine on CD1b HC expression in (A) FO-1 and (B) 293 cells. Transfected or untransfected FO-1 and 293 cells were starved in methionine- and cysteine-deficient medium for 60 min in the absence or presence of 0.3 mM castanospermine (Cas). The cells were radiolabeled with [35S]methionine and [35S]cysteine for 20 min, chased for the indicated time periods by addition of an excess of cold methionine and cysteine, and lysed in NP-40 lysis buffer. CD1bpTag and CD147pTag were precipitated with the H902 mAb from the respective transfectants, and MHC class I HC with mAb HC-10 from untransfected cells. SDSPAGE analysis was performed under reducing conditions. The data of one out of three independent experiments are shown.
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Tagged CD1b HC associate with calnexin and calreticulin in ß2m-negative FO-1 cells
Due to the lack of ß2m, transport of CD1b to the cell surface is blocked in FO-1 cells resulting in accumulation of CD1b in the ER (8,10). To investigate the molecular interactions responsible for the intracellular retainment and stabilization of ß2m-unassociated CD1b HC, we isolated CD1bpTag, CD147pTag and MHC class I HC from metabolically labeled FO-1 cells, and analyzed the samples by SDSPAGE and autofluorography (Fig. 2
). Strong bands corresponding to the relative mol. wt of these molecules demonstrated that CD1bpTag, CD147pTag and MHC class I were efficiently immunoprecipitated. In search of molecules interacting with CD1b HC, we detected a 90 kDa co-precipitated protein. A band of similar size was also seen in the precipitate of MHC class I, but barely visible in that of CD147pTag. Further molecules associating with CD1b HC were not found by this approach, possibly due to a slow turn-over of such molecules and therefore an ineffective incorporation of radioactive amino acids or, alternatively, due to an overlap with proteins unspecifically co-precipitated with CD1b HC.
It has been recently published that CD1b HC associate with the molecular chaperone calnexin (10), which has an apparent molecular mass of 90 kDa. Therefore, we assumed that the 90 kDa protein co-isolated with recombinant CD1b is identical to calnexin (Fig. 2
). To verify this, we precipitated CD1bpTag, CD147pTag and MHC class I HC, and probed the blotted samples with a calnexin-specific polyclonal rabbit antibody (Fig. 3A
). We found CD1b HC and MHC class I HC associated with calnexin. A reactivity of the anti-calnexin antibody with the CD147pTag precipitate was barely detectable. Thus, these data indicate that pTag fused to CD1b and CD147 neither interferes with nor induces a calnexin association, suggesting that pTag does not modify molecular interactions.

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Fig. 3. Analysis of CD1b HC and MHC class I HC association with calnexin and calreticulin. CD1bpTag transfected or untransfected (untransf.) (A) FO-1 cells and (B) 293 cells were lysed in lysis buffer containing Brij 58 as detergent. As a pTag control, the cells were transfected with CD147pTag and lysed in the same way. The pTagged molecules and MHC class I HC were isolated using mAb H902 and HC-10 respectively. Anti-insulin mAb IN-06 was used as control. The precipitates were resolved by non-reducing SDSPAGE and immunoblotted by using antibodies recognizing calnexin and calreticulin. Similar results were obtained by three further experiments.
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The calnexin-related chaperone calreticulin has been shown to be also involved in the processing of MHC class I molecules (17,32). To investigate a possible association of this chaperone with ß2m-free CD1b HC, we blotted the CD1bpTag isolate with a calreticulin specific antibody (Fig. 3A
). The precipitate of pTagged CD1b HC reacted with the calreticulin antibody, but not the isolates of MHC class I HC and of pTagged CD147 molecules. Thus, in contrast to ß2m-free MHC class I HC, ß2m-free CD1b HC seem to associate with both calnexin and calreticulin in FO-1 cells.
