Mutations in specific I-Ak {alpha}2 and ß2 domain residues affect surface expression

Mark L. Lang, Shyam Yadati, E. Scott Seeley, Thom Nydam, Terri K. Wade, Jerome L. Gabriel1, Grant Yeaman, B. George Barisas2 and William F. Wade

Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756, USA
1 Department of Biochemistry, Temple University, Philadelphia, PA 19140, USA
2 Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA

Correspondence to: W. F. Wade


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
A previous investigation demonstrated that several mutations in class II dimer-of-dimers contact residues interfere with antigen presentation by transfectants but not with plasma membrane expression of the mutant class II. In the present study we examined other class II mutations in this region that did inhibit plasma membrane expression of mutant class II molecules. Molecules containing both mutations H{alpha}181D in the {alpha}2 domain and Eß170K in the ß2 domain exhibited low plasma membrane expression, but molecules with only one of these mutations were expressed normally. The mutant class II molecules were transported to organelles that were accessible to a fluid-phase protein, hen egg lysozyme (HEL). Culture of transfectants with lysozyme enhanced the amount of class II compact dimer ({alpha}ß plus peptide; CD), and this was especially marked for the class II mutant H{alpha}181D/Eß170K and for other molecules possessing both mutations. Formation of class II CD was not paralleled by an increase in class II surface expression. Thus the joint mutation of H{alpha}181 and Eß170 has two effects. In the absence of high concentrations of exogenous peptide, it prevents efficient CD formation, possibly by affecting invariant chain (Ii) proteolysis and/or the stability of the class II after Ii/CLIP is removed. At high peptide concentrations supplied by exogenous HEL, the mutations allow CD formation, but not expression of class II on the plasma membrane. Molecular modeling of the possible interaction of class II and Ii suggests that the mutant amino acids H{alpha}181D and Eß170K, besides affecting the overall stability of class II, might also interact with Ii via two loops in class II's {alpha}2 and ß2 domains respectively.

Keywords: antigen presentation, endocytic pathway, invariant chain, MHC class II


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
MHC class II antigens, cell surface proteins expressed on antigen-presenting cells, bind peptide fragments derived from endogenous and exogenous proteins for presentation to CD4+ T cells. The 33–36 kDa {alpha} and 29 kDa ß chains of class II are assembled in the endoplasmic reticulum (ER) and then transported through the Golgi complex where the mature carbohydrate moieties are acquired (reviewed in 1). Chaperone proteins such as GRP94, ER72 and calnexin bind nascent {alpha} and ß chain in the ER prior to their association with invariant chain (Ii) (2,3). The Ii chain plays a major role in assembly of a nine chain complex (three {alpha}ß dimers and three Ii chains) that is required for efficient transport of class II from the ER through the endocytic–lysosomal pathway and to the plasma membrane (4). The Ii chain has multiple roles. Endocytic localization signals in Ii chain direct the associated class II dimers through the transport pathway where Ii chain is exchanged for antigenic and self-peptides (5,6). A segment of the Ii chain, consisting of residues 81–104 and termed CLIP, is thought to stabilize class II to ensure proper transport and to protect the binding cleft from acquisition of self peptide (7).

Potentially unique membrane compartments with lysozyme-like characteristics have been identified and postulated to be the site where peptides charge class II prior to expression of the complex on the plasma membrane (8,9). In B lymphoblasts, class II molecules may be charged with peptide in a number of vesicles that differ by their densities and other endocytic markers (6). The exact pathway class II takes to the plasma membrane is not known; however, recent evidence by Castellino and Germain indicates that, for B cells, newly synthesized class II enters the endocytic pathway and acquires peptide prior to plasma membrane expression (6). Regardless of the route class II takes to the compartment(s) where peptide is loaded, after Ii is proteolytically removed and CLIP is exchanged for peptide perhaps facilitated by interactions with DM (10,11), mature, peptide-bound class II acquires its characteristic semi-SDS-resistant conformation termed the compact dimer (CD) (12,13). The percentage of class II CD in a cell can be increased by culture of the cells with exogenous proteins (6).

The transmembrane and cytoplasmic domains of class II affect its assembly and transport (1416). The polymorphic residues in the {alpha}1 and ß1 domains that define the MHC haplotype are also known to affect assembly, transport and plasma membrane expression (12,18). Chervonsky et al. have shown that residues 80–83 in the ß1 domain can either positively or negatively affect movement of class II to the endocytic vesicle (19). The occupancy of the binding site by CLIP or exogenous peptide is important for stability of class II as lack of occupancy results in aggregation of class II (6). Thus, multiple residues and/or domains of the class II molecule are necessary for effective expression.

