The MHC class II ß chain cytoplasmic tail overcomes the invariant chain p35-encoded endoplasmic reticulum retention signal

Hayssam Khalil1, Alexandre Brunet1, Ingrid Saba1, Rafik Terra1, Rafick Pierre Sékaly2,4 and Jacques Thibodeau1

1 Laboratoire d’Immunologie Moléculaire and 2 Laboratoire d’Immunologie, Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, CP 6128 Succursale Centre-Ville, Montréal, Québec H3C 3J7, Canada 3 Department of Experimental Medicine, McGill School of Medicine, Montréal H3G 1A4, Canada 4 Laboratoire d’Immunologie, Hôpital Hôtel-Dieu, Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montreal H2W 1T8, Canada

Correspondence to: J. Thibodeau; E-mail: Jacques.Thibodeau{at}umontreal.ca
Transmitting editor: C. J. Paige


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The human-specific p35 isoform of the invariant chain (Ii) includes an R-X-R endoplasmic reticulum (ER) retention motif that is inactivated upon HLA-DR binding. Although the masking is assumed to involve the cytoplasmic tails of class II molecules, the mechanism underlying this function remains to be investigated. Moreover, in light of the polymorphic nature of the class II cytosolic tails, little is known about the capacity of various isotypes or alleles to overcome the retention signal of Iip35. To gain further insights into these issues, we first addressed the proposed role of the HLA-DR cytoplasmic tails. As shown by flow cytometry, the presence of Iip35 in transfected HeLa cells prevented surface expression of HLA-DR molecules lacking their cytoplasmic tails (DR{alpha}TM/ßTM). These truncated class II molecules and Iip35 accumulated in the ER, and co-localized with calnexin, as determined by confocal microscopy. Sensitivity of DR{alpha}TM/ßTM to endoglycosidase H treatment confirmed that these molecules do not reach the trans-Golgi network when associated with Iip35. Further characterization revealed that the ß chain cytosolic tail is critical for efficient ER egress of class II/Iip35 complexes. Interestingly, our results clearly demonstrate for the first time that DP and DQ isotypes can also overcome the retention motif of Iip35 through a mechanism involving their very distinctive polymorphic ß chain cytoplasmic tails. Altogether, these results further dissect the masking of di-basic retention signals, and emphasize the interplay between class II molecules and Ii for the transport of the complex to the endocytic pathway.

Keywords: antigen presentation, di-arginine, HLA, Iip35, R-X-R


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
MHC class II molecules are cell-surface heterodimeric glycoproteins which present antigenic peptides to CD4+ T cells. HLA-DR, -DP and -DQ represent co-dominantly expressed isotypes each composed of polymorphic {alpha} (~35 kDa) and ß (~27 kDa) transmembrane (TM) polypeptides (1). Following their synthesis, the {alpha} and ß subunits associate in the endoplasmic reticulum (ER) together with pre-formed trimers of the invariant chain (Ii) to produce ({alpha}ßIi)3 nonamers (24). Ii is a non-polymorphic type II protein that folds in part through the groove of the class II molecule, stabilizing the {alpha}ß heterodimer and preventing the undesirable binding of ER polypeptides (58). Another function of Ii is to direct MHC class II molecules to the endocytic antigen-loading compartments. Two short leucine-based signals located in the cytoplasmic tail of Ii are responsible for sorting the complex to the endocytic pathway: the membrane-distal sorting signal (Leu–Ile residues at positions 7 and 8) and the membrane-proximal signal (Met–Leu residues at positions 16 and 17). Either signal is sufficient for endosomal localization of Ii (9,10). Such leucine-based signals were first identified in the CD3 {gamma} chain and the cation-dependent mannose 6-phosphate receptor (11,12). When these endosomal localization signals are obliterated, Ii is found mostly at the cell surface and has a much longer half-life (1317).

Once within the acidic compartments, the Ii luminal domain is progressively degraded by various proteinases, and a residual class II-associated Ii peptide (CLIP) must be removed from the groove to allow the binding of an antigen and the export of the MHC molecule to the cell surface (1820). Removal of CLIP is catalyzed by the non-classical HLA-DM heterodimer (2123).

Ii is a membrane glycoprotein with two highly conserved N-linked glycosylation motifs (2426). However, Arunachalam et al. have shown that inhibition of Ii glycosylation with tunicamycin did not prevent calnexin association (27). The role of this post-transcriptional modification is not completely understood. Recently, a sequence C-terminal to those modified asparagines was shown to interact with the ribosome-associated membrane protein 4 which controls Ii glycosylation and may contribute to the efficiency of antigen processing (28).

Four isoforms (p33, p35, p43 and p45) of human Ii have been described (2931). Iip43 and p45 originate from translation of an alternatively spliced exon encoding a C-terminal domain responsible for the inhibition of cathepsins in endosomes (32). Iip35 and p45 differ from Iip33 and p43 respectively due to the use of a minor 5' in-phase start codon on the mRNA. As a consequence, Iip35 and p45 isoforms bear an N-terminal cytosolic extension of 16 amino acids containing a strong di-arginine ER retention motif (ERM) (14,33). Although the mechanism of retention is controversial, it would involve the retrograde transport of Iip35 from the Golgi to the ER (33,34). Several proteins encoding an R-X-R motif have been characterized. For example, the ATP-sensitive potassium channel (KATP) subunits Kir6.1, Kir6.2 and SUR1 contain R-X-R-like motifs that are sequentially masked during subunit oligomerization. This assures that only correctly assembled channels reach the plasma membrane (35). A similar R-X-R motif present in the GABAB receptor GB1 subunit is masked by assembly with GB2, ensuring expression of functional heterodimers (36). Also, surface expression of calcium channels is facilitated by ß subunits which mask ER retention domains in {alpha} subunits (37). Finally, an R-X-R-type motif is found in the C-terminal tail of the NMDA receptor subunit NR1 that regulates surface expression in heterologous cells and in neurons (38).

