Soluble HLA-DQ2 expressed in S2 cells copurifies with a high affinity insect cell derived protein
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We here describe that soluble HLA-DQ2 (sDQ2) molecules, when expressed in Drosophila melanogaster S2 insect cells without a covalently tethered peptide, associate tightly with the D. melanogaster calcium binding protein DCB-45. The interaction between the proteins is stable in S2 cell culture and during affinity purification, which is done at high salt concentrations and pH 11.5. After affinity purification, the sDQ2/DCB-45 complex exists in substantial quantities next to a small amount of free heterodimeric sDQ2 and large amounts of aggregated sDQ2 free of DCB-45. Motivated by the stable complex formation and our interest in the development of reagents which inhibit HLA-DQ2 peptide binding, we have further characterized the sDQ2/DCB-45 interaction. Several lines of evidence indicate that an N-terminal fragment of DCB-45 is involved in the interaction with the peptide binding groove of sDQ2. Further mapping of this fragment of 54 residues identified a pentadecapeptide with high affinity for sDQ2 which may serve as a lead compound for the design of HLA-DQ2 blockers.
KeywordsHLA sDQ2 DCB-45 S2 Protein interactions Peptide binding
HLA-DQ2 (DQA1*0501/DQB1*0201) is associated with several immune-mediated disorders including celiac disease (Sollid et al. 1989; Thorsby and Ronningen 1993; Todd et al. 1987). In order to develop specific blockers as a potential future treatment for celiac disease and other HLA-DQ2 associated diseases, there is a need to define high affinity ligands for HLA-DQ2. The development of such blockers will be facilitated by the production of recombinant HLA-DQ2 that can be made in high quantities and utilized for binding studies. On this background, we have produced recombinant, soluble HLA-DQ2 (sDQ2) molecules in either stably transfected S2 cells or in baculovirus infected Sf9 cells. Interestingly, when expressing sDQ2 in S2 cells without a covalently bound peptide that occupies the binding groove, we obtained remarkably high amounts of stable molecules. Further analysis of these sDQ2 molecules revealed that a major proportion of the molecules copurifies with the Drosophila melanogaster calcium binding protein DCB-45, a homolog to the D. melanogaster supercoiling factor (SCF; Kobayashi et al. 1998). Given the apparent ability of the protein to stabilize sDQ2, we hypothesized that it could contain a peptide which binds efficiently to the binding groove of sDQ2 and which could function as a lead peptide for the development of HLA-DQ2 blockers. Here, we report on the identification and characterization of the DCB-45 sequence which mediates binding to sDQ2. Next to characterizing the interaction between sDQ2 and DCB-45, our observations are of general relevance for expression of recombinant major histocompatibility complex (MHC) class II molecules in S2 cells.
Materials and methods
HLA expression and purification
Water soluble HLA-DQ2 (DQA1*0501/DQB1*0201) was expressed in a D. melanogaster S2 cell line by cotransfection of three vectors: a pMtal vector with the sequence encoding for the extracellular part of the DQα-chain fused to the Fos zipper, a pMtal vector with the sequence encoding the extracellular part of the DQβ-chain fused to the Jun zipper, and a pCoHYGRO resistance vector (Paulsen et al., unpublished). The construct did not include a sequence for a high affinity peptide ligand tethered to the β-chain. The stably transfected S2 cells were grown in 1L cell spin flasks at 22°C in serum-free media (Insect-XPRESS™, BioWhittaker, Walkersville, MD, USA), containing 300 μg/ml hygromycin B (Invitrogen, Carlsbad, CA, USA) and 25 μg/ml garamycin (Schering-Plough, Kenilworth, NJ, USA). The pMtal vectors contain a metallothionein promoter and the production of sDQ2 was induced by 100 mM CuSO4 over 3 days. From cell culture supernatants, the sDQ2 molecules were affinity-purified like previously described (Quarsten et al. 2001). HLA-DM molecules were produced as soluble molecules in transfected S2 cells (kind gift of Elizabeth Mellins) and purified by FLAG-tag immunoaffinity chromatography and size exclusion chromatography as described (Sloan et al. 1995). Detergent-solubilized HLA-DQ2 (DQA1*0501/DQB1*0201; EBV-DQ2) molecules were purified from Epstein–Barr virus-transformed B lymphoblastoid cell lines as previously described (Johansen et al. 1994). The protein concentration of the various HLA molecules was determined by a BCA protein assay kit (Pierce, Rockford, IL, USA).
