Differential post-translational modification of CD63 molecules during maturation of human dendritic cells Anneke Engering 1,3 , Lotte Kuhn 1,2 , Donna Fluitsma 3 , Elisabeth Hoefsmit 3 and Jean Pieters 1,2 1 Basel Institute for Immunology, Basel, Switzerland; 2 Biozentrum, University of Basel, Basel, Switzerland; 3 Department of Cell Biology and Immunology, Vrije Universiteit, Amsterdam, The Netherlands The capacity of dendritic cells to initiate T cell responses is related to their ability to redistribute MHC class II mole- cules from the intracellular MHC class II compartments to the cell surface. This redistribution occurs during dendritic cell development as they are converted from an antigen capturing, immature dendritic cell into an MHC class II- peptide presenting mature dendritic cell. During this matu- ration, antigen uptake and processing are down-regulated and peptide-loaded class II complexes become expressed in a stable manner on the cell surface. Here we report that the tetraspanin CD63, that associates with intracellularly localized MHC class II molecules in immature dendritic cells, was modified post-translationally by poly N-acetyl lactosamine addition during maturation. This modification of CD63 was accompanied by a change in morphology of MHC class II compartments from typical multivesicular organelles to structures containing densely packed lipid moieties. Post-translational modification of CD63 may be involved in the functional and morphological changes of MHC class II compartments that occur during dendritic cell maturation. Keywords: antigen presentation; poly N-acetyl lactosamine addition; dendritic cells; tetraspanins; MHC class II. Dendritic cells have the unique feature to induce T cell responses in lymphoid organs against antigens captured in peripheral tissues (for review, see [1,2]). Immature tissue dendritic cells use several mechanisms to internalize a broad array of antigens via the endosomal–lysosomal pathway including fluid phase endocytosis, macropinocytosis and several receptor-dependent mechanisms [3–5]. Peptides derived from internalized antigens are loaded onto class II molecules in MHC class II compartments [6–9]. After migration to lymph nodes upon inflammation or an infection, mature dendritic cells present these MHC class II-peptide complexes to T lymphocytes. Several co-ordinated changes enable efficient presenta- tion of epitopes generated at sites of inflammation to T lymphocytes for prolonged periods of time [10,11]. During maturation of dendritic cells, the number of MHC class II-peptide complexes that are generated is increased, both by a transient up-regulation of synthesis as well as by an increase in half-life of MHC class II molecules [10]. In addition, uptake and processing of antigen is down- regulated and MHC class II molecules are redistributed to the cell surface. Trafficking of MHC class II-peptide com- plexes is in part regulated by the protease, cathepsin S, that is activated upon maturation of dendritic cells [12,13]. This protease removes the sorting signal in the cytoplasmic tail of the invariant chain, a protein involved in targeting newly synthesized MHC class II molecules to MHC class II com- partments [14–16], thus allowing MHC class II molecules to exit these organelles. Moreover, maturation induces a reduction of internalization and degradation of cell surface MHC class II molecules [17]. These mechanisms result in the stable expression of MHC class II-peptide complexes on the cell surface of mature dendritic cells [10,11,18]. Recently, more insight has been gained into the transport-routes of MHC class II molecules to the plasma membrane [19,20]. Direct fusion of multivesicular MHC class II compartments has been shown to occur, resulting in secretion of the internal MHC class II-con- taining vesicles, so-called exosomes [21,22]. However, this route is down-regulated upon maturation of dendritic cells and may represent only a minor pathway of MHC class II transport to the cell surface [22,23]. Recently, using GFP- tagged MHC class II molecules in living murine dendritic cells, it was shown that upon a maturation stimulus, tubular MHC class II-containing endosomes extend from MHC class II compartments and can fuse directly with the plasma membrane [24,25]. Interestingly, these tubules were directed towards the contact phase with a T cell in an antigen-dependent