Disappearance of CD1b HC upon prevention of chaperone binding by castanospermine in ß2m-negative FO-1 cells
Interaction of calnexin and calreticulin with several target molecules has been shown to be prevented by the glucosidase-inhibitor castanospermine, resulting in destabilization of the respective molecules (12,33,34). To analyze whether this holds also true for CD1b, we performed a pulsechase experiment. The expression of newly synthesized CD1bpTag, CD147pTag and MHC class I HC was investigated during a 2 h chase period in the presence or absence of castanospermine (Fig. 4A
). In ß2m-negative FO-1 cells, CD1b HC and MHC class I HC almost completely disappeared within 2 h in the presence of castanospermine. However, castanospermine induced no disappearance of CD147pTag. These data indicate that similarly processed oligosaccharides are involved in the interaction of chaperones with CD1b HC as with MHC class I HC. Furthermore, the experiment suggests that interaction of calnexin and calreticulin with CD1b HC is essential for the stabilization of ß2m-unassociated CD1b HC.
Castanospermine-induced disappearance of CD1b HC involves mannosidases and degradation by the proteasome in FO-1 cells
Abnormally folded or unassembled proteins from the ER are subjected to destruction by the cytosolic proteasome (35). A specific irreversible inhibitor of the proteasome is lactacystin which covalently binds to the unique threonine active sites of the proteasome (36). To analyze the involvement of the proteasome in the disappearance of CD1b upon castano- spermine treatment, we preincubated the FO-1 cells with different combinations of castanospermine and lactacystin and performed a pulsechase experiment (Fig. 5
). The castanospermine-induced disappearance of CD1b HC and MHC class I HC was almost completely prevented by lactacystin. CD1b HC had an increased mol. wt when isolated from castanospermine and lactacystin incubated cells in comparison to cells incubated with lactacystin only. This demonstrated that oligosaccharide trimming was prevented by castanospermine and excluded a hypothetical disturbance of the castanospermine action by lactacystin. In conclusion, prevention of chaperone binding by castanospermine seems to cause CD1b HC disappearance through degradation by the proteasome. A recent study reported an involvement of mannosidases in degradation of CD3
(37). To investigate a possible influence of these enzymes in the degradation of CD1b HC and MHC class I HC, we combined castanospermine and dMNJ, a specific inhibitor of ER and Golgi mannosidases, in the pulsechase experiment shown in Fig. 5
. We found that dMNJ partially prevented the castanospermine-induced degradation of CD1b HC and MHC class I HC, demonstrating that mannosidase activity is necessary for the proteasomal degradation of CD1b HC and MHC class I HC in FO-1 cells.

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Fig. 5. Effect of lactacystin and dMNJ on castanospermine-destabilized CD1b HC. FO-1 cells were starved in methionine- and cysteine-deficient medium for 60 min in the absence or presence of 0.3 mM castanospermine, 10 µM lactacystin (Lac) and 2 mM dMNJ. The cells were radiolabeled with [35S]methionine and [35S]cysteine for 20 min, chased for 2 h, and lysed in NP-40 lysis buffer. CD1bpTag was precipitated with the H902 mAb from CD1bpTag FO-1 transfectants and MHC class I HC with the HC-10 mAb from untransfected FO-1 cells. Precipitated proteins were resolved under reducing SDSPAGE conditions and visualized by autofluorography. The data of one out of three independent experiments are shown.