Recently the crystal structures of murine and human class II molecules have been reported (2023). Published structures indicate a MHC class II dimer-of-dimers in contrast to that reported for MHC class I molecules. Guided by a class II dimer-of-dimers model structure, we mutated presumed contact residues of the I-Ak dimer-of-dimers to study effects on antigen presentation (24) and prepared some 20 different class II mutant phenotypes. We found that phenotypes combining the two substitutions H{alpha}181D and Eß170K had particularly low surface expression when expressed in B cells, and that this low expression was consistent among multiple clones of each phenotype. Only the paired mutations, H{alpha}181D with Eß170K, resulted in intracellular retention of class II. Other class II molecules containing only one of these mutations paired with various other one- or two-residue changes were efficiently expressed. In this study we compare transfectants expressing selected class II phenotypes with respect to class II expression levels and cellular localization, CD formation, peptide binding and Ii interactions, and interpret these results to suggest how the specific mutations may reduce class II surface expression.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
Antibodies and transfectants
The I-Ak-specific mouse mAb 39J (serologic specificity Ia.19, {alpha} chain), H116.32 (serologic specificity Ia.19, {alpha} chain), 40M (serologic specificity Ia.1, ß chain) and 10.2.16 (serologic specificity Ia.17, ß chain) were purified by Protein A chromatography from hybridoma supernatants produced in our laboratory (2527). 39J, which is conformationally specific for Ak{alpha}Akß, was obtained from Dr D. McKean (Mayo Clinic, Rochester, MN). 40M and 10.2.16 are specific for the I-Ak ß chain and are not dependent on class II conformation for binding. The mAb H116.32 is dependent on the intact conformation of the I-Ak for binding. All three I-Ak mAb were obtained from Dr John Freed (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO). FACS analysis was performed using FITC-conjugated 39J and 40M that were produced by direct coupling of FITC (Sigma, St Louis, MO) to the mAb. H116.32 and 40M were labeled with Cy3 using the Fluorolink kit (Amersham Life Sciences, Arlington Heights, IL) according to the manufacturer's instructions. Alexa 568-conjugated 39J antibody was prepared with labeling kits obtained from Molecular Probes (Beaverton, OR) and used according to the manufacturer's instructions.

M12.C3 (I-A, I-E) cell transfectants were produced and cultured as previously described (14,26). Briefly, PCR mutagenesis (splicing by overlap extension) was used to introduce amino acid changes in several of the class II dimer-of-dimers contact amino acids that comprise contact regions 3 and 4 (24). The Ak{alpha}_ and Akß cDNA constructs, a gift from Dr Terry Potter (National Jewish Center for Immunology and Respiratory Medicine), have been described previously (14). Oligonucleotide sequences used as primers and the individual PCR amplification conditions are available upon request. To confirm the fidelity of the mutagenesis, nucleotide changes were confirmed by double-stranded DNA sequencing. M12.C3 cells were co-transfected by electroporation with cDNA encoding the Ak{alpha} and Akß chains plus DNA from the plasmid pSV2-neo (28). Resulting G418 resistant clones were analyzed for class II surface expression by FACS using 39J and 40M. The class II mutation phenotypes in the {alpha}ß dimer are specified by amino acid changes, e.g. wt/Eß170K is a class II molecule with a wild-type {alpha} chain and a ß chain with the Glu residue at position 170 changed to Lys. The class II mutation phenotypes that are described in this study are: wt/Eß170K, H{alpha}181D/wt; H{alpha}181D/Eß170K; H{alpha}181D/Hß112D, Hß113D,Eß170K; E{alpha}183D/Eß170K. The wt/wt transfectants were 9D4 or F6 which have been described previously (15,28).

Flow cytometry
A total of 300,000 cells in a final volume of 100 µl PBS was incubated for 30 min on ice with 5 µg of FITC-conjugated 39J or 40M. Cells were diluted in PBS and centrifuged at 1500 r.p.m. for 10 min at 4°C. Cells were resuspended in 300 µl of PBS and data collected on a Becton Dickinson FACScan (Franklin Lakes, NJ) followed by analysis using CellQuest software (Becton Dickinson). HEL-treated cells were recovered from overnight cultures and washed once in PBS, and then stained as above with either FITC-conjugated 39J or 40M.