For nonameric Ii–class II complexes, efficient sorting to the endocytic pathway or the cell surface is dependent on the phosphorylation of p35-specific cytoplasmic serines as well as on the DR-induced ‘inactivation’ of the Iip35 ERM (3,3941). However, the efficacy of other class II isotypes in masking the di-arginine motif remains to be determined. As opposed to Iip35, the p33 isoform rapidly reaches the endocytic pathway and has a short half-life, even in the absence of class II molecules (4,13,42). Interestingly, mixed trimers consisting of p33 and at least one p35 molecule accumulate in the ER (2,4). Thus, Iip35 plays an important role in coordinating the assembly of newly synthesized Ii with class II molecules and in the transport of the complex to endosomal compartments. In this report, we have used wild-type, truncated and chimeric versions of MHC class II molecules to demonstrate that the strong di-arginine ER retention signal of Iip35 is inactivated by the cytosolic tail of MHC class II ß chains.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
cDNAs and mutagenesis
cDNAs encoding both the p33 and p35 isoforms of the human Ii (43), HLA-DR{alpha} (43), HLA-DRß 0101 (DRß008) (44) as well as DPw2 {alpha} and ß cDNAs cloned from 45.1 cells were a kind gift from Dr Eric O. Long (NIH). DP{alpha} (43) was cloned in the RSV.5 vector (45) and DPß (44) in the RSV.7 vector, a fusion between RSV.3 (45) and pHEBO (46). DQ{alpha}1 (0102) and DQß1 (0602) cDNAs cloned into the pLNCX retroviral vector were obtained from Dr William W. Kwok. All mutations were introduced by the PCR overlap extension method (47) and the sequence of the mutagenic oligonucleotides is available upon request. A mutation was introduced in the Ii cDNA in order to eliminate the second start codon (Met17 -> Ala) and translate exclusively the long p35 isoform. The mutated cDNA was cloned into the BamHI site of pBluescript KS+ vector (Stratagene, Cedar Creek, TX) and sequenced. The BamHI insert was subcloned into SR{alpha} (48) eukaryotic expression vector bearing the puromycin resistance gene (a kind gift from Dr F. Denis). Iip35 was also subcloned as a KpnI–NotI fragment into pREP4 (Invitrogen, San Diego, CA). Serines 6 and 8 on Ii were changed to lysine and histidine respectively (pRep4 Iip35 S6K,S8H). The HLA-DRß chain cDNA coding for a truncated protein (see Fig. 1A) was obtained after introducing a stop codon instead of the phenylalanine-coding triplet at position 221. This DRßTM cDNA was cloned into the RSV.3 vector. The cDNA coding for the truncated DR{alpha} chain (Fig. 1A) was described previously (49). The two leucine-based sorting signals of the p35 Ii were mutated to alanines and cloned into the SR{alpha} vector (SR{alpha}puro Iip35 LI/ML). The p33 isoform of Ii was generated by mutating the first start ATG triplet of Ii cDNA cloned into pREP4 (pREP Iip33).



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Fig. 1. Antigen-presentation-related molecules used in this study. (A) Schematic representation of the DR{alpha} and DRß chain combinations expressed in HeLa cells. (B) HeLa cells stably transfected with the wild-type or truncated DR molecules were sorted and analyzed by flow cytometry using L243. (C) N-terminal amino acid sequence and schematic representation of Iip35 and Iip33. The glycosylation sites are depicted next to the CLIP region. The two methionine residues corresponding to the two initiation sites are boxed. The arginine residues forming the ERM are underlined; the leucine-based targeting signals and the phosphorylated serines 6 and 8 are indicated.

 
The cytoplasmic tail of HLA-DRß was replaced by that of either HLA-DPß or -DQß. An intermediate DRß molecule (DRß RN:RS) had to be constructed to facilitate cloning. This mutation creates a XbaI restriction site near the junction between the TM and the cytosolic region. A first fragment was amplified from pBS KS 3'DRß008.14 using a DRß 3' BamHI:ClaI.D primer which harbors BamHI and ClaI restriction sites for latter cloning steps, and a mutagenic primer encoding the RN:RS mutation (DRßRN:RS.BglI.c). A second reaction was made on pBS KS 3'DRß008.14 using a complementary fusion primer (DRßRN:RS.BglI.b). Following the overlap reaction, the PCR product was subcloned into StyI and ClaI sites of pBS KS 3'DRß008.14 (pBS DRßRN:RS). The DNA sequence was confirmed by sequencing.

Prior to cloning the cytoplasmic tails of DPß and DQß, pBS DRßRN:RS was digested with BamHI and cloned in the opposite orientation. Overlapping oligonucleotides corresponding to the sequence of DP or DQ cytosolic tails were cloned in pBS DRßRN:RS.1 vector digested with BglII and XbaI. The oligonucleotides used were: DRß/DPßb (5'-CT AGA CTA TGC AGA TCC TCG TTG AAC TTT CTT A-3'), DRß/DPßc (5'-GA TCT AAG AAA GTT CAA CGA GGA TCT GCA TAG T-3'), DRß/DQßCyto-B401.b (5'-CT AGA CTA AT GCA GTA GAC CTT TCT GA-3') and DRß/DQßCyto-B401.c (5'-GA TCT CAG AAA GGT CTA CTG CAT TAG T-3'). The correct sequence was confirmed by sequencing. The chimeric cDNA was cloned as a XbaI–HincII fragment into the XbaI–SmaI site of SR{alpha}puro.

Cell lines and transfections
MHC class II epithelial HeLa (ATCC CCL-2) cells were cultured in DMEM (Wisent, St-Bruno, Quebec, Canada) containing 10% FBS (Wisent) and appropriate selective agents (see below). HeLa cells expressing the wild-type HLA-DR molecule (DR{alpha}+ DRß 0101) (HeLa DR) or the wild-type DRß chain together with a truncated DR{alpha} chain devoid of most of its cytoplasmic tail (see Fig. 1) (HeLa DR{alpha}TM/DRß) have been described elsewhere (49). Stable transfection of the mutated class II molecules was performed by the calcium phosphate co-precipitation technique as described (50). Cells resistant to G-418 (Life Technologies) and expressing class II {alpha}ß heterodimers were sorted by flow cytometry using the L243 mAb. The Ii cDNAs were transfected using Fugene6 (Roche, Mississauga, Canada) and selected in 400 µg/ml puromycin (Sigma, Oakville, Canada) or 50 U/ml hygromycin (Cederlane, Hornby, Canada).