manner [24]. In human dendritic cells, immuno-electron microscopy also demonstrated the appearance of MHC class II-containing tubules and vesi- cles upon induction of maturation [26]. These structures were suggested to represent transport intermediates between MHC class II compartments and the plasma membrane [26,27]. The underlying mechanisms of trans- porting MHC class II-containing vesicles to the cell surface remain unclear. Correspondence to J. Pieters, Biozentrum, University of Basel, Klingelbergstrasse 50–70, 4056 CH Basel, Switzerland. Fax: + 41 61 267 21 49, Tel.: + 41 61 267 21 51, E-mail: jean.pieters@unibas.ch Abbreviations: CD, cluster of differentiation; endo H, endoglyco- sidase H; LAMP, lysosomal-associated membrane proteins; LPS, lipopolysaccharide; MHC, major histocompatibility complex. (Received 18 February 2003, accepted 7 April 2003) Eur. J. Biochem. 270, 2412–2420 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03609.x We have reported recently that distinct tetraspanins associate with MHC class II molecules at different sites in immature dendritic cells [28]. CD9, CD53 and CD81 associate with surface MHC class II molecules, whereas CD63-MHC class II complexes are present exclusively in MHC class II compartments. Similarly, in B cells, CD63 as well as the tetraspanin CD82 were shown to associate with MHC class II molecules intracellularly [29]. In this paper, we describe that CD63 is modified post-translationally upon maturation of dendritic cells. This modification was attri- buted to the addition of poly N-acetyl lactosamine groups onto CD63. Interestingly, CD63-positive organelles chan- ged morphologically during dendritic cell maturation – from compartments with a multilaminar appearance into structures containing multiple condensed lipid layers. The increase in lactosaminoglycans on CD63 may be involved in the changes that occur in MHC class II compartments during dendritic cell maturation. Materials and methods Antibodies, reagents and cells The following antibodies were used: I98 (anti-CD63, IgG 1 ), anti-CD63 (IgG 1 , CLB, Amsterdam), and a polyclonal antibody against MHC class II (kind gift of H. Ploegh, Harvard Medical School, Boston, MA, USA). Dendritic cells were generated from human peripheral blood monocytes cultured for 4–8 days in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone), 50 ngÆmL )1 recombinant GM-CSF (Leucomax, Sandoz) and 1000 UÆmL )1 recombinant IL-4 [4,30]. Buffy coats were from healthy blood donors, upon written consent. To induce maturation, dendritic cells were stimu- lated with LPS for 40 h (1 lgÆmL )1 , S. abortus equi,Sebak). The human melanoma cell line, Mel JuSo [31] was grown in RPMI-1640 supplemented with 10% fetal bovine serum; cells were stimulated by culturing for 48 h in 500 UÆmL )1 Interferon-c (IFN-c)(Pharmingen). Metabolic labeling and immunoprecipitation Prior to metabolic labeling, cells were cultured in RPMI without methionine and cysteine for 20 min. Cells were labeled for the times indicated in the same medium containing 0.1–0.2 mCiÆmL )1 [ 35 S]methionine/cysteine and 10% dialyzed fetal bovine serum. Cells were washed and chased in complete medium, supplemented with 2 m M methionine and cysteine, or lysed directly. Lysis buffer contained 20 m M Hepes (pH 7.5) with 100 m M NaCl, 5 m M MgCl 2 , 1% Triton X-100 with protease inhibitors [32]. For immunoprecipitation, lysates were incubated with the indicated antibodies for 2–12 h at 4 °C, followed by 1 h incubation with 30 lL protein A-Sepharose (Pharmacia). The immune complexes were washed as described [33], eluted from the protein A-Sepharose beads by incubation at 95 °C for 5 min in Laemmli sample buffer [34] and subjected to SDS/PAGE, fluorography and autoradio- graphy. When indicated, half of the immune complexes were incubated prior to elution with 10 mU endo-b- galactosidase (Bacteroides fragilis, Boehringer Mannheim) in 50 m M sodium acetate (pH 5.8) with 0.2 mgÆmL )1 bovine serum albumin for 24 h at 37 °C. As a control, the enzyme was omitted. Two-dimensional gel electrophoresis Two-dimensional IEF/SDS/PAGE was performed accord- ing to O’Farrell [35] with described modifications [36]. Resolyte pH 4–8 (BDH) was used for IEF. Immunocytochemistry Dendritic cells were fixed at room temperature in 2% paraformaldehyde and 0.5% glutaraldehyde in NaCl/P i for 2 h. Cells were pelleted and resuspended