Immunoprecipitation and Western blot
HEL-treated and untreated transfectants (1x107 cells) were washed once in PBS and lysed in 200 µl of ice-cold NP-40 lysis buffer consisting of 50 mM Tris–HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.2 mg/ml PMSF and 25 µg/ml aprotinin (2). Cell debris and nuclei were removed following the 1 h incubation at 4°C by centrifugation at 1000 r.p.m. for 10 min at 4°C. The resulting supernatants were centrifuged at 16,000 g at 4°C for 30 min. Two million cell equivalents of lysate from each cell line were precleared of IgG by incubating with 10 µl of Protein G–Sepharose beads (Sigma, St Louis, MO) for 3 h at 4°C and of IgM by incubation for 3 h at 4°C with 10 µl of Protein G–Sepharose beads conjugated with 0.5 µg of anti-mouse IgM. Pre-cleared samples were immunoprecipitated by incubation with 5 µl (1.1 mg/ml) of 10.2.16-coupled Sepharose 4B beads at 4°C overnight followed by three sets of two 5 min washes. The first wash buffer contained 10 mM Tris (pH 7.5), 150 mM NaCl, 0.2% NP-40 and 2 mM EDTA; the second, 10 mM Tris (pH 7.5), 500 mM NaCl, 0.2% NP-40 and 2 mM EDTA; and the third 10 mM Tris (pH 7.5).

Washed immunoprecipitates were resuspended in 80 µl of respective PAGE loading buffer containing, for boiled samples, 500 mM Tris–HCl (pH 6.8), 50% (v/v) glycerol, 10% (w/v) SDS and 5% ß-mercaptoethanol or, for non-boiled samples, 500 mM Tris–HCl (pH 6.8), 50% (v/v) glycerol, 10% (w/v) SDS and 100 mM iodoacetemide. Samples were boiled for 10 min or left unboiled. Two hundred and fifty thousand cell equivalents of the non-boiled samples and 6.3x104 cell equivalents of boiled samples were subjected to SDS–PAGE in 10% acrylamide gel. Gels were transferred to 0.45 µm pore nitrocellulose (Schleicher & Schuell, Keene, NH) using a semi-dry blotter (BioRad, Hercules, Ca) at 23 V for 30 min. The nitrocellulose was blocked for 1 h at room temperature with 5% non-fat dry milk and 0.5% Tween 20 in PBS. Following blocking, membranes were reacted overnight at 4°C with 10.2.16 mAb (2 µg/ml in 5% non-fat dry milk and 0.5% Tween 20 in PBS). The membrane was washed 6 times, 5 min each, with PBS, and then incubated with an anti-mouse IgG-horseradish peroxidase-conjugated antibody (1:2500) in 3% non-fat dry milk and 0.05% Tween 20 in PBS for 2 h at room temperature. After incubation with the secondary antibody the blots were washed 6 times in PBS (5 min each wash) and antibody complexes were detected with the ECL kit (Amersham, Little Chalfont, UK). Class II Western blots were scanned into a PowerMac computer using Adobe Photoshop (Mountain View, CA) version 4.0 software. The images were cropped to a standard size and converted to PICT files for manipulation in NIH Image version 1.60. Using NIH Image, the area of the image, i.e. total pixels of each band to be analyzed, was measured and used to determine the fold increase of the CD following culture of transfectants with HEL.

Confocal analysis
Cells (5x105) were permeabilized for 1 h at 4°C in 0.5% w/v saponin, 0.1% w/v BSA and 0.1% w/v azide, then incubated with 1 µg of Alexa 568-conjugated 39J antibody in a 100 µl final volume. The isotype control was a non-specific mouse IgG1. Cells were incubated with 39J antibody or isotype control for 1 h at 4°C. Cells were then washed 3 times by centrifugation in permeabilization buffer (200 g, 5 min, 4°C). Cells were then fixed for 30 min at 4°C with 0.5% w/v paraformaldehyde in PBS, resuspended in PBS, and mounted on slides and prepared for confocal microscopy. Cells were analyzed using a BioRad MRC1024 laser-scanning confocal microscope in conjunction with a x40 objective. Random fields were imaged and 50–100 cells acquired for each condition. Mid-section images were obtained to allow differentiation between cell surface and internal staining. Confocal settings were maintained constant throughout the experiment to allow comparison between different transfectants. Images were analyzed with Adobe Photoshop software. Cells were assessed as having surface 39J staining when distinct peripheral staining was observed.