HEK 293T cells were kindly provided by Dr Eric Cohen. For transient expression, cells were co-transfected by the calcium phosphate precipitation method using 2 µg of each DNA (50). Cells were analyzed 2–3 days post-transfection.

Antibodies
L243 (IgG2a) is an anti-DR mAb which recognizes a conformational epitope on the DR{alpha} chain (51). XD5.117 (IgG1) recognizes a linear epitope in the ß1 domain of all class II molecules (52). ISCR3, a kind gift from Dr Robert Busch, recognizes the {alpha}ß heterodimer through an epitope on the DR{alpha} chain (53). R.Ip35N, a kind gift from Dr Peter Cresswell, is a rabbit antisera specific for a synthetic peptide corresponding to amino acids 1–16 of the N-terminal extension present on the p35 isoform of Ii (4). BU45 (IgG1) is a mouse mAb specific for a C-terminal epitope of human Ii (The Binding Site, Birmingham, UK). Pin.1 mAb (3) recognizes the N-terminal cytoplasmic domain of Iip33 and p35. mAb CerCLIP.1 (IgG1) is directed against the N-terminal segment of CLIP (PharMingen, San Diego, CA). The polyclonal rabbit antibody against calnexin recognizes the C-terminal part of the protein (Stressgen, Victoria, Canada). Goat anti-mouse and goat anti-rabbit IgG coupled to Alexa Fluor or phycoerythrin (PE) were obtained from Molecular Probes (Eugene, OR).

Flow cytometry and fluorescence microscopy
Intracellular staining for class II molecules or Ii was performed on saponin-permeabilized cells as previously described (50). For double stainings, cells were first stained at the surface using biotinylated L243 and avidin–PE or in some cases with ISCR3 and an IgG2b-specific antibody coupled to PE (Molecular Probes). Then, cells were washed, fixed in 4% paraformaldehyde, permeabilized with saponin (50), and stained for Ii using BU45 and an IgG1-specific secondary antibody (PharMingen) coupled to FITC. Cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Mississauga, Canada). For confocal microscopy analyses, HeLa cells were grown on coverslips, fixed in 4% paraformaldehyde and permeabilized with saponin (50) before staining. HEK 293T cells were first fixed in 4% paraformaldehyde and permeabilized with saponin (50) before staining with Pin.1 mAb followed by a goat anti-mouse IgG coupled to Alexa Fluor 488. HEK 293T cells were centrifuged onto microscope slides and examined with a Leica TCS-SP1 confocal microscope using a x100 planapochromat objective.

Western blotting and immunoprecipitations
Cells were trypsinized, washed with cold PBS and sonicated in 400 µl lysis buffer (20 mM Tris, pH 7.5 and 150 mM NaCl) containing protease inhibitors (Roche). Following sonication, 100 µl of 5% Triton X-100 was added to the supernatant and incubated at 4°C for 30 min. After centrifugation, supernatants were harvested and incubated for 2 h with goat anti-mouse IgG-coated magnetic beads (BioMag; Polysciences, Warrington, PA) coupled to the appropriate antibody. Following washes in lysis buffer, samples were resuspended in reducing buffer, boiled and subjected to SDS–PAGE. After transfer to nylon membranes (Amersham Biosciences, Quebec, Canada), proteins were blotted with the appropriate antibody. Secondary antibody (peroxidase-coupled goat anti-mouse; BIO/CAN Scientific, Mississauga, Canada) was used at a 1:1000 dilution for 2 h and the signal was detected using ECL (Amersham Biosciences). For endoglycosidase H (Endo H; New England Biolabs, Beverly, MA) and acetyl-neuraminyl hydrolase (neuraminidase; New England Biolabs) treatment, immunoprecipitated material or whole-cell lysates were digested according to the manufacturer’s protocols.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Iip35 prevents surface expression of truncated HLA-DR molecules
Previous reports have suggested that the association of class II molecules with Iip35 is needed to conceal the ER retention motif on Ii (4,54). To gain insights into the mechanism and molecular determinants involved in this phenomenon, we used transfected HeLa cells expressing HLA-DR molecules devoid of their {alpha}ß cytoplasmic domains (Fig. 1A). HeLa cells do not normally express Ii or class II molecules and have been extensively used to study their trafficking upon transfection (9,10,14,39). First, a stop codon was introduced at the interface between the TM helix and the cytoplasmic tail coding regions. In general, aromatic amino acids are preferentially located next to TM regions, with tyrosine being restricted to the carboxyl boundary (55). Tyrosine and tryptophan residues may serve to position the TM helix with respect to the lipid bilayer. These residues have been proposed to behave as ‘floats’ with their polar atoms facing water. On the other hand, positively charged residues such as arginine and lysine play a major role in determining the orientation of the TM region as predicted by the ‘positive-inside rule’ (56,57). Based on these studies, we inserted a stop codon after the Y220 residue in HLA-DRß. As for the DR{alpha} chain, which lacks a similar aromatic residue, the positively charged lysine residue 215 was kept to ensure the stabilization of the TM helix. In the absence of Ii, truncation of the {alpha} and/or ß cytosolic tails did not hinder cell-surface expression of class II molecules. Figure 1(B) shows the expression of various {alpha}/ß combinations at the surface of stably transfected HeLa cells sorted with L243.