in 2% para- formaldehyde at 4 °C. Subsequently, samples were infused with 2.3 M sucrose and frozen quickly in liquid nitrogen. Ultrathin cryosections were labeled with specific primary antibodies as indicated, followed by colloidal gold particles coupled to protein A. To minimize cross reactivity of protein A-gold particles, sections were fixed briefly using 1% glutaraldehyde before double-labeling. Cryosections were analyzed on a CM 100 electron microscope (Philips). Subcellular fractionation Subcellular fractionation of dendritic cells was performed essentially as described [4,7,37]. Dendritic cells were harves- ted, washed and resuspended in homogenization buffer (10 m M triethanolamine, 10 m M acetic acid, 1 m M EDTA, 0.25 M sucrose, pH 7.4) at 10 7 cellsÆmL )1 . The cells were homogenized at 4 °C by passing through a 27G3/4 needle. After removal of the nuclei by centrifugation (840 g, 15 min), the postnuclear supernatant was incubated with trypsin (25 lgÆmg )1 protein) for 5 min at 37 °C. Digestion was stopped by addition of ice-cold soybean trypsin inhibitor (625 lgÆmg )1 protein). Membranes were sediment- ed by centrifugation for 45 min at 100 000 g, resuspended in 6% Ficoll-70 (Pharmacia) in homogenization buffer and electrophoresed for 90 min at 10.4 mA [7]. Fractions of 0.5 mL were collected from the top and analyzed for the different markers. Protein levels were analyzed according to the Bradford method [38]. The activity of b-hexosaminidase was assayed as described [39]. Results Analysis of CD63 expression during dendritic cell maturation Upon maturation, dendritic cells undergo a number of changes that contribute to their capacity to induce T cell responses [10,11,24–26]. These changes include biochemical as well as morphological alterations in organelles and molecules involved in MHC class II-restricted T cell activation. The tetraspanin CD63 associates with MHC class II molecules within class II compartments in a number of antigen presenting cells [29,40], including human immature dendritic cells [28]. To analyze CD63 expression during maturation, immature and mature dendritic cells were pulse-labeled with [ 35 S]methionine/cysteine, and CD63 molecules immunoprecipitated and analyzed by SDS/ PAGE and fluorography. After a 20-min labeling period, Ó FEBS 2003 CD63 glycosylation during dendritic cell maturation (Eur. J. Biochem. 270) 2413 in both immature and mature dendritic cells, a protein of 34 kDa was resolved (Fig. 1A, lane 1 and 3). However, after a 4-h pulse, in immature dendritic cells, anti-CD63 Igs immunoprecipitated alongside the 34 kDa polypeptide, an  50 kDa protein, the molecular mass of which increased to  70 kDa after maturation of the cells induced by LPS (Fig. 1A, lane 2 and 4). The increase in molecular mass of CD63 was independent of the stimulus used to induce maturation of dendritic cells (not shown). In all other cell types tested (including B lymphoblastoid, HeLa, Mel JuSo cells, with or without stimulation by LPS or IFN-c)CD63 antibodies precipitated a 34 and 50 kDa protein (shown for Mel JuSo, Fig. 1A, lanes 5–8). This indicates that the 34 kDa protein of CD63 either became associated with distinct proteins during maturation, or that CD63 was differentially modified post-translationally in immature vs. mature dendritic cells. To distinguish between these two possibilities, pulse/chase analysis was performed. As shown in Fig. 1B, in both immature and mature dendritic cells, the amount of the 34 kDa form of CD63 disappeared gradually during the chase period, concomitantly with the appearance of isoforms of CD63 of  50 and  70 kDa, respectively. Together, these data indicate that the 70 kDa isoform of CD63 is a result of post-translational modifications of the 34 kDa isoform that occurred exclusively in mature dendritic cells. Characterization of post-translational modifications on CD63 during maturation of dendritic cells CD63 contains three putative acceptor sites for N-linked glycosylation [41]. To analyze the addition of carbohydrates to these sites in CD63 molecules from immature and mature dendritic cells, both cell types were labeled for 10 min with [ 35 S]methionine and -cysteine, lysed and proteins were precipitated using anti-CD63 Igs. Immuno-isolated com- plexes were treated with endoglycosidase H (endo H). This enzyme cleaves N-linked glycans of the high mannose form that are