Molecular modeling of interactions between I-Ak and Ii
Possible interactions of Ii with an I-Ak {alpha}ß heterodimer were modeled as described previously (24). The parent structure for I-Ak was the dimer-of-dimers (22) derived from the DR3 or DR1 crystal structures. Only residues 36–110 of Ii (P04441) were used, including the following domains: (i) transmembrane (residues 36–56), (ii) putative MHC class II interaction sites (residues 57–80), (iii) the CLIP domain (residues 81–104) and (iv) part of the class II–Ii trimerization site (residues 105–110). The actual modeling was performed in two stages (29). In the first stage a peptide consisting of Ii residues 36–80 was docked to an I-Ak heterodimer through the following step sequence: (i) initial modeling of this peptide around a transmembrane helix, (ii) energy minimization, (iii) 200 ps molecular dynamics refinement, (iv) energy minimization, (v) docking of the peptide to an I-Ak {alpha}ß heterodimer, (vi) energy minimization, (vii) 100 ps molecular dynamics refinement and (viii) energy minimization of the entire Ii–I-Ak complex. In the second phase, the bound Ii peptide was extended to include Ii residues 81–110 and interaction with the {alpha}ß heterodimer re-optimized (30). The steps were: (i) independent modeling of Ii residues 81–110, (ii) docking of this fragment as a continuation of the Ii chain on the previous complex, (iii) energy minimization, (iv) molecular dynamics refinement and (v) energy minimization of the full complex, i.e. I-Ak with the 36–110 residue Ii peptide.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
Surface expression of MHC class II is compromised only for molecules possessing both the H{alpha}181D and Eß170K mutations
During the course of our experiments to define the role of class II dimer-of-dimers contact residues in antigen presentation, we generated over 45 stable transfectants expressing some 20 mutant class II phenotypes (24). Surprisingly two mutant phenotypes were poorly expressed on the plasma membrane. The level of surface expression of the transfectants with phenotypes H{alpha}181D/Eß170K and H{alpha}181D/Hß112D,Hß113D, Eß170K was significantly lower than other transfectants where neither or only one of the class II amino acids (H{alpha}181 or Eß170) was substituted with an oppositely charged residue. Figure 1Go shows the surface expression of class II, evaluated by FACS analysis of 39J-labeled cells, for several clones each of various mutant phenotypes from the initial analysis of the transfected clones. The wt/wt control for class II expression in Fig. 1Go is F6. Transfectants that express the mutant phenotypes H{alpha}181D/Eß170K and H{alpha}181D/Hß112D,Hß113D,Eß170K had markedly low class II cell surface staining. We then stained representative transfectant clones of each phenotype with the conformation-dependent mAb 39J. Compared to 9D4, a wt/wt M12.C3 transfectant with characteristic level of class II expression, H{alpha}181D/Eß170K had an average of only 28% of wt/wt and H{alpha}181D/Hß112D,Hß113D,Eß170K had 19% (Table IGo). 40M staining of H{alpha}181D/Eß170K transfectants revealed similar low expression. The defect in surface expression required both the H{alpha}181D and the Eß170K mutations since mutants expressing only one of these mutations, e.g. H{alpha}181D/Eß163K, E{alpha}183K/Eß170K, H{alpha}181D/wt or wt/Eß170K, had class II expression comparable to 9D4.



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Fig. 1. Plasma membrane expression of mutant class II molecules. Several class II mutations we generated were poorly expressed on the plasma membrane as evidenced by flow cytometric analysis and comparison to the F6 wt/wt transfectant. Transfectants expressing the class II mutation phenotypes H{alpha}181D/Eß170K (top bar) and H{alpha}181D/Hß112D,Hß113D,Eß170K (second bar from top) have lower class II surface expression than did 9D4 or other class II mutations. Other class II phenotypes (remaining bars in descending order) are: E{alpha}183K/Eß170K, H{alpha}181D/wt, wt/Eß170K and wt/wt (F6 cells).

 

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Table 1. Surface expression and CD formation of mutant class II molecules
 
Mutations in H{alpha}181D and Eß170K result in intracellularly expressed MHC class II
Confocal microscopic images representative of the population of transfectants and showing the cellular distribution of wild-type and mutant class II are presented in Fig. 2Go(a). Images show 9D4 cells expressing wild-type I-Ak (top left), 22.32.3 cells expressing H{alpha}181D/Eß170K (Fig. 2aGo, top right), 14.2.3.17 cells expressing H{alpha}181D/Hß112D,Hß113D,Eß170K (Fig. 2aGo, bottom left) and I-Ak-negative M12.C3 cells (Fig. 2aGo, bottom right). The lower right panel of Fig. 2Go(a) shows an absence of background when the I-Ak-negative host cell line is stained for I-Ak, so that the other panels unambiguously show class II localization. The difference in cellular localization between the mutant class IIs and wild-type molecules is quite striking. Wild-type class II is expressed primarily on the plasma membrane with little or no internal staining. By contrast, mutant class II is equally strongly labeled and thus expressed at levels comparable to the wild-type molecule, but is predominantly retained intracellularly. Figure 2Go(b) shows the cellular distribution (i.e. plasma membrane versus intracellular) of class II among cell populations. Substantial plasma membrane staining is seen in 90% of cells expressing wild-type class II compared with only 35% of H{alpha}181D/Eß170K cells and 25% of H{alpha}181D/Hß112D,Hß113D,Eß170K cells. By contrast, substantial cytoplasmic staining appears in 80% of H{alpha}181D/Eß170K calls and 85% of H{alpha}181D/Hß112D,Hß113D,Eß170K cells compared with only 40% of wild-type cells.