Cells stably expressing high levels of control wild-type DR{alpha}/DRß molecules (DR) were super-transfected with the Iip35 cDNA (Fig. 1C) and placed under selection for 2–3 weeks. According to previous reports, transfection of Iip35 in these cells should not preclude class II surface expression due to the efficient masking of the ERM (4). This was confirmed by flow cytometry on cells first stained at the surface for HLA-DR, then permeabilized and stained for Ii. Intracellular staining is required to identify Iip35+ cells since this isoform does not accumulate at the cell surface (58). As expected, control wild-type DR molecules efficiently overcome the ERM of Iip35 (3,40) and no decrease in cell-surface expression of class II molecules was observed in Ii+ cells when compared to the negative cell population (Fig. 2A). Similar results were obtained using the control Iip33 isoform devoid of the strong ERM. It is important to note that these flow cytometry analyses are internally controlled as all stably transfected populations always include a proportion of cells becoming resistant to the selective agent, but which are negative for Ii expression. This allows the direct, simultaneous and unbiased comparison of class II surface expression between the Ii+ and Ii subpopulations. Thus, in our flow cytometry analyses, any Iip35-induced intracellular retention of class II molecules unable to mask the ERM would result in a specific decrease of surface HLA-DR staining for cells in the Ii+ quadrants as compared to those in the Ii quadrants.



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Fig. 2. Iip35 prevents the surface expression of class II molecules devoid of their cytoplasmic tails. (A) Cells expressing homogenous levels of class II molecules were stably transfected with either Iip33, Iip35 or Iip35 (S6K,S8H) cDNAs cloned in pREP4. Expression of surface class II molecules and total Ii was monitored by flow cytometry. Cells were stained at the surface with the conformational antibody L243, and then permeabilized with saponin and stained for Ii using BU45. Similar results were obtained in independent transfections using Iip35 cloned in SR{alpha}puro (data not shown). (B) HeLa DR{alpha}TM/ßTM transfected with Iip35 were stained as in (A) using either L243 (upper panel) or ISCR3 (lower panel). (C) HeLa DR{alpha}TM/ßTM cells expressing Iip35 were stained with ISCR3 (bold lines) for cell-surface (top histogram) or total (bottom histogram) class II molecules after permeabilization. The ISCR3 antibody was used for this experiment instead of L243 because the latter mAb would not recognize immature class II molecules bound to Ii in the ER (60). Control cells were stained only with the goat anti-mouse secondary antibody coupled to Alexa Fluor 488 (thin line). (D) Cell-surface staining using the CerCLIP.1 antibody (bold line). Control cells were stained only with the secondary antibody (thin line).

 
The utility of this system for the detection of Iip35-induced retention of class II molecules was tested using the control Iip35 S6K,S8H molecule having the two N-terminal phosphorylated serine residues substituted for positively charged amino acids (Fig. 2A). Although the precise effect of these mutations remains to be established, it is clear that these serine residues are critical for the Ii delivery to the endocytic pathway (34,39,40). Indeed, a sharp decrease in class II expression was observed at the surface of Ii+ cells (Fig. 2A). This intracellular retention by the S6K,S8H mutant confirms the DR/Iip35 interaction and validates our experimental system for monitoring the masking of the ERM.

We then assessed the role of the HLA-DR cytoplasmic tails on the ER egress of Iip35. HeLa cells expressing both truncated HLA-DR {alpha} and ß chains (DR{alpha}TM/ßTM, Fig. 1A and B) were stably transfected with Iip35. Cell-surface expression of class II molecules was monitored by flow cytometry as above. Figure 2(A) demonstrates that those cells expressing Iip35 have less surface class II molecules as compared to Ii cells and that the decrease appears proportional to the level of Ii expression. However, this reduction in class II surface expression was not observed when co-expressed with Iip33 (Fig. 2A). The Iip35-induced down-modulation of truncated HLA-DR expression was confirmed using different class II-specific antibodies (Fig. 2B and data not shown). Importantly, we ruled out the trivial possibility that Ii-expressing cells selectively lost one or both of the {alpha}TM/ßTM chains, precluding cell-surface expression. Figure 2(C) shows that despite heterogeneous staining for surface class II molecules, the Ii-transfected total population of DR{alpha}TM/ßTM cells expresses homogeneous levels of total class II molecules.

Due to the lack of HLA-DM in these transfectants, CLIP is not removed from the class II molecules. Therefore, the presence of CLIP at the cell surface reflects the efficient DR/Ii interaction as well as ER egress and targeting of the complex to endosomes where Ii is degraded. As shown by flow cytometry using the CerCLIP.1 mAb, only the full-length class II molecules are bound with CLIP at the cell surface (Fig. 2D). Moreover, no CerCLIP.1 staining was observed in permeabilized cells expressing Iip35 and the truncated HLA-DR mutant (data not shown). Altogether, these results show that DR{alpha}TM/ßTM are prevented from reaching the cell surface in the presence of Iip35, suggesting that the complex is retained in the ER.

DR{alpha}TM/ßTM accumulates in the ER in the presence of Iip35
To further characterize the mechanism responsible for the reduced surface expression of DR{alpha}TM/ßTM in Iip35+ cells, the subcellular localization of DR and DR{alpha}TM/ßTM molecules was compared by confocal microscopy. In HeLa DR cells expressing Iip35 or p33, the intracellular class II staining was characterized by the presence of well-defined vesicles reminiscent of endosomal /lysosomal compartments (Fig. 3a and b). Many of these vesicles were also found positive for Ii expression, but negative for calnexin, an ER resident chaperone (Fig. 3m' and n'). On the other hand, no such small vesicle containing class II and/or Ii was seen in cells expressing DR{alpha}TM/ßTM and Iip35 (Fig. 3c and g) Moreover, the distribution of these molecules overlapped the one of calnexin (Fig. 3k, o and o'). Control DR{alpha}TM/ßTM cells expressing Iip33 showed large endocytic DR+, Ii+ vesicles devoid of calnexin (Fig. 3p'). These results demonstrate that the Iip35 ERM is active and retains in the ER those tailless class II molecules.