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Influence of ß2m on stability of CD1b HC
ß2m was shown to be an essential factor in the biogenesis of CD1 (8). We therefore investigated the influence of castanospermine on CD1bpTag expression in the presence of ß2m. We stably transfected ß2m-positive 293 cells with CD1bpTag encoding cDNA. In a pre-experiment, we precipitated CD1bpTag and probed the blotted samples with a calnexin and a calreticulin specific antibody (Fig. 3B
). Calnexin and calreticulin were detected in the CD1b HC-precipitate indicating that this complex is also present in 293 cells. However, in contrast to FO-1 cells, the degradation of CD1bpTag proteins was significantly delayed upon castanospermine treatment in 293 cells (Fig. 4B
); the same held true for MHC class I HC. Expression of CD147pTag, which was used as a pTag control, was not altered. We could exclude an inactivation or exclusion of castanospermine from the 293 cells in this experiment, because this treatment resulted in the generation of a higher mol. wt CD1bpTag molecule indicating that the glucose trimming was inhibited. These data suggested that ß2m has a stabilizing effect on CD1b HC and also MHC class I HC in the absence of the lectin-like chaperone interactions of calnexin and calreticulin. However, we were not sure whether the lower degradation of CD1b HC in ß2m-positive 293 cells compared to ß2m-negative FO-1 cells is not due to another cellular factor rather than a consequence of ß2m. To analyze this, we transiently expressed ß2m in CD1bpTag FO-1 transfectants using lipofection and incubated the cells for 24 h with or without castanospermine. To monitor expression of ß2m and CD1bpTag simultaneously at the single-cell level, we used a FITC-labeled anti-ß2m mAb and biotinylated anti-pTag mAb H902, which was visualized by streptavidinPerCP. By cytoplasmic flow cytometry, we gated three different cell populations which expressed no, medium or high levels of ß2m (Fig. 6A
) and measured the CD1bpTag expression of these cell populations (Fig. 6B
). In the non-ß2m-expressing cell population, the mean fluorescence intensity (MFI) of CD1b staining was >40% reduced in castanospermine-treated compared to untreated cells; at medium ß2m levels CD1b was decreased only by 16%, and at high ß2m-levels almost no difference in the CD1b staining between untreated and treated cells was observed. Furthermore, the relative amount of CD1bpTag was 3 times higher in the ß2m-high versus ß2m-negative cell population. This difference was even more pronounced in the castanospermine-treated samples, because of the low level of CD1b expression in the ß2m-negative population. These data demonstrate a stabilizing effect of ß2m on CD1b HC in the absence of chaperone interactions mediated by monoglucosylated N-linked oligosaccharides.

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Fig. 6. Effect of ß2m on the expression level of castanospermine-destabilized CD1b HC. FO-1 cells expressing CD1bpTag were transiently transfected with ß2m by lipofection. Immediately after transfection, the sample was divided and one part was treated with 0.5 mM castanospermine (Cas) 24 and 4 h before cytoplasmic flow cytometric analysis. (A) Using FITC-conjugated anti-ß2m mAb BBM.1, three distinct cell populations (R1, R2 and R3) differing in their ß2m expression level were gated. (B) The three gated cell populations of castanospermine-treated (+Cas) and untreated (Cas) samples were analyzed for their CD1bpTag expression level by biotinylated mAb H902 and streptavidinPerCP. Irrelevant biotinylated and FITC-conjugated mAb were used as negative control. Shown are the MFI values of one out of two independent experiments.
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Effect of castanospermine on expression of native CD1b
Finally, we analyzed the role of castanospermine-sensitive chaperones for expression of native CD1b. For this approach, we isolated monocytes from human PBMC and incubated the cells with GM-CSF and IL-4. This induced the differentiation to dendritic cells, which is accompanied by CD1 neoexpression (38,39). As can be seen in Fig. 7
, the MFI of CD1b staining was 71% reduced in castanospermine-treated compared to untreated cells; the MHC class I staining 44%. Expression of CD80, which is also up-regulated upon differentiation of monocytes by GM-CSF and IL-4, was increased by the castanospermine treatment. The latter finding excludes a general negative effect of castanospermine on the expression level of molecules. Thus, this experiment shows that expression of native CD1b and MHC class I is specifically reduced by castanospermine treatment. Together with our studies on transfectants, we conclude from this experiment that the chaperones calnexin and calreticulin have an important function for the processing of CD1b and MHC class I.

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Fig. 7. Effect of castanospermine on CD1b HC neoexpression upon differentiation of monocytes to dendritic cells. Monocytes isolated from PBMC were incubated for 24 h in medium containing GM-CSF and IL-4 with or without 0.5 mM castanospermine. The cells were stained by using the indicated mAb. (A) The fluorescence profiles of the untreated cells (dark area), the castanospermine-treated cells (dark line) and the negative control (dashed line) are shown. (B) The MFI values of the stainings, from which the MFI values of the negative controls were subtracted, are shown. The data represent one out of two independent experiments.