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Fig. 2. Cells were permeabilized with saponin as described under Methods. I-Ak was then stained directly using Alexa 568-conjugated 39J antibody. Cells were then fixed in 0.5% w/v paraformaldehyde and analyzed by confocal microscopy (A). Images show 9D4 cells expressing wild-type I-Ak (top left), 22.32.3 cells expressing H{alpha}181D/Eß170K (top right), 14.2.3.17 cells expressing H{alpha}181D/Hß112D, ß113D,Eß170K (bottom left) and I-Ak-negative M12.C3 cells (bottom right). In each case, cells were incubated with an isotype control antibody and no staining was observed (not shown). This experiment was repeated twice with similar results. Surface staining is significantly weaker in mutants than in 9D4 cells while, conversely, internal staining in 9D4 was weaker than in the mutants. (B) Images such as shown in (A) were analyzed using Adobe Photoshop version 4.0 software to assess 39J surface staining (black bars) and internal 39J staining (cross hatched bars). Data are expressed as the percentage of cells demonstrating surface and/or internal staining from random fields. At least 50 cells were analyzed for each data point. In some cells both internal and surface staining was observed.

 
Transfectants with both H{alpha}181D/Eß170K exhibit very low fractional contents of class II CD
Mature class II molecules that have peptide bound assume the SDS-resistant CD form of class II (12,13). Therefore, the percent of CD expressed in a particular transfectant is a measure either of class II transport to the particular point in the pathway where CD can be formed or of the stability of the CD itself. Cell lysates were immunoprecipitated with anti-class II mAb coupled to beads and Western blot analysis was performed to assess the CD content of the transfectants of the various mutant phenotypes (Fig. 3aGo). The CD and ß chain were clearly seen in the 9D4 immunoprecipitate but not in the M12.C3 or 10.2.16 bead samples (cf. Fig. 3aGo, lane 3 to 1 and 2). Immunoprecipitates from transfectants that expressed H{alpha}181D/Eß170K or H{alpha}181D/Hß112D,Hß113D,Eß170K had significantly less CD compared to 9D4, 15- and 92- fold less respectively (Table 1Go). Other mutation phenotypes with either H{alpha}181D or Eß170K (i.e. H{alpha}181D/Eß163K and E{alpha}183K/Eß170K) had amounts of CD comparable to, or exceeding that of, 9D4. H{alpha}181D/wt and wt/Eß170K transfectants had lower amounts of CD and more free ß chain compared to 9D4. As would be expected, and as previous studies have shown for other class II molecules, the CD form of class II, regardless of class II phenotype, was not stable to boiling (not shown).



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Fig. 3. Effect of HEL on CD formation. Transfectants were incubated with HEL for 4 h at 37°C or left untreated before lysis, immunoprecipitated and samples analyzed by Western blot for class II expression. Only results for unboiled samples are shown since boiled samples showed the expected complete absence of CD. Lane correspondence in Western blot (A and B): lane 1, Ia (M12.C3); lane 2, 10.2.16 immunoprecipitation beads; lane 3, wt/wt (9D4); lane 4, H{alpha}181D/Eß170K (clone 14.23.17); lane 5, wt/Eß170K (clone 15.14); lane 6, H{alpha}181D/wt (clone 17.17.8); lane 7, H{alpha}181D/Hß112D,Hß113D,Eß170K (clone 22.32.3); lane 8, E{alpha}183K/Eß170K (clone 35.23.1) and lane 9, H{alpha}181D/Eß163K (clone 51.7). (A) Immunoprecipitates of class II from non-HEL-treated cells. (B) Immunoprecipitates of class II from HEL-treated cells. The relative increase in CD was determined from Western blots of cell lysates from untreated and HEL-treated cells using NIH Image. Results are representative of two experiments.