Size exclusion chromatography and gel electrophoresis
Preparative size exclusion chromatography was performed on an Äkta purifier system (Amersham Biosciences Corp., Piscataway, NJ, USA) using a Superdex 200 10/300 GL column (Amersham Bioscience). Proteins were separated by isocratic elution (flow rate 0.75 ml/min) using phosphate buffered saline (PBS; pH 7.3) and monitored at 280 nm. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 6–12% polyacrylamide gels. Samples were reduced by heating in Laemmli buffer containing 1% β-mercaptoethanol (95°C, 5 min). Proteins were stained by Coomassie Blue.
For protein identification by peptide mass fingerprinting, protein bands were excised from the Coomassie Blue stained gel and in-gel digested as previously described (Fleckenstein et al. 2004). For the acetylation of primary amino groups, the gel pieces were incubated in 3% acetanhydride/0.1 M NaHCO3 pH 8.4 for 2 h at room temperature, followed by several washing steps and tryptic digestion. Partial digestion of the sDQ2/DCB-45 complex in solution was performed with N-tosyl l-phenylalanyl chloromethyl ketone (TPCK) trypsin agarose beads (Pierce). For 24 μg protein, 4.8 μl bead suspension in 0.1 M NH4HCO3 buffer in a total volume of 22.8 μl was used. The samples were incubated for 17 min at 37°C under rotation. For size exclusion chromatography, the beads were removed by filtration.
Matrix-assisted laser desorption–ionization time-of-flight mass spectra (MALDI-TOF MS) were acquired on a MALDI-TOF/TOF instrument (Ultraflex II, Bruker Daltonics, Bremen, Germany). Tryptic peptide mixtures and peptides eluted from sDQ2 were desalted and concentrated on Poros 20 R2 reverse-phase packing sorbent (Applied Biosystems, Foster City, CA, USA) packed in 20-μl GELoader tips (Eppendorf, Hamburg, Germany). Peptides were eluted onto a stainless steel target plate using 70% acetonitrile, containing 0.1% trifluoroacetic acid (TFA) and 10 g/l α-cyano-4-hydroxycinnamic acid. Synthetic peptides were also analyzed using α-cyano-4-hydroxycinnamic acid, but then samples were applied as a dried droplet.
Peptides with a length of 11–20 amino acid residues were synthesized on Rink amid methylbenzhydrylamine-resin using Fmoc/2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) chemistry and a pipetting robot (Syro I, MultiSynTech, Bochum, Germany). Coupling was performed with a tenfold molar excess of each Fmoc-l-amino acid and HBTU and a 15-fold excess of N,N-diisopropylethylamine. Piperidine (20% in dimethylformamide) was used for Fmoc deprotection and 96% TFA containing 2% triisopropylsilane and 2% water were used for side chain deprotection and cleavage from the resin. The identity was confirmed by MALDI-TOF MS, and the purity was determined by analytical reversed-phase high performance liquid chromatography (HPLC; Agilent 1100 system, Agilent Technologies, Santa Clara, CA, USA) using a Zorbax C18 column (Agilent Technologies). The 54 residue long peptide was synthesized on 10 mg NovaPEG Rink Amide resin LL (Novabiochem®, Merck KGaA, Darmstadt, Germany) with a low loading capacity of 0.16 mmol/g. The pseudoproline dipeptides Fmoc-Gly-(Hmb)Gly-OH (Novabiochem) in position 24 and Fmoc-Glu(OtBu)-Ser(ψMe,Mepro)-OH (Novabiochem) in position 41 and 52 were used to substitute for Gly-Gly and Glu-Ser, respectively. The secondary amide bond of these amino acid surrogates is reversibly protected and cleaved during deprotection with 96% TFA. The first 30 cycles were run under standard conditions as described above. From cycle 30 on, all coupling steps were repeated and 30% piperidine in dimethylformamide for Fmoc deprotection was used. Side chain deprotection and cleavage from the resin was performed as described above. From the crude peptide, the observed TFA ester on the N-terminal Ser side chain was removed by treatment with 1 M NaOH for 30 min, followed by neutralization with equimolar amounts of HCl. The identity and purity were determined like described above.