acquired upon synthesis in the endoplasmic reticulum. As shown in Fig. 2A, endo H treatment resulted in a reduction of the apparent molecular mass to  25 kDa in immature as well as in mature dendritic cells, indicating identical N-linked glycosylation of CD63 in immature as well as mature dendritic cells. To analyze the type of modification on CD63 occurring during maturation, immature and mature dendritic cells were labeled metabolically for 16 h and immunoprecipi- tated material analyzed by two-dimensional IEF/SDS/ PAGE followed by fluorography. In immature cells, besides the 34 kDa form of CD63, eight additional spots of  50 kDa were resolved. The shapes of these spots indicated that they represent forms of CD63 that have been modified post-translationally with carbohydrate residues. Interest- ingly, after maturation of dendritic cells, the apparent molecular mass of each of these eight carbohydrate containing polypeptides was increased by  10 kDa (Fig. 2B). In mature dendritic cells, the 34 kDa form of CD63 was not detectable anymore, indicating a more complete conversion of CD63 to the complex-type carbo- hydrate form than in immature dendritic cells during the 16 h labeling period. Together, these results indicate that not only the type or complexity, but the degree of CD63 glycosylation differed in immature vs. mature dendritic cells. A modification known to result in differences in mole- cular mass, rather than charge, is addition of poly N-acetyl lactosamine to N-linked glycans, and this post- translational modification is known to occur on lysosomal associated membrane glycoproteins (LAMP) [42,43]. Poly γ Fig. 1. CD63 isoforms in immature and mature dendritic cells. (A) Immature dendritic cells, mature dendritic cells (DC) and Mel JuSo cells incubated for 48 h in the absence or presence of IFN-c were labeled metabolically for 20 min or 4 h using [ 35 S]methionine/cysteine and lysed. Proteins were immunoprecipitated with anti-CD63 Igs. (B) Immature and mature dendritic cells were labeled metabolically for 10 min, washed and cultured for the times indicated before lysis and immunoprecipitation with anti-CD63. Shown are autoradiographs after SDS/PAGE and fluorography. 2414 A. Engering et al. (Eur. J. Biochem. 270) Ó FEBS 2003 N-lactosamine groups are sensitive to endo-b-galactosidase [43]. To analyze the presence of polylactosaminoglycans, immature dendritic cells and maturing cells, cultured for 12 h in the presence of LPS, were labeled metabolically for 4 h, followed by lysis and immunoprecipitation using anti- CD63 or anti-LAMP Igs. Immune complexes were incuba- ted for 24 h at 37 °C in the presence or absence of 10 mU endo-b-galactosidase. As can be seen in Fig. 3A and B in both immature and maturing dendritic cells, CD63 as well as LAMP molecules are susceptible to digestion with endo- b-galactosidase. Therefore, these results indicate that poly- lactosaminoglycan addition already occurs in immature cells, and that during maturation these molecules acquire additional polylactosaminoglycans. Indeed, when immature dendritic cells were induced to mature by LPS, a gradual increase in the molecular masss of both CD63 and LAMP was observed, reaching a maximum after 12–24 h, with a subsequent decrease of the apparent molecular mass (Figs 3C,D). As can be seen in Fig. 3C,D, both the biosynthesis and the polylactosaminoglycan addition is dependent on the state of maturation, as has also been observed to occur for other molecules during maturation [10]. Stability and subcellular localization of CD63 molecules in immature and mature dendritic cells Maturation of dendritic cells is known to result in a differential stability of MHC class II [10,11]. Given the association of CD63 molecules with MHC class II mole- cules in MHC class II compartments [28,29], we analyzed whether the addition of polylactosaminoglycan moieties on CD63 molecules resulted in an altered stability of CD63. To that end, immature and mature dendritic cells were pulsed for1 hwith[ 35 S]methionine/cysteine followed by a chase for thetimesindicatedinFig.4.AsshowninFig.4,bothin immature as well as mature dendritic cells, CD63 molecules displayed a similar half life of  15 h, indicating that the degree of polylactosaminoglycan modification does not alter CD63 stability. In immature dendritic cells, CD63 is largely located intracellularly within MHC class II