 
HEL treatment of H{alpha}181D/Eß170K increases CD content almost to wt/wt levels
The culture of B cells with immunogenic protein is known to increase the percentage of CD (10). Fluid-phase endocytic vesicles intersect with class II-containing vesicles facilitating exchange of CLIP for processed peptides. Thus an increase in CD following culture with HEL would indicate transport of mutant class II to a point in the pathway that was accessible to fluid phase, extracellular proteins. We treated transfectants with 2 mg/ml HEL for 4 h and assessed its effect on CD formation (Fig. 3bGo). The conditions for the Western blot and subsequent ECL exposures were identical to that of Fig. 3Go(a). Incubation with HEL enhances CD formation even in the transfectants that express the H{alpha}181D/Eß170K or H{alpha}181D/Hß112D,Hß113D,Eß170K mutant phenotypes. The relative difference in CD expression between the HEL-treated and untreated samples was determined by image analysis as described previously. The average CD content of various mutant phenotypes, with and without HEL treatment, is shown in Table 1Go where content is normalized to that of untreated 9D4. The HEL-induced increase in the CD conformer for wt/wt class II was 1.4-fold. The transfectants that express H{alpha}181D/Eß170K or H{alpha}181D/Hß112D,Hß113D,Eß170K mutant class II showed 13- and 56-fold increases in CD respectively. This result indicates that {alpha} and ß chains of class II of the H{alpha}181D/Eß170K or H{alpha}181D/Hß112D,Hß113D,Eß170K mutation phenotypes were assembled and present in the transport pathway past the Golgi. After HEL treatment, the CD contents of transfectants expressing H{alpha}181D/Eß170K or H{alpha}181D/Hß112D,Hß113D,Eß170K were comparable to that of untreated 9D4 cells and were >50% of HEL-treated 9D4 cells, indicating that the amount of mutant protein H{alpha}181D/Eß170K could be stabilized virtually to wild-type levels. Thus mutant class IIs must be synthesized at levels comparable to wild-type. Differences in CD levels among cell lines in the absence of HEL must reflect differences in the ability of the different class II molecules to attain the CD state. There may also be some degree of HEL-induced change in wild-type class II expression (1). However, our concern is how, under various conditions, mutant class II expression compares with that of wild-type molecules. Figure 3Go makes clear that mutant CD levels relative to wild-type are significantly elevated by HEL treatment and this observation stands on its own, independent of what effects HEL treatment has on wild-type class II.

By contrast HEL treatment does not increase class II surface expression in H{alpha}181D/Eß170K mutants
Since HEL treatment was able to increase the CD conformer in H{alpha}181D/Eß170K and H{alpha}181D/Hß112D,Hß113D,Eß170K transfectants, we analyzed class II surface expression on transfectants treated overnight (16 h; Fig. 4Go) with 4 mg/ml HEL. Figure 4Go presents actual FACS results. HEL treatment increased 39J staining of wt/wt (9D4) cells 1.1-fold, H{alpha}181D/Eß170K cells 1.6-fold and H{alpha}181D/Hß112D,Hß113D,Eß170K cells 1.3-fold. Transfectants stained with 40M showed similar results (Table 1Go). The relatively minor changes in the surface expression did not correlate with the increase in CD for H{alpha}181D/Eß170K or H{alpha}181D/Hß112D,Hß113D,Eß170K. These results suggest that ability of H{alpha}181D/Eß170K class II to exchange Ii for exogenous peptide and to subsequently assume the CD conformation are not sufficient to insure increased transport to the plasma membrane. Unanue has used the Aw3.18 mAb specific for I-Ak binding HEL(46–61) peptide to show that, if M12.C3 transfectants are treated with HEL for 30 min, they have maximal membrane expression of new class II binding HEL(46–61) peptide at 4 h (31). Thus any class II present as CD at 4 h should appear on the membrane at least by 16 h if at all. Clearly, 16 h HEL treatment does not substantively increase plasma membrane levels of the class II mutants. Thus, conditions that greatly facilitate formation of mutant class II CD still cannot drive the mutant class II to the PM.



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Fig. 4. Plasma membrane expression of class II after 16 h HEL treatment. Transfectants were stained with FITC-conjugated antibody, 39J or 40M, and analyzed by flow cytometry. The 39J staining is shown for 9D4 expressing wt/wt (right panel), for clone 14.23.17 expressing H{alpha}181D/Eß170K (middle panel) and for clone 22.27.3 expressing H{alpha}181D/Hß112D,Hß113D,Eß170K (left panel). A slight shift in the histogram to the right from the untreated to the treated (bold line) can be seen in all three cell lines. Similar results were received from 40M staining. The increase in cell surface expression of class II expressed on 9D4, 14.23.17 and 22.32.3 as measured by 39J staining was 1.1-, 1.6- and 1.4-fold respectively. The results when staining with 40M for the same three cell lines were 1.6-, 1.3- and 1.4-fold respectively.