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Discussion
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In this study we investigated early processing steps of CD1b HC. In contrast to ß2m-unassociated CD1b HC which were found to complex with both calnexin and calreticulin (Fig. 3A
), human MHC class I HC associate with calnexin in the absence of ß2m and with calreticulin upon ß2m binding (17,32). MHC class I HC contain a single N-glycosylation site, which seems to be occupied by calnexin in the absence of ß2m and by calreticulin in the presence of ß2m. Thus, one can assume that binding of ß2m changes the three-dimensional structure of the chaperone binding site, which results in the replacement of calnexin by calreticulin. This assumption is supported by reports describing that the location of the same oligosaccharide on target molecules determines the binding of calnexin or calreticulin (1921). In contrast to MHC class I HC, CD1b HC contain four putative N-glycosylation sites (40). These N-glycosylation sites may differ in their pReferences for calnexin or calreticulin or may have affinity for both. CD1b HC may therefore be able to associate either simultaneously or alternatively with calnexin and calreticulin.
Calnexin and calreticulin recognize processed N-linked oligosaccharides present on newly synthesized proteins in the ER. It was demonstrated that castanospermine inhibits this oligosaccharide processing and thereby prevents calnexin and calreticulin binding to newly synthesized proteins (13,32). We found that castanospermine incubation of ß2m-negative FO-1 transfectants led to nearly complete disappearance of newly synthesized CD1b HC and MHC class I HC within 2 h (Fig. 4A
). Furthermore, we demonstrated that castano- spermine inhibited neoexpression of native CD1b by 71% upon differentiation of monocytes to dendritic cells. Thus, our findings indicate that the oligosaccharide-dependent interactions of calnexin and calreticulin are critical for the stabilization of CD1b HC. Although we did not observe any other dominant protein co-isolated with recombinant CD1b HC from FO-1 cells (Fig. 2
), we cannot exclude that other identified and/or unidentified lectin-like chaperones additionally participate in the stabilization and biogenesis of CD1b HC. One such candidate could be thiol-dependent reductase ER-60 (ERp57), recently found to be involved in MHC class I assembly (41). Such unidentified complexes could be involved in the calnexin- and calreticulin-independent partial neoexpression of native CD1b in dendritic cells which were generated from monocytes in the presence of castanospermine (Fig. 7
).
Another factor involved in the calnexin- and calreticulin-independent partial neoexpression of native CD1b might be ß2m. In ß2m-positive 293 cells, the castanospermine-induced disappearance of CD1b HC and MHC class I HC was significantly delayed (Fig. 4B
). This suggests that ß2m can stabilize CD1b HC under conditions which prevent binding of calnexin and calreticulin. However, we could not exclude cell-type-specific differences between FO-1 and 293 cells resulting in slower degradation of CD1b HC in 293 cells. We therefore expressed ß2m in the CD1b FO-1 transfectants and investigated the ß2m effect in cell populations expressing different ß2m levels. This experiment revealed that the castanospermine-induced disappearance of CD1b HC was continuously abrogated upon increasing expression levels of ß2m. This suggested that CD1b HC were rescued from disappearance by the formation of CD1bß2m complexes in FO-1 cells (Fig. 6
).
It has been already observed in ß2m-negative Daudi cells that upon transfection, ß2m is able to promote folding and disulfide-bond formation of MHC class I HC in the absence of calnexin interactions (42). In this study it was reported that ß2m binds to MHC class I HC only when all disulfide bonds are formed, whereas the calnexin interaction is independent of this structural feature. This suggests that ß2m interacts with and stabilizes only correctly folded MHC class I HC. It might be possible that calnexin- and calreticulin-unassociated CD1b HC are stabilized by ß2m in the same way. However, it is not clear how the correct conformation of CD1b HC could be formed in the absence of calnexin and calreticulin interactions. One possibility would be random folding or, alternatively, by the help of castanospermine-insensitive chaperone interactions.