compartments [28,29,40]. To investigate whether the post-translational modification of CD63 molecules by polylactosaminoglycans may coincide with an altered subcellular localization, organelles from [ 35 S]methionine/cysteine metabolically labe- led immature as well as mature dendritic cells were separated by organelle electrophoresis. Upon electropho- resis, MHC class II compartments and late endosomal lysosomal organelles shift towards the anode, whereas the plasma membrane and most other subcellular organelles do not migrate significantly [4,7]. After fractionation, the fractions containing the lysosomal marker b-hexosamini- dase were pooled (ÔPool IÕ) as well as the nonshifted fractions (ÔPool IIÕ; see Fig. 5A). From these pooled fractions, CD63 as well as LAMP molecules were immunoprecipitated and analyzed by SDS/PAGE and fluorography. As shown in Fig. 5B in both immature and mature dendritic cells, CD63 as well as LAMP were largely present within pool I, indicating that before and after maturation of den- dritic cells, CD63 remained localized within MHC class II compartments. Immunocytochemical localization of CD63 in immature and mature dendritic cells The subcellular distribution of CD63 molecules was analyzed further by immunocytochemistry using immature and mature dendritic cells. In immature dendritic cells, CD63 colocalized with class II molecules in MHC class II compartments, predominantly with a multilaminar appear- ance (Fig. 6Aa). CD63 was occasionally detected in small Fig. 2. Two dimensional gel electrophoresis of CD63 isoforms. (A) Core-glycosylation of CD63 molecules. Immature (imm. DC) and mature dendritic cells (mat. DC) were labeled metabolically with [ 35 S]methionine/cysteine for 10 min and lysed. Immune-isolated complexes after preci- pitation with anti-CD63 Igs were incubated with (+) or without (–) endo-glycosidase H (endo H) for 14 h at 37 °C. An asterix indicates the deglycosylated forms of CD63. (B) Immature and mature dendritic cells were labeled with [ 35 S]methionine/cysteine for 14 h. After lysis, proteins were immunoprecipitated with anti-CD63 Igs and analyzed by IEF/SDS/PAGE. Shown are autoradiographs after SDS/PAGE and fluorography, basic end, right; acidic end, left. Ó FEBS 2003 CD63 glycosylation during dendritic cell maturation (Eur. J. Biochem. 270) 2415 electron dense vesicles, devoid of MHC class II molecules (Figs 6Aa,c), potentially representing a transport vesicle. After maturation, MHC class II molecules are redistributed to the cell surface, whereas CD63 was still predominantly found intracellularly (Figs 6Bb,d). Interestingly, the mor- phology of CD63 positive organelles changed upon matur- ation from the typical multilaminar structure into an organelle that appeared to contain densely packed lipid moieties (Fig. 6A). This difference in morphology is further illustrated in Fig. 6B, that shows several sections labeled for MHC class II molecules. In mature dendritic cells, densely packed structures were abundantly present (Figs 6Bb–d) but were rarely found in immature dendritic cells (Fig. 6Ba). Taken together, these results indicate that concomitant with the acquisition of polylactosaminoglycans on CD63, the MHC class II compartments became altered, both with respect to their morphology as well as the occupancy. Discussion Dendritic cells efficiently present epitopes from antigens captured at sites of inflammation to naive T lymphocytes in lymphoid organs by regulating MHC class II distribution and antigen-internalization mechanisms [1–3,18]. In imma- ture dendritic cells, antigens are efficiently internalized and antigenic peptides loaded onto MHC class II complexes intracellularly [44]. Maturation stimuli induce a transient enhancement of antigen uptake and peptide loading, whereas in fully matured dendritic cells, antigen uptake is down-regulated and peptide-loaded class II complexes are redistributed to the cell surface [10,11,44]. The molecular mechanisms regulating these processes are not well under- stood. In this paper, we describe that during maturation of dendritic cells, CD63 acquired additional polylactos- aminoglycans, concomitant with morphological changes β Fig. 3. CD63 and LAMP glycosylation in immature and mature dendritic cells Poly N-acetyl lactosaminoglycans on CD63 and LAMP molecules during maturation of dendritic cells. (A,B): Dendritic cells were incubated with LPS