Labeling of synthetic peptides
The HLA-DQ2 high affinity ligand P198 (KPLLIIAEDVEGEY, Mycobacterium bovis 65 kDa Hsp 243–255Y) was either fluorescently labeled or radiolabeled. The radiolabeling with 125I was done with the chloramine T method (Greenwood et al. 1963). Fluorescence labeling was done by N-terminally coupling of 5(6)-carboxyfluorescein (CF) using 5 equivalents CF and 6 equivalents N,N-diisopropylcarbodiimide. The coupling step was repeated several times until a negative Kaisertest was obtained (Kaiser et al. 1970). The fluorescently labeled peptide was cleaved from the resin and analyzed as described above.
Peptide binding assays
Detergent-solubilized HLA-DQ2 (DQA1*0501/DQB1*0201; EBV-DQ2) at concentrations of 0.1–0.2 μM was incubated with the radioactively labeled P198 indicator peptide and synthetic peptides at various concentrations as described earlier (Johansen et al. 1994). The EBV-DQ2-peptide complexes were subsequently separated from unbound peptides by size exclusion chromatography in a spin column system as described (Buus et al. 1995). The radioactivity in the void volume and in the column material was counted by a γ-counter (Wallac, Turku, Finland) and IC50 values were calculated. sDQ2 (6.25 μM) or trypsin digested sDQ2 (6.25 μM) was incubated with CF-labeled P198 indicator peptide (10 μM). In some instances, soluble HLA-DM (sDM) was added in a concentration of 0.38 μM. The incubation was performed in a total volume of 13 μl of a citrate phosphate buffer (pH 5.3) at 37°C over 48 h. Next, 5 μl of each sample were transferred on a Zorbax GF 450 4.9 × 250-mm column and eluted with 150 mM PBS at a flow rate of 1 ml/min using an Agilent 1100 HPLC system. The UV signal (214 nm) and the fluorescence signal (Ex. 490 nm, Em. 520 nm) were monitored. Peptide binding to sDQ2 was quantified by measuring the area under the curve (AUC) of the fluorescence signal for peaks with a maximum between 9.5 and 9.7 min (corresponding to sDQ2 with bound indicator peptide). The mean values from at least two independent experiments were plotted.
Characterization of the sDQ2 preparation by SDS-PAGE and size exclusion chromatography
Identification of the 50-kDa protein by peptide mass fingerprinting
In order to identify the nature of the 50-kDa contamination, the protein was subjected to tryptic in-gel digestion. The digest was analyzed on a MALDI-TOF/TOF mass spectrometer and a MASCOT search reported the identification of a D. melanogaster-derived SCF (accession number Q9W0H8_DROME). Acetylation prior to digestion and analysis by MALDI-TOF/TOF MS identified the tryptic peptide SSIPEELPHNPLEHDPLHPR as the N terminus of the protein. The observed 50-kDa protein therefore represents a N-terminally truncated derivate of the D. melanogaster calcium binding protein (DCB-45), which is described as an elongated homolog of SCF (Kobayashi et al. 1998). The identified protein is highly acidic with a pI of 4.27.