Unassembled or abnormally folded proteins are targeted to the cytosol, where proteolysis is catalyzed by the proteasome (4347). We demonstrated that castanospermine-induced disappearance of CD1b HC in FO-1 cells was sensitive to the proteasomal inhibitor lactacystin (Fig. 5
). This indicates that calnexin-, calreticulin- and ß2m-unassociated CD1b HC are degraded by the proteasome. Proteasomal degradation of CD1b HC or MHC class I HC could occur when two situations happen at the same time: (i) when calnexin and calreticulin interactions are prevented (due to incorrect oligosaccharide processing of CD1b HC or MHC class I HC, wrong function or down-regulation of calnexin and calreticulin, CD1b HC or MHC class I HC overexpression), and (ii) when the relative expression level of ß2m compared to CD1b HC or MHC class I HC is low (due to ß2m down-regulation, or CD1b HC or MHC class I HC overexpression).
Degradation of many short-lived proteins by the proteasome requires ubiquitin conjugation (48). To investigate the possible involvement of ubiquitination in CD1b HC and MHC class I HC degradation, we preincubated FO-1 cells with the proteasomal inhibitor lactacystin, immunoprecipitated CD1b HC and MHC class I HC, and probed the isolates with an ubiquitin antibody. However, no ubiquitinated CD1b HC and MHC class I HC intermediates were detected (data not shown). This finding corroborates published data for MHC class I HC (46).
Further analysis of the degradation pathway upon castanospermine incubation revealed that the mannosidase inhibitor dMNJ blocked CD1b HC and MHC class I HC degradation (Fig. 5
). This experiment demonstrates that removal of one or more mannose residues from N-linked oligosaccharides is necessary to render CD1b HC and MHC class I HC accessible for proteasomal degradation. The inhibition of the mannose modification blocks proteasomal degradation also in the absence of calnexin/calreticulin interactions. This differs from the degradation mechanism described for a mutant form of
1-antitrypsin, which was mannosidase-dependent in the presence of intact calnexin interactions, but mannosidase-independent when calnexin interactions were blocked by castanospermine (49).
In conclusion, our data show that ß2m-unassociated CD1b HC interact with the chaperones calnexin and calreticulin for the generation of a stable intermediate, whereas MHC class I HC complex with calnexin only. Apart from this difference, the early processing pathways of CD1b HC and MHC class I HC share several common characteristics: in the absence of ß2m, prevention of the chaperone interactions by castanospermine subjects both molecules to a degradation process which requires mannosidase activity and finally results in the degradation by the proteasome. ß2m is able to protect both molecules from this degradation process independently of calnexin and calreticulin. However, this escape mechanism seems to play a minor role under physiological conditions as prevention of chaperone interactions significantly inhibited neoexpression of CD1b and MHC class I upon differentiation of monocytes to dendritic cells, underlining the importance of chaperones for expression of CD1b and also MHC class I.
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Acknowledgments
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The authors would like to thank Drs Gottfried Baier, Soldano Ferrone, Daniel Olive, Hidde Ploegh and Vaclav Horejsi for their generous gift of the pTag/CMV-neo plasmid, the FO-1 cells, the HC-10 mAb and the IN-06 mAb respectively. This work was supported by the Novartis Research Institute Vienna.
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Abbreviations
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ß2m | ß2-microglobulin |
dMNJ | deoxymannojirimycin |
ER | endoplasmic reticulum |
GM-CSF | granulocyte macrophage colony stimulating factor |
HC | heavy chain |
NTA | Ni2+-nitrilo-tri-acetic acid |
PBMC | peripheral blood mononuclear cells |
TBS | Tris-buffered saline |
 |
Notes
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Transmitting editor: S. H. E. Kaufmann
Received 11 September 1998,
accepted 10 June 1999.
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