Directed evolution of a single-chain class II MHC product by yeast display

Scott E. Starwalt1, Emma L. Masteller2, Jeffrey A. Bluestone2 and David M. Kranz1,3

1 Department of Biochemistry, University of Illinois, 600 S. Matthews Avenue, Urbana, IL 61801 and 2 The Diabetes Center, University of California–San Francisco, San Francisco, CA 94143, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC). The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus. For example, T cells from non-obese diabetic mice recognize specific ‘diabetogenic’ peptides bound to a class II MHC allele called I-Ag7. The I-Ag7 molecule is noted for being unstable and difficult to work with, especially in soluble form. In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-Ag7. A library containing mutations at two residues (positions 56 and 57 of the I-Ag7 ß-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57. Random mutation of I-Ag7 followed by selection with an anti-I-Ag7 antibody also yielded stabilized variants with mutations in other residues. The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases.

Keywords: antigens/class II/major histocompatibility complex/protein stability/yeast display


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A number of autoimmune diseases have been linked to class II proteins encoded by the major histocompatibility complex (MHC). Type 1 diabetes, or insulin-dependent diabetes mellitus, is a T cell-mediated disease that results in autoimmune destruction of pancreatic ß cells leading to hyperglycemia. A breakdown in T cell tolerance or suppression of self-reactivity appears to allow the proliferation of autoreactive T cells against ß cell self-antigens. There are several self-antigen candidates that could initiate the disease, including insulin B-chain (Daniel and Wegmann, 1996aGo,Daniel and Wegmann, 1996bGo; Yu et al., 2000Go), glutamic acid decarboxylase 65 (GAD65) (Herman et al., 1999Go; Jean-Francois and Lucienne, 2001Go; Judkowski et al., 2001Go), heat shock protein 60 (Bockova et al., 1997Go; Elias et al., 1990Go, 1991Go) and I-A2 (Lampasona et al., 1996Go; Bearzatto et al., 2002Go), a tyrosine phosphatase associated with the ß cell membrane. Peptides from these self-antigens are processed, bound to membrane-associated proteins encoded by the MHC and the peptide–MHC complex is presented to T cells. Insulitis, or the infiltration of lymphocytes into the pancreatic islets, is observed prior to the destruction of the ß cells, although the precise etiology remains to be elucidated. Similar symptoms are observed in non-obese diabetic (NOD) mice (Makino et al., 1980Go; Kikutani and Makino, 1992Go), the mouse model of type 1 diabetes.

Genetic linkages to the heterodimeric {alpha}ß class II MHC alleles, I-Ag7 (Wicker et al., 1995Go) and DQ8/DQ2 (Todd et al., 1988Go; She, 1996Go; Sonderstrup and McDevitt, 2001Go) in the NOD mouse and humans, respectively, have been characterized. I-Ag7 shares the same {alpha}-chain as the non-linked murine class II MHC allele, I-Ad (Singer et al., 1993Go) but it differs from I-Ad by 17 residues in the ß-chain. Two of these residues, ß56 and ß57, have been shown to be essential for disease susceptibility (Acha-Orbea and McDevitt, 1987Go). Replacement of Hisß56 and Serß57 in I-Ag7 with the non-disease-linked I-Ad residues, Proß56 and Aspß57 reduced the incidence of disease in NOD mice (Lund et al., 1990Go; Quartey-Papafio et al., 1995Go). It has also been shown that an aspartic acid residue at position ß57 of human class II MHC alleles confers resistance to disease onset, whereas an alanine residue at this position is associated with disease susceptibility (Todd et al., 1987Go).

It has been suggested that the association of class II genes with disease is related to the instability of the class II proteins. SDS stability studies showed that I-Ag7 is unstable compared with non-disease-linked class II alleles and that it has a shorter half-life when expressed on antigen presenting cells (Carrasco-Marin et al., 1996Go). Structural studies revealed that a salt bridge exists between Aspß57 and Arg{alpha}76 in non-disease-linked alleles (Scott et al., 1998Go). The absence of this salt bridge in I-Ag7 was proposed to destabilize the {alpha}ß heterodimer, perhaps contributing to its diabetogenic character. Recent crystal structures (Corper et al., 2000Go; Latek et al., 2000Go) and peptide binding studies (Gregori et al., 2000Go; Stratmann et al., 2000Go; Judkowski et al., 2001Go) prompted the suggestion that other features might also be important for disease susceptibility. For example, the unique peptide-binding groove of I-Ag7, most noticeably the P9 pocket, influences the presentation of diabetogenic peptides to autoreactive CD4+ T cells in the NOD mouse (Suri et al., 2002Go). The DQ8 peptide-binding groove is similar to the peptide-binding groove of I-Ag7 (Lee et al., 2001Go), providing structural support for the presentation of similar diabetogenic self-antigens in humans and NOD mice.

Although progress has been made in deciphering aspects of I-Ag7 peptide specificity, little is known about the autoreactive CD4+ T cell subsets that trigger the disease. Current approaches have used I-Ag7/GAD tethered peptide tetramers to isolate GAD-specific autoreactive CD4+ T cells (Liu et al., 2000Go). GAD specific T cells, however, were only detected in vivo upon expansion of autoreactive T cell subsets following peptide immunization. The ability to rationally design and engineer peptide-class II MHC complexes could facilitate a number of avenues of investigation. For example, stable I-Ag7 tetramers or Ig dimers (Howard et al., 1999Go) tethered to various diabetogenic peptides might be capable of detecting autoreactive T cell populations in unprimed NOD mice. Peptide-MHC class II chimeras, linked to an IgG scaffold, have recently been shown to induce tolerance in an I-Ed restricted type 1 diabetes mouse model (Casares et al., 2002Go) and the ability to engineer peptide-tethered I-Ag7 chimeras of increased stability could be useful in such tolerogenic studies. In other diseases, functional human, HLA-DR1 (Zhu et al., 1997Go) and rat RT1.B (Burrows et al., 2000Go) single-chain class II MHC molecules (peptide–linker–ß-chain–linker–{alpha}-chain) have been shown to inhibit superantigen-mediated septic shock and encephalitogenic T cell proliferation, respectively. Directed evolution of single-chain class II MHC molecules could potentially enhance the effectiveness of these agents.