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Fig. 3. Intracellular localization of class II molecules in the presence of Iip35. Cells were grown on coverslips for 24–48 h before permeabilization and simultaneous staining for class II (ISCR3) (a–d), Ii (Pin.1) (c–h) and calnexin (i–l). The secondary antibody used were goat anti-mouse IgG2b coupled to PE, goat anti-mouse IgG1 coupled to Alexa Fluor 488 and goat anti-rabbit coupled to Alexa Fluor 633 respectively. Cells were analyzed by confocal microscopy. Shown in (m)–(p) are overlays of class II, Ii and calnexin staining for the representative cells. White boxes indicate the areas shown at higher magnification in (m') –(p'). Co-localization of class II and Ii is seen in yellow, whereas co-localization of class II, Ii and calnexin is seen in white. The arrows indicate vesicles containing class II and Ii, but devoid of calnexin.

 
Iip35 prevents the sialylation of DR{alpha}TM/ßTM
To further confirm that the truncated class II molecules are retained in the ER when associated with Iip35, we assessed the sensitivity of their glycans to Endo H and neuraminidase. Endo H removes high mannose, but not complex forms, of N-linked glycans that are acquired following passage through the medial- and trans-Golgi compartments. Sensitivity to neuraminidase, which catalyses the hydrolysis of N-acetyl-neuraminic acid (sialic acid), reflects an encounter with sialyl-transferase in the trans-Golgi compartment.

Figure 4(A) shows that in the presence of Iip33 or Iip35, wild-type class II molecules acquire Endo H-resistant carbohydrates (black arrow). This maturation is reminiscent of a passage beyond the cis-Golgi compartment and therefore reflects an efficient egress from the ER. The Endo H-sensitive DRß chains (arrow head) most likely represent those molecules in the ER that have yet to associate with an {alpha} chain or which are part of newly associated heterodimers. On the other hand, truncated class II molecules acquired Endo H-resistant carbohydrates when expressed with Iip33, but not with Iip35 (Fig. 4B). The sensitivity to neuraminidase of these p33-associated DRßTM molecules suggests that they have acquired sialic acid. Surprisingly, we could not detect any Endo H-resistant Ii molecules, even in cells expressing wild-type HLA-DR (Fig. 4C). This was true also for Iip35 molecules co-immunoprecipitated with class II molecules using a DR-specific mAb (data not shown). These results suggest that the maturation of Ii-bound sugars cannot be used as a read-out for monitoring ER egress in this system (see below). Altogether, these experiments provide biochemical evidence that truncated class II complexes are retained in the ER in the presence of Iip35.



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Fig. 4. Maturation of class II molecules and Ii in HeLa cells. Cells expressing Ii and surface class II molecules were sorted positively using CerCLIP.1 in order to obtain homogenous expression of Ii. Cells expressing DR{alpha}TM/ßTM together with Iip35 were sorted negatively using L243. (A) HeLa cells were lysed in 1% Triton X-100 and either mock digested or digested with Endo H before SDS–PAGE and Western blot analysis. Proteins were immunoblotted with a DRß-specific mAb (XD5.117). (B) Cells were lysed, treated with Endo H or with neuraminidase and analyzed by Western blotting as above. (C) Cells were lysed, treated with Endo H and analyzed by Western blotting using the Ii-specific mAb Pin.1 Black arrows depict proteins containing Endo H-resistant sialic acid. A filled arrowhead indicates molecules that have acquired high mannose or complex type oligosaccharides and an open arrow indicates Endo H-sensitive species.

 
DR{alpha}TM/ßTM prevents the cell-surface expression of Iip35 LI/ML
In order to unequivocally demonstrate that Iip35 does not access compartments beyond the Golgi or ER, we have generated a mutant molecule devoid of its two leucine-based sorting signals (Fig. 1C). This modification was shown previously to direct Ii to the plasma membrane rather than to the endocytic pathway (9,10). Thus, in our system, an efficient masking of the Iip35 ERM should lead to the cell-surface expression of Ii. First, HeLa cells stably expressing DR or DR{alpha}TM/ßTM were transfected with the mutant Iip35 LI/ML and analyzed by flow cytometry as above. The level of DR{alpha}TM/ßTM at the cell surface sharply decreases upon co-expression of Iip35 LI/ML, whereas wild-type class II molecules are not affected (Fig. 5A). This result confirms the capacity of Iip35 to prevent expression of truncated class II molecules. Then, we stained transfected cells for the surface expression of Iip35 LI/ML molecules. As expected, when expressed in the absence of class II molecules, no cell-surface accumulation of this Ii mutant can be detected (Fig. 5B). However, when Iip35 LI/ML was expressed in HeLa DR cells, a significant level of Ii was detected at the cell surface. On the other hand, truncated DR{alpha}TM/ßTM did not support cell-surface expression of Iip35 LI/ML, corroborating our previous observations that the cytoplasmic tails of HLA-DR are required to overcome the Iip35 ERM (Fig. 5B). Accordingly, in these cells, the DRßTM chain did not acquire Endo H-resistant sialylated sugars (Fig. 5C, upper panel).



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Fig. 5. Iip35 LI/ML is not expressed at the cell surface in the presence of DR{alpha}TM/ßTM. (A) HeLa cells expressing homogenous levels of class II molecules were stably transfected with the SR{alpha}puro Iip35 LI/ML cDNA. Expression of surface class II molecules and Ii was monitored as in Fig. 2. (B) HeLa cells were stably transfected with the indicated molecules and analyzed by flow cytometry using BU45 either before (upper panel) or after permeabilization with saponin (lower panel). (C) Cells were sorted positively (DR1) using CerClip.1 or negatively (DR{alpha}TM/ßTM) using L243 to obtain a homogenous expression of Ii (data not shown). After lysis in 1% Triton X-100, the DRß chain was analyzed by Western blotting as in Fig. 4 using mAb XD5.117 or the Iip35-specific rabbit antiserum (R.Ip35N) which recognizes the LI/ML Ii mutant. The R.Ip35N antibody was used for this experiment instead of Pin.1 because the latter mAb would not recognize the LI/ML Ii mutant. (D) HeLa cells were stably transfected with pREP4 Iip33 or a mock pREP4 vector. Cell lysates were treated or not with Endo H and analyzed by Western blotting using Pin.1. (E) 293T cells were stably transfected with the indicated molecules and analyzed by flow cytometry using BU45 either before (upper panel) or after permeabilization with saponin (lower panel).