 
Mutant class II molecules associate effectively with Ii
To demonstrate that mutant class II could in fact associate with Ii chain, we immunoprecipitated class II from cell lysates of transfectants expressing either wt/wt, H{alpha}181D/Eß170K or H{alpha}181D/Hß112D,Hß113D,Eß170K class II and assessed class II-associated Ii chain (Fig. 5Go). The mutant H{alpha}181D/Eß170K or H{alpha}181D/Hß112D,Hß113D,Eß170K can associate with Ii chain and Ii can be partially degraded. Interestingly, the H{alpha}181D/Eß170K class II has more Ii chain associated with it than does the wild-type class II. The H{alpha}181D/Hß112D,Hß113D,Eß170K mutant class II can also associate with Ii chain. However, these complexes may be less stable or formed less efficiently since, relative to wt/wt class II, twice as many cell equivalents had to be loaded to visualize the Ii chain associated with the mutants.



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Fig. 5. Western blot analysis of Ii co-precipitating with class II. Cells were lysed and class II immunoprecipitated with 10.2.16 Akß chain-specific antibody conjugated to Sepharose beads. Lanes 1–4 contain immunoprecipitates of 2.0x106 cell equivalents of Ia M12.C3 cells (lane 1), 9D4 cells expressing wt/wt (lane 2), clone 14.23.17 expressing H{alpha}181D/Eß170K (lane 3) and clone 22.32.3 expressing H{alpha}181D/Hß112D,Hß113D,Eß170K (lane 4). Lanes 5 and 6 contain immunoprecipitates from 3.0x106 and 4.0x106 cell equivalents respectively of clone 22.32.3 expressing H{alpha}181D/Hß112D, Hß113D,Eß170K.

 
Molecular modeling suggests how residues H{alpha}181 and Eß170 may affect class II stability and/or interactions with Ii
The structure of the peptide-binding I-Ak heterodimer has been determined crystallographically (23) and we have modeled the dimer-of-dimers structure which the class II molecule may adopt under certain conditions (24). In the mature surface-expressed class II, H{alpha}181 normally forms a salt-bridge with Dß163 in the class II dimer-of-dimers (22,24). Similarly, Eß170 appears to interact with a cluster of positive charges formed by Rß134, Rß166 and Rß167. Thus both residues seem likely to play major roles in the overall stabilization of I-Ak. In the present study we have modeled a structure for residues 36–110 of the Ii, docked to our published structure for the I-Ak dimer-of-dimers (24) and optimized the structure of the resulting {alpha}ßIi complex. The Ii residues examined include part of the transmembrane domain and residues C-terminal to the CLIP peptide. The model (Fig. 6Go) suggests an additional role for class II residues H{alpha}181 and Eß170 in mediating interactions between I-Ak and Ii. The class II {alpha} chain is shown in yellow, the ß chain in red and the Ii in white. The class II {alpha} chain contains two ß strands comprised of amino acids {alpha}161–{alpha}182 that may represent a binding pocket for the Ii chain. Residue H{alpha}181 (green, space-filling) lies within the binding pocket. In the mature surface-expressed class II H{alpha}181 normally forms a salt-bridge with Dß163 in the class II dimer-of-dimers (22,24). In our class II–Ii chain model, which is based on a single {alpha}ß- heterodimer-of-class II rather than the dimer-of-dimers, we predict a salt-bridge between the H{alpha}181 and Ii chain E74 (white, space-filling) when Ii is bound. This salt-bridge would provide a strong interaction between H{alpha}181 and Ii E74 in wild-type class II. Two differing class II structures, heterodimers aggregated into dimer-of-dimers and heterodimer binding Ii, both show H{alpha}181 paired with another amino acid, either from the ß chain or the Ii, and this may suggest that unpaired H{alpha}181 is unstable and thus unfavored. The ß chain also interacts with Ii in the model we generated. As shown in Fig. 6Go, ß chain residues Lß104–Nß114 form a loop structure (red dots) which may interact with the N-terminal portion of Ii from residues 36–57 (white helix). The flexibility of this loop, and thus its interaction with Ii, may depend on electrostatic interactions between Eß170 (cyan, space-filling) and Rß167 (magenta, space-filling) which stabilize the turn of an adjacent ß chain loop. Thus molecular modeling suggests that both the class II residues found critical for expression, i.e. H{alpha}181 and Eß170, could potentially modulate class II stability and/or interactions with Ii. This model is also consistent with reports that residues ß80–ß82 of the ß1 domain regulate class II access to the endocytic compartment (19). These residues are located at the C-terminal end of the ß chain {alpha} helix of class II which forms the peptide-binding groove. In our model, Rß80 and Hß81 seem capable of electrostatic interaction with the C-terminal residue (P110) of the Ii as it exits the peptide-binding groove.