Limited digestion of sDQ2/DCB-45 complexes
Peptide binding ability of sDQ2/DCB-45 before and after limited tryptic digestion
Identification of the HLA-DQ2 peptide binding frame and binding affinity of DCB-45-derived peptides
Binding of synthetic peptides to EBV-DQ2 given as IC50 values
Peptide binding specificity of sDQ2 compared to EBV-DQ2
In this study, we show that soluble HLA-DQ2 molecules without a covalently tethered peptide ligand copurify with the D. melanogaster-derived protein DCB-45, when expressed in S2 insect cells. We identified the fragment of DCB-45 which mediates the strong interaction with sDQ2 and obtained evidence indicating that a part of this fragment is binding to the sDQ2 binding groove with high affinity. The sDQ2 stabilizing effect of the DCB-45 protein motivated us to unravel the basis for this interaction as part of an effort to design compounds that can block HLA-DQ2-mediated antigen presentation.
Expression of HLA class II molecules in insect cells is a commonly used method for the production of recombinant molecules (De Wall et al. 2006; Stern and Wiley 1992; Wallny et al. 1995). Initially, DR1 and other MHC class II molecules were expressed without a covalently tethered peptide as so called “empty” class II molecules (Stern and Wiley 1992). The expression of recombinant molecules with a high affinity ligand covalently tethered to the N terminus of the β-chain (Crawford et al. 1998), however, gave improved expression and stable molecules and this strategy thus developed as a preferred expression modality. Our early work to express sDQ2 without a covalently tethered peptide in the S2 cell system gave unusually high protein yields and stable molecules what is in contrast to a poor expression yield and unstable molecules obtained with similar baculoviral constructs in Sf9 cells (Quarsten et al., unpublished). When analyzing and comparing peptide binding specificity of these sDQ2 molecules with detergent-solubilized HLA-DQ2 purified from EBV-transformed B cells (EBV-DQ2), we found the same peptide binding preferences but approximately a tenfold increase of the IC50 values for each peptide when using the sDQ2 molecules (Fig. 5).
In mammalian antigen presenting cells the assembly of HLA class II α- and β-chains is dependent on the invariant chain (Ii) protein. In the ER, Ii is protecting and stabilizing the class II binding groove with its class II-associated invariant chain peptide (CLIP) sequence, and the Ii is directing the class II molecules to the late endosomes where HLA-DM catalyzes the exchange of CLIP for antigenic peptides (Busch et al. 2005; Cresswell 1994). In S2 cells, this mechanism for sDQ2 assembly and loading is missing. One might thus expect that the unoccupied amphiphilic peptide binding groove is rendering the class II molecules unstable and prone to aggregation (Rabinowitz et al. 1998; Vogt et al. 1997). For this reason, it has been argued that so-called “empty” class II molecules are not truly empty but filled with loosely bound peptides. We thus wanted to characterize sDQ2 molecules produced in S2 cells in more detail.
By SDS-PAGE analysis under reducing conditions, we observed a strong band at 50 kDa in addition to the bands of the sDQ2 α- and β-chains. A similar type of contamination has not been observed with other soluble HLA molecules produced in baculovirus infected Sf9 insect cells, nor in the wild type EBV-DQ2 produced by EBV-transformed B cells. This contaminating protein was identified as the D. melanogaster-derived DCB-45 protein and our findings suggest a tight interaction between sDQ2 and DCB-45. The complex does not dissociate under the condition of high pH and high salt concentrations used for elution from the antibody affinity column (pH 11.5 and 2 M Tris), and treatment at low pH resulted in precipitation rather in dissociation of the two proteins. In contrast, the sDQ2 molecules found in the large aggregates (>600 kDa) apparently are not associated with DCB-45, suggesting that sDQ2 is unstable in the absence of DCB-45. The sDQ2/DCB-45 interaction thus likely explains the observed high expression yield of peptide-receptive sDQ2.