for the times indicated, followed by metabolic labeling with [ 35 S]methionine/cysteine for 4 h. Cell lysates were immunoprecipitated with anti-CD63 (A,C) or anti-LAMP (B,D) Igs. In A and B, immuno- isolated complexes were incubated in the presence (+) or absence (–) of endo-b-galactosidase (endo-b)for24hat37°C. The differences in metabolic labeling are probably to be due to the different efficiencies in labeling at the different times after LPS addition. Shown are autoradio- graphs after SDS/PAGE (A,C: 12% acrylamide gels; B,D: 7.5% acrylamide gels) and fluorography. Fig. 4. Stability of CD63 molecules in immature and mature dendritic cells. Immature and mature dendritic cells were labeled metabolically with [ 35 S]methionine/cysteine followed be a chase in normal medium for the times indicated. At each chase time, cells were lysed and CD63 molecules immunoprecipitated from the detergent lysates followed by analysis by SDS/PAGE, fluorography and autoradiography. 2416 A. Engering et al. (Eur. J. Biochem. 270) Ó FEBS 2003 in MHC class II compartments from a characteristic multivesicular/multilaminar appearance to structures with dense lipid moieties. CD63 is generally considered as one of the best markers of MHC class II compartments besides MHC class II mole- cules themselves [45,46]. CD63 is a member of the tetraspanin superfamily, consisting of polypeptides with four membrane spanning domains that associate with a variety of proteins [47]. On the cell surface, tetraspanins were found to colocalize in distinct membrane microdomains that are enriched for specific MHC class II-peptide complexes as well as costim- ulatory molecules, thereby facilitating T cell activation [29,48,49]. It has been shown recently that in human immature dendritic cells and in B cells, CD63 and CD82 form complexes with MHC class II molecules in class II compartments, whereas another set of tetraspanins associ- ate with surface MHC class II molecules [28,29]. Here, we show that in both immature and mature dendritic cells, the subcellular localization of CD63 molecules was confined to the intracellular MHC class II compartment. At later time points of maturation, MHC class II synthesis and recycling of MHC class II molecules from the cell surface is shut off [10,17]. In accordance with this, CD63 positive organelles in mature dendritic cells were found to be depleted from MHC class II molecules. CD63 has been localized to a wide variety of distinct intracellular organelles whose content or membrane mole- cules are discharged after appropriate stimuli. These include the cytolytic granules of cytotoxic T lymphocytes [50,51], the Weibel–Palade bodies of vascular endothelial cells [52], the secretory granules of neutrophils and basophils [53,54], as well as those from megakaryocytes and platelets [55]. Interestingly, all of these organelles rely on stimulated processes to release their content, a process that may be similar to the regulated redistribution of MHC class II molecules from the MHC class II compartment to the plasma membrane, as occurs during dendritic cell develop- ment [10]. Indeed, dendritic cells secrete exosomes upon fusion of multivesicular organelles with the cell surface [21,23]. Exosomes contain high levels of tetraspanins, including CD63, in agreement with the localization of CD63 to internal membranes of multivesicular bodies [40]. Dendritic cells, however, contain mainly multilaminar MHC class II compartments and probably only a minor part of the MHC class II molecules are secreted in exosomes. Recent data revealed an additional pathway of transport of MHC class II molecules to the cell surface, namely via tubules that emerge from MHC class II com- partment and fuse with the plasma membrane [24–26]. CD63 was found to be absent from these tubules and remained associated with MHC class II compartments [26], in agreement with our results. Although we did not observe tubular structures in our immuno-electronmicroscopy stud- ies, both the described modification of CD63 (this study) and the previously reported appearance of tubules occurs 12–20 h after maturation [26]. Whether there is a direct role for CD63 in the formation of tubules remains to be investigated. Maturation of dendritic cells resulted in an increase in the CD63 molecular mass by  20 kDa, that could be accoun- ted for by poly N-lactosaminoglycan addition. This modi- fication