 
Our biochemical analysis presented above failed to detect Endo H-resistant sugars on Iip35 in DR+ cells (Fig. 4C). One possibility to explain this result is that Iip35 is degraded rapidly after leaving the Golgi and gaining access to the endosomes. We took advantage of the fact that Iip35 LI/ML accumulates at the cell surface of DR+ cells to further characterize the nature of its glycans. Nonetheless, we could not detect Endo H-resistant forms of Ii in DR+ cells (Fig. 5C, lower panel), suggesting that upon association with class II molecules in the ER, Ii glycans become inaccessible and are no longer processed to complex forms in the Golgi. The same was observed for the p33 isoform in class II+ cells (data not shown). A control experiment revealed that this lack of sugar maturation on Ii is caused by class II molecules and is not inherent to HeLa cells. Indeed, we could clearly show the acquisition of complex type carbohydrates and even sialic acid moieties on Iip33 which egress the ER in class II cells (Fig. 5D). These observations are in agreement with previous reports showing that the N-linked oligosaccharides of the {alpha} and ß chains were processed to the complex form, while those on the associated Ii remained predominantly Endo-H sensitive (59). Furthermore, Schaiff et al. have shown that only one of the two N-linked carbohydrates on HLA-DR{alpha} is processed to complex-type oligosaccharides in the presence of Ii, whereas both are processed in Ii transfectants (60). Taken together these results demonstrate the reciprocal impact of MHC class II molecules and Ii, especially p35, on their maturation.

HLA-DRß cytosolic tail is responsible for masking the Iip35 ERM
In order to determine which cytoplasmic tail of the HLA-DR heterodimer is involved in the ER egress of Iip35, mixed pairs containing either a truncated {alpha} or ß chain were expressed in HeLa cells (see Fig. 1A and B). Cell-surface expression of these truncated molecules in HeLa cells confirmed their proper folding and membrane anchoring (Fig. 1B). As shown in Fig. 6(A), the expression profile of DR{alpha}TM/ß in the presence of Iip35 was similar to the one observed for Ii cells. On the other hand, we observed a markedly decreased cell-surface expression of DR{alpha}/ßTM in Ii+ cells. These results suggest that the DRß chain is required to overcome the Ii ERM.



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Fig. 6. The DRß chain cytoplasmic tail is required to overcome the Iip35 ER retention motif. (A) HeLa cells expressing truncated class II molecules were stably transfected with pREP4 Iip35 and double stained as in Fig. 2(A). Independent transfections using SR{alpha}puro Iip35 gave similar results (data not shown). (B) Unsorted populations shown in (A) were used. The Ii was immunoprecipitated from cell lysates using BU45. The samples were treated or not with Endo H and analyzed by Western blotting using the HLA-DRß-specific mAb (XD5.117). Black arrows depict proteins containing Endo H-resistant sialic acid. A filled arrowhead indicates molecules that have acquired high mannose or complex type oligosaccharides and an open arrow indicates Endo H-sensitive species. (C) Cells were grown on coverslips for 24–48 h before permeabilization and simultaneous staining for class II (ISCR3) (a–d), Ii (Pin.1) (c–h) and calnexin (i–l). The secondary antibodies used were as in Fig. 3. Cells were analyzed by confocal microscopy. Shown in (m)–(p) are overlays of class II, Ii and calnexin staining for the representative cells. White boxes indicates the areas shown at higher magnification in (m')–(p'). Co-localization of class II and Ii is seen in yellow, whereas co-localization of class II, Ii and calnexin is seen in white. The arrows point to vesicles containing class II and Ii but devoid of calnexin.

 
The critical role of the ß chain in masking the Ii ERM was validated by looking at the maturation of class II and Iip35 glycans. Figure 6(B) shows that a significant proportion of the Ii-associated DR{alpha}TM/ß class II molecules became Endo H-resistant. Coupled to the near complete absence of complex sugars on DR{alpha}/ßTM molecules, these results further support the fact that the ß chain cytoplasmic tail masks the Iip35 ERM.

The intracellular localization of the truncated molecules was analyzed by confocal microscopy. In DR{alpha}TM/ß cells, class II molecules co-localized with Iip35 or Iip33 in small endosomal/lysosomal vesicles (Fig. 6C). On the other hand, when the ß chain cytoplasmic tail is truncated, DR{alpha}/ßTM and Iip35 molecules co-localized with the ER marker calnexin and no punctuated, scattered vesicles could be observed (Fig. 6C, o'). This pattern contrasts with the vesicular co-localization observed between the ß-truncated class II molecule and control Iip33 (Fig. 6C, p').

Finally, to decisively demonstrate that only the DRß cytoplasmic tail can mask the R-X-R motif, we tested the ability of truncated {alpha}ß combinations to support cell-surface expression of Iip35 LI/ML. DR and Iip35 molecules were co-expressed stably in HeLa cells or transiently in HEK 293T cells. As predicted from the above-described experiments using Iip35, we observed the surface expression of Iip35 LI/ML on cells transfected with DR or DR{alpha}TM/ß but not on those expressing DR{alpha}/ßTM (Fig. 7). These results conclusively pinpoint the DRß cytosolic tail as the key element needed to overcome the Iip35 ERM.



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Fig. 7. Iip35 LI/ML is not expressed at the cell surface in the presence of DR{alpha}/ßTM. HeLa cells expressing class II molecules were stably transfected with pREP4 Iip35 LI/ML. HEK 293T cells were transiently co-transfected with the indicated DR molecules and with pREP4 Iip35 LI/ML, and tested after 48 h. Cells were analyzed by flow cytometry using BU45 either before (surface Ii) or after permeabilization with saponin (total Ii). The surface:total Ii ratios are shown. The XD5.117 mean fluorescent values (MFV) for DR, DR {alpha}TM/ß, DR {alpha}/ßTM expression were 411, 331, 109 in HeLa cells, and 298, 229, 290 in HEK 293T cells respectively. The MFV for the secondary antibody alone was 4 (data not shown).