DCB-45 is a highly acidic protein (pI 4.27) and HLA-DQ2 has a strong preference for binding negatively charged residues in several of its anchor positions (Kim et al. 2004). We therefore investigated whether the sDQ2/DCB-45 binding is also involving the peptide binding groove of sDQ2. The characterization of such a strong interaction could be helpful to design new lead structures for high affinity HLA-DQ2 blockers. Such reagents have been discussed in the treatment of celiac disease to inhibit HLA-DQ2-rectricted presentation of gluten derived epitopes (Bergseng et al. 2005; Xia et al. 2006, 2007).
In the presence of HLA-DM, binding of the known HLA-DQ2 ligand P198 to the isolated sDQ2/DCB-45 complex increased. The same effect of HLA-DM was observed after limited proteolysis of this complex leading to almost exclusive degradation of DCB-45 and a markedly increase of heterodimeric sDQ2 (Fig. 2). Analysis by size exclusion chromatography showed that this fraction was stable. Interestingly, DCB-45-derived peptides could be released at low pH and were found to originate from the N-terminal 54-mer (Fig. 3). As these findings strongly indicate a specific interaction of this region with the sDQ2 peptide binding groove, a more detailed mapping was performed. Using detergent-solubilized EBV-DQ2, the pentadecapeptide HNAQFDHEAFLGPDE was found as the best binder with an affinity similar to that of the P198 ligand. The data also suggest the sequence AQFDHEAFL as the 9-mer core region with negative charges in positions P4 and P6 and a large hydrophobic residue in position P9 that are favorable for interaction with the sDQ2 peptide binding groove. This sequence contains alanine in position 1, which is not expected to be favorable due to its small size and the preference that HLA-DQ2 has for bulky hydrophobic anchor residues at this position (van de Wal et al. 1996; Vartdal et al. 1996). Notably, peptide binding to HLA-DQ2 is characterized by contributions from anchor residues in position 1, 4, 6, 7, and 9 (Kim et al. 2004). This is in contrast to peptide binding to HLA-DR molecules which has a dominant contribution of the P1 anchor (Jardetzky et al. 1990; Stern et al. 1994). Given that the peptide AQFDHEAFL has optimal anchor residues at positions P4 (D), P6 (E), and P9 (L), an alanine residue at P1 with no repulsive effect may well be compatible with the high affinity binding of this ligand. Moreover, in silico searching for possible binding registers based on binding studies with peptide libraries (Jüse et al., unpublished) gave no better candidates for high affinity HLA-DQ2 ligands within the 54-mer region. The DCB-45-derived 15-mer HNAQFDHEAFLGPDE is a good HLA-DQ2 binder, and we found that it is binding to HLA-DQ2 with a higher affinity than the HLA-DQ2-α-Ι-gliadin peptide that is an immunodominant epitope of celiac lesion derived CD4 T cells. Work parallel to this study (Xia et al. 2007) has demonstrated that even higher binding affinity than that of the DCB-45-derived pentadecapeptide as well as high proteolytic stability are required to obtain HLA-DQ2 blockers that effectively prevent activation of gliadin reactive T cells. Several chemical modifications that improve HLA-DQ2 binding affinity and proteolytic resistance would thus be required to make the DCB-45-derived ligand becoming an effective HLA-DQ2 blocker.
The observations presented in this paper are relevant for anyone aiming to express MHC class II molecules in S2 cells. DCB-45 is influencing the availability of free and functional peptide binding site of recombinant sDQ2, resulting in a decreased peptide binding capacity of the expressed molecules. Our findings illustrate that de novo interaction of recombinant MHC class II molecules with proteins derived from the expression system can occur. Prior to large scale use in peptide binding studies, such MHC class II molecules should be carefully characterized and compared with respect to peptide binding specificity and capacity to the wild type molecules, which are, e.g., purified from Epstein–Barr virus-transformed B lymphoblastoid cell lines.
This work has been funded as part of a European Commission Marie Curie Research Training Network (MRTN-CT-2004-512385). We would like to thank Elizabeth D. Mellins, Stanford University, for providing S2 cells producing HLA-DM and Chu-Young Kim, Stanford University, for synthesizing some of the peptides.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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