differs from the usual complex-type N-linked saccharides and is characterized by having side chains of Galb1–4GlcNAcb1–3 repeats. Poly N-lactosaminoglycans are present on several membrane proteins, including the lysosomal associated membrane proteins 1 and 2 (LAMP-1 Fig. 5. Subcellular localization of CD63 and LAMP molecules in immature and mature dendritic cells. Immature and mature dendritic cells were labeled metabolically with [ 35 S]methionine/cysteine prior to homogeni- zation and subcellular fractionation by organelle electrophoresis. After electropho- resis, fractions were pooled as indicated in A, lysed and CD63 or LAMP molecules immunoprecipitated and analyzed by SDS/PAGE. Shown are autoradiographs after fluorography (B). Ó FEBS 2003 CD63 glycosylation during dendritic cell maturation (Eur. J. Biochem. 270) 2417 and LAMP-2 [42,43]), but the physiological significance of this post-translational modification has remained unclear. In human dendritic cells, LAMP also acquired additional lactosaminoglycans upon dendritic cell maturation (this study), with similar kinetics as CD63, whereas cell-surface localized tetraspanins were not modified (not shown). It will be interesting to analyze the post-translational modifica- tions on the dendritic cell-specific lysosomal protein DC- LAMP during dendritic cell maturation, since this protein was found to colocalize with MHC class II in tubules in contrast to LAMP and CD63 [26]. One possible function of the extensive glycosylation of lysosomal membrane proteins is to protect the lysosomes as well as lysosome-like organelles from excessive degradative activities. The changes observed in MHC class II compart- ments from multilaminar organelles to vesicles with densely packed membranes might be accompanied with changes in the function of these organelles. In mature dendritic cells, macropinocytosis has ceased, but receptor-mediated endo- cytosis is still ongoing, although it is unclear if internalized antigens can reach degradative organelles. Interestingly, preliminary studies revealed reduction in the activity of the lysosomal enzyme b-hexosaminidase in MHC class II com- partments, in accordance with the previously reported disappearance of acidic organelles during dendritic cell maturation [56]. Neither the stability nor the subcellular localization of CD63 molecules was altered upon the addition of poly N-lactosaminoglycans during dendritic cell maturation. However, the extensive poly N-lactosaminoglycan addition on CD63 molecules during dendritic cell development, may be functionally important to maintain the endo–lysosomal system during dendritic cell development and accompanies the dramatic change in lysosomal morphology (see Fig. 6). Although post-translational modification could occur as a result from the morphological changes observed upon dendritic cell maturation, the addition of lactosamino- glycans on CD63 molecules could be involved in the modulation of class II peptide loading events, possibly through contributing to a differential distribution of associated MHC class II molecules during maturation of dendritic cells. Fig. 6. Distribution of CD63 and MHC class II molecules in immature and mature dendritic cells by immunocytochemistry. (A) Immature (a) and mature (b–d) dendritic cells were fixed and embedded as described in Materials and methods. Ultrathin cryosections were labeled with anti-CD63 and anti-MHC class II Igs, followed by 10 nm and 15 nm protein A-gold, respectively. (a–c) Detail of intracellular organelles, (d) overview. Bar, 50 nm. (B) Morphology of MHC class II-containing organelles in immature and mature dendritic cells analyzed by immunocytochemistry. Immature (a) and mature (b–d) dendritic cells were fixed and embedded and ultrathin cryosections were labeled with anti-MHC class II Igs, followed by 15 nm protein A-gold. Bar, 50 nm. 2418 A. Engering et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Acknowledgements We thank M. Cella and A. Lanzavecchia for discussion and critical review of the manuscript, S. Paniry for photography and H. L. Ploegh for antibodies. The Basel Institute for Immunology was founded and supported by F. Hoffmann-La Roche & Co., Ltd, Basel, Switzerland. This work was supported in part by the Swiss National Science Foundation. References 1. Lanzavecchia, A. & Sallusto, F. 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