 
All class II isotypes mask the Iip35 di-arginine motif
In humans, the family of classical MHC class II molecules comprises three isotypes called HLA-DR, -DP and -DQ. Overall, the crystal structures of the extracellular domains of DR and DQ are very much alike (61), and DP is likely to adopt a similar quaternary structure (62). Isotypic polymorphisms are found in all regions of the proteins including the cytoplasmic portions of both the {alpha} and ß chains (63). Interestingly, allelic polymorphisms in DQß allotypes have been described, and some arise from the differential splicing of exon 5 in DQB1*06011 and DQB1*05031. Thus, in addition to variations in their own primary structure, most DQ molecules are 8 and 2 amino acids shorter than DR and DP respectively. This significant polymorphism prompted us to test the capacity of each isotype to mask the R-X-R motif of Iip35.

Having demonstrated the utility of screening for cell-surface expression of Iip35 LI/ML (Figs 5 and 7), we turned to transient transfections to rapidly assess the capacity of any class II molecule or mutant to mask the R-X-R motif. Moreover, we used human HEK 293T cells to circumvent our repeated inability to generate stable HeLa cells expressing HLA-DQ. All three isotypes, in addition to the DR{alpha}/ßTM molecule, were efficiently expressed after 48 h (see legend, Fig. 8A). As described above, class II molecules lacking the ß chain cytoplasmic tail could not support the maturation and sorting of Iip35 LI/ML. Indeed, the ratio of surface over total Ii was low for cells expressing such class II molecules. On the other hand, the ratio was ~10 times higher for cells expressing DR and DP molecules, showing that both isotypes mask the Iip35 R-X-R motif. Surprisingly, cells expressing HLA-DQ were consistently found to be nearly negative for Iip35 LI/ML surface expression. However, the possibility remained that the di-leucine motif found in the cytoplasmic tail of DQ [(64) and see below] could compensate for the mutation of the two sorting signals of Ii, thus preventing accumulation of the complex at the cell surface. To get around this problem, we repeated the experiment with Iip35 and monitored, by fluorescence microscopy using Pin.1, the presence of Ii in the endocytic pathway. As a matter of fact, the results clearly showed the presence of Ii+ vesicles in cells expressing the DQ molecules, suggesting that this isotype can efficiently associate with Ii and mask its R-X-R motif (Fig. 8B). In cells devoid of a ß chain cytoplasmic tail, Iip35 staining did not produce vesicles, but rather a diffuse pattern reminiscent of the ER (see Fig. 6). We reasoned that the presence of Ii in the endocytic pathway should result in its processing and the expression of class II/CLIP complexes at the cell surface. To test this hypothesis, the cells were stained using the CerCLIP.1 antibody that recognizes DR/CLIP as well as DQ/CLIP complexes (Fig. 8C). The strong expression of DQ/CLIP complexes at the cell surface confirmed that this isotype can overcome the R-X-R motif of Ii and allow its egress from the ER.



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Fig. 8. HLA-DP and -DQ mask the Iip35 R-X-R motif. (A) HEK 293T cells were transfected with either DR, DP, DQ or DR {alpha}/ßTM together with pREP4 Iip35 LI/ML and analyzed after 48 h by flow cytometry using BU45 either before (surface Ii) or after permeabilization with saponin (total Ii). The surface/total Ii ratios are shown. The XD5.117 MFV for DR, DP, DQ and DR {alpha}/ßTM expression were 298, 257, 88 and 229, respectively. The MFV for mock-transfected cells was 5 (data not shown). (B) HEK 293T cells transfected with either a mock vector or the DR, DQ and DR {alpha}/ßTM cDNAs together with pBUD Iip35 were permeabilized with saponin after 72 h and stained for the Ii using Pin.1. Cells were centrifuged onto microscope slides and analyzed by confocal microscopy. (C) These transfected HEK 293T cells were also analyzed by flow cytometry for surface class II (XD5.117) and CLIP (CerCLIP.1) expression. Cells were also permeabilized with saponin and stained with BU45 to monitor total Ii content. Filled histograms represent the background obtained using the secondary antibody alone.

 
The possibility remains that the DP and DQ ß chain polymorphisms preclude masking, but that the {alpha} chain compensates in these isotypes. To decisively demonstrate the activity of these short cytosolic tails, we used chimeric DR molecules in which the ß chain cytoplasmic region was replaced by the one of DP or DQ. These chains were expressed transiently in HEK 293T cells together with the truncated DR{alpha} chain (DR{alpha}TM/ßcytoDP and DR{alpha}TM/ßcytoDQ) and Iip35 (Fig. 9A). Positive cell-surface staining for CLIP expression suggested that all three cytoplasmic tails could efficiently mask the Ii-encoded retention motif (Fig. 9B). Again, as judged by the presence of Ii in distinct vesicles spread out in these cells (Fig. 9B), we assume that the CLIP staining detected by flow cytometry originates from the processing of Iip35 by acid proteases and the subsequent export of class II/CLIP complexes to the cell surface. Similar results were obtained for stably transfected HeLa cells and no sign of class II ER retention was detected in bi-parametric analyses (data not shown). Interestingly, in HeLa cells, sorting of the DR{alpha}TM/ßcytoDQ molecules to endosomes from the Golgi seems less stringent than in HEK 293T cells (Fig. 8A) and we were able to detect expression of the Iip35 LI-ML molecule at the cell surface (Fig. 9C). Although we cannot formally rule out a small contribution of the {alpha} chain of DP or DQ in the context of the wild-type heterodimers, our results strongly support a role for the ß chain of all class II isotypes in masking the Iip35 ERM.