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Fig. 6. Molecular modeling of interaction of class II and Ii. Ii (white) is docked to class II ({alpha} chain yellow and ß chain red). Two ß strands in the {alpha} chain defined by amino acids {alpha}161–{alpha}182 may represent a binding pocket for the Ii chain. This {alpha}2 domain region contains the residue H{alpha}181 (green, space filling) which forms a salt-bridge with Ii residue E74 (white, space filling). ß chain residues Lß104 through Nß114 form a loop structure (red dots) which may interact with the N-terminal portion of Ii from residues 36–57 (white helix). The flexibility of this loop, and thus its interaction with Ii, may depend on electrostatic interactions between Eß170 (cyan, space-filling) and Rß167 (magenta, space-filling) which stabilize the turn of an adjacent ß chain loop. Special structural significance of H{alpha}181 and Eß170 is suggested by H{alpha}181 being very highly conserved in other class II molecules and Eß170 being either an E or a D (34). The CLIP peptide (white) lies between the {alpha} and ß chain helices of the peptide-binding groove at the top of the molecule.

 

    Conclusions
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
In this report we describe two mutant class II phenotypes, H{alpha}181D/Eß170K and H{alpha}181D/Hß112D,Hß113D, Eß170K, that were not well expressed on the plasma membrane. The effect on expression was specific for the pair of mutations H{alpha}181D and Eß170K as other mutants paired with either were expressed as well if not better than the wt/wt class II. The mechanism of effect of these two specific mutations on class II expression is not known. It is, however, likely to be very specific. The other double-mutant class II phenotypes that are well-expressed, H{alpha}181D/Eß163K and E{alpha}183K/Eß170K, contain a second amino acid change located very close in the primary sequence to the residues that we report as being involved in surface expression. Thus, for example, E{alpha}183K is only 2 amino acids away from H{alpha}181D, yet the first mutation paired with Eß170K yields normal membrane expression while the second paired again with Eß170K is arrested in transport as has been described. Thus it seems to us likely that effects of the H{alpha}181D/Eß170K mutations on transport arise from quite specific interactions involving these residues which maintain either class II's structure and or its ability to interact with other transport proteins. Our other study involving mutation of residues ({alpha}166, {alpha}183, {alpha}185, ß106, ß112, ß113 and ß163) in this region of class II revealed no single or combined mutations in the {alpha}2 and ß2 domains which significantly affected membrane expression (23).

Our experiments with HEL indicate that class II molecules involving the paired mutations can form CD given adequate exogenous HEL but that this CD formation is inadequate to elevate plasma membrane class II expression. Thus the mutation of H{alpha}181 and Eß170 has two effects. In the absence of high concentrations of exogenous peptide, it prevents efficient CD formation, possibly by affecting Ii proteolysis and/or the stability of the class II after Ii/CLIP is removed. At high peptide concentrations supplied by exogenous HEL, the mutations allow CD formation, but not effective expression of class II on the plasma membrane. The first effect could arise if the mutations slightly impair Ii–{alpha}ß interactions and thus class II folding. In such a case, the resulting class II might be unstable after Ii proteolysis in the endosomal/lysosomal compartment unless rapidly stabilized by high peptide concentrations. The second effect implies that the mutations H{alpha}181D/Eß170K together have a separate structural effect preventing CD from progressing on to maximum plasma membrane expression. The specific mechanism for such behavior is unknown, but class II structure is well known to be sensitive to allosteric changes that can alter its transport and expression (1). One possible explanation of our results would be that stability of the mutant class II depends strongly on pH so that mutant CD is stable while in endosomes but rapidly aggregates upon reaching the plasma membrane.


    Acknowledgments
 
This work was supported in part by NIH grants AI36306 and CA 23108, and by equipment grants from the Fannie E. Ripple Foundation to the Dartmouth Medical School.


    Abbreviations
 
CD compact dimer
ER endoplasmic reticulum
HEL hen egg lysozyme
Ii invariant chain
MFI mean fluorescence intensity

    Notes
 
Transmitting editor: I. Pecht

Received 30 September 1999, accepted 7 September 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 

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