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Fig. 9. The DPß and DQß chain cytoplasmic tails are sufficient to overcome Iip35 ER retention motif. (A) Schematic representation of the chimeric class II {alpha} and ß chain combinations expressed in HeLa cells. The C-terminal amino acid sequence of the recombinant DRß chains is shown. (B) HEK 293T cells transfected with pBUD Iip35 and either a mock vector or a combination of chimeric chains, as indicated. Cells were analyzed by flow cytometry for either surface class II (XD5.117) or CLIP (CerCLIP.1). A fraction of the transfected cells was also permeabilized with saponin and stained with BU45. Total Ii expression was monitored by flow cytometry and the subcellular localization of Ii was analyzed by confocal microscopy. (C) HeLa cells stably expressing combinations of chimeric DR chains were transfected with pREP4 Iip35 LI/ML and analyzed by flow cytometry for the surface expression of class II molecules (XD5.117) or Ii (BU45). Cells were also permeabilized with saponin and stained for Ii using BU45 (total Iip35 LI/ML). Filled histograms represent the background obtained using the secondary antibody alone.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ii plays a critical role in maintaining the specificity of class II molecules for antigens of the endocytic pathway (65,66). It prevents peptides from binding to class II molecules in the early stages of biosynthesis (42,54), and directs the complex to a post-Golgi intracellular compartment containing internalized antigens and proteases (67). However, the exact function of the ERM found in Iip35 and p45 remains enigmatic. In most antigen-presenting cells, Ii is present in excess over class II {alpha} and ß chains, probably to efficiently inhibit peptide uptake in the ER. In various class II-expressing cell types, the Iip35 isoform accounts for 20% of the total Ii pool and an estimated 48% of all Ii exists as heterotrimers containing at least one Iip35 molecule (2,68). This could maximize the chance of an encounter between Ii and class II molecules, and/or prevent the sorting of an Ii overload capable of fusing endosomal compartments (6973). Recently, down-regulation of Iip35 expression in diabetic B cell lines was proposed to impair antigen processing and presentation (74). On the other hand, the impact of the Iip35 overexpression on antigen presentation in tumors such as hairy cell leukemia (7577) and B-CLL (78,79) remains to be determined.

Individual subunits of multimeric protein complexes are often retained in the ER by arginine- or lysine-based signals (33,80). These retention motifs could be part of quality control mechanisms preventing cell-surface expression of partially assembled or misfolded complexes. For example, the R-X-R motif would coordinate the assembly and proper stoichiometry of mature KATP channels, GABAB receptors or NMDA receptors (35,36,81). By analogy, proper assembly of the class II/Ii nonameric protein complex would result in the shielding of the Iip35 retention motif, allowing ER egress. Interestingly, as opposed to its human p33 counterpart, the murine Ii contains an R-X-R sequence in its cytoplasmic tail. Although not as stringent as the di-arginine motif found in the human Iip35, an ER retention mechanism might also operate on the murine Iip33. Characterization of this motif will be required before establishing any functional correlation with the human Iip35 ERM.

Our results clearly identified the ß chain as the critical class II determinant allowing egress of Iip35. However, the mechanism by which this cytosolic tail overcomes the Ii ERM remains to be determined. Di-basic signals were found to bind COP I proteins which mediate retrieval from the Golgi (82,83). Very recently, the coupling of such signals to a ‘release’ sequence has been proposed to explain the masking and ER egress (34). This release motif encompasses the phosphorylated serine at position 8 and binds the 14-3-3ß adaptor protein. Although the binding of COP I and 14-3-3 is mutually exclusive, interaction with the latter is certainly not sufficient to allow phosphorylated Iip35 molecules to leave the ER (39). Thus, we can speculate that unphosphorylated Iip35 bound to class II molecules are retained in the ER because of a COP I-dependent retrograde transport from the cis-Golgi. On the other hand, in the absence of class II molecules, PKC-phosphorylated Iip35 would bind 14-3-3 and be retained by a still unidentified chaperone. The ß chain cytoplasmic tail could allow ER egress of Ii by displacing this chaperone.

We are presently investigating if a specific molecular determinant embedded in the cytoplasmic tail of the ß chain is needed to prevail over the Ii ERM. Although a critical motif in the ß chain could assist the folding of the Ii cytoplasmic tail and release of a retention chaperone, the poor conservation between DR, DP and DQ isotypes in this region suggests otherwise (63). Such variability would rather be compatible with a steric hindrance mechanism of masking, similar to the one described for the assembled Fc{epsilon} receptor (84). The fact that the short (10 amino acids) cytosolic tail of DQ is capable of hiding the retention motif suggests that the N-terminal region of Ii folds in such a manner to keep the R-X-R motif close to the membrane. Such folding could be in line with reported quaternary structure constraints leading to the formation of a co-planar triple-stranded {alpha}-helical bundle of Ii cytosolic tails (85). Site-directed mutagenesis in the ß chain and Ii cytoplasmic domains will shed some light on the mechanism by which the complex egress the ER. Finally, it was recently shown that Ii associates with CD1d and regulates its trafficking to endosomal compartments (86). It will be interesting to test the ability of this molecule to egress the ER when bound to the p35 isoform.


    Acknowledgements
 
We would like to thank Dr Eric O. Long, Dr William W. Kwok and Dr Peter Cresswell for providing cDNAs, antibodies and hybridomas. We thank Dr Robert Nabi for the use of the confocal microscopy facility. We thank Nathalie Simard for helping in the construction of the Iip35 LI/ML cDNA. H. K. and A. B. are supported by studentships from the Fonds pour la Formation de Chercheurs et l’aide à la recherche. R. P. S holds a scientific award from the Canadian Institute of Health Research (CIHR). J. T. is the recipient of a CIHR scholarship. This work was supported by grants from Boehringer Ingelheim Canada to J. T. and Valorisation Recherche Québec to R. P. S. and J. T.


    Abbreviations
 
CLIP—class II-associated Ii peptide

Endo H—endoglycosidase H

ER—endoplasmic reticulum

ERM—endoplasmic reticulum retention motif

Ii—invariant chain

MFV—mean fluorescent value

PE—phycoerythrin

TM—transmembrane


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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