Yeast display is a system for directed evolution in which a protein of interest is fused as a single-chain polypeptide to the yeast AGA2 agglutinin mating factor (Boder and Wittrup, 1997Go, 2000Go). The ability to use yeast display to manipulate and isolate proteins in vitro using fluorescence-activated cell sorting (FACS) offers some advantages over other directed evolution strategies such as phage display (Wittrup, 2001Go). Yeast display has been used recently to engineer single-chain antibodies (scFvs) (Kieke et al., 1997Go; Boder et al., 2000Go) and single-chain T cell receptors (scTCRs) (Shusta et al., 1999Go; Holler et al., 2000Go; 2002Go) of increased affinity and stability. The direct relationship between stability and yeast cell surface levels of the fused protein (Shusta et al., 1999Go, 2000Go) provides an attractive method for the genetic engineering of stabilized single-chain class II MHC molecules.

In this work, the yeast display system was used to isolate single-chain I-Ag7 variants that were expressed at higher surface levels than the wild-type (wt) I-Ag7. The variants were produced either by focusing libraries of mutants on residues 56ß and 57ß or on all residues randomly throughout the entire class II construction. Mutants were selected by flow cytometric sorting with a conformational antibody, 10.2.16, against I-Ag7. Substitutions of preferred residues (e.g. Glu56 and Leu57) at positions 56ß and 57ß yielded stabilized variants. None of these stabilized variants were recognized by a different antibody, AG2.42.7, that is specific for the I-Ag7 allele, indicating that these residues are within the epitope of this antibody and thus explaining its specificity for I-Ag7. Several randomly mutated variants selected with antibody 10.2.16 were expressed at higher surface levels and were also reactive with antibody AG2.42.7. These variants represent useful candidates for retaining the peptide binding and T cell stimulatory capabilities of wt I-Ag7, yet with enhanced stability. The findings show that residues 56ß and 57ß are directly associated with class II MHC stability and that yeast display can be used to engineer class II MHC molecules. The identification of stabilized class II MHC molecules can provide insight into the molecular basis of type 1 diabetes and could serve as diagnostic and therapeutic agents.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines and antibodies

Ltk cells transfected with genes encoding the I-Ag7 ß- and I-Ad {alpha}-chains (Peterson and Sant, 1998Go) were kindly provided by Andrea Sant (University of Chicago). Transfected and non-transfected Ltk cell lines were cultured in DMEM medium containing 5% fetal bovine serum, 100 U/ml penicillin/streptomycin, 3% HEPES buffer and 0.5% glutamine at 37°C in 10% CO2. Transfectants were maintained in blasticidin S HCl (Invitrogen, Carlsbad, CA) at 10 µg/ml. Monoclonal antibody 10.2.16 (Oi et al., 1978Go), specific for I-Ag7 and I-Ak, was purified from hybridoma supernatants by filtering through a 0.22 µm filter followed by purification over protein A agarose (Invitrogen). Monoclonal antibodies AG2.42.7 and AG2.27.5 (Suri et al., 2002Go), which recognize the I-Ag7 ß-chain, were kindly provided by Paul Allen (Washington University, St. Louis, MO). Antibody AG2.42.7 is specific for the I-Ag7 allele, whereas AG2.275 is cross-reactive with I-Ag7, I-Ak, I-Az, I-Au and I-As alleles. Biotin-labeled anti-c-myc monoclonal antibody 9E10 was purchased from Berkeley Antibody (Richmond, CA). Anti-hemagglutinin (HA) monoclonal antibody 12CA5 was purchased from Boehringer Mannheim (Indianapolis, IN). FITC-labeled F(ab')2 goat anti-mouse, {gamma}2b chain specific, IgG2b was purchased from Southern Biotechnology Associates (Birmingham, AL) and streptavidin–phycoerythrin (SA–PE) conjugate was purchased from PharMingen (San Diego, CA).

Engineering single-chain class II MHC, I-Ag7

Drosophila pRMHa3 plasmids that encode the I-Ag7 ß- and I-Ad {alpha}-chains were provided by Nicolas Glaichenhaus (Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne, France) and served as templates for PCR cloning. The I-Ag7 ß-chain was PCR amplified using a forward primer (5'-ATT GCA GCT AGC GGT GGA CTT AAG GGT GGC GGC GGT TCT TTA GTT CCA AGA GGT TCT GGT GGC GGT GGC TCT GGA GAC TCC GAA AGG CAT TT-3') incorporating an NheI restriction site and a 16 amino acid residue linker (Gly4–Ser–Leu–Val–Pro–Arg–Gly–Ser–Gly4–Ser) (Kozono et al., 1994Go) upstream of the ß-chain. The reverse primer (5'-TCC GCC ACC TCC AGA ACC TCC TCC GCC CCT CCA CTC CAC AGT GAT GGG–3') encoded a 9 amino acid residue linker (Gly4–Ser–Gly4) (Zhu et al., 1997Go) downstream of the ß-chain. The I-Ag7 {alpha}-chain was amplified using a forward primer (5'-GGC GGA GGA GGT TCT GGA GGT GGC GGA GAA GAC GAC ATT GAG GCC-3') that encodes the same 9 amino acid residue linker upstream of the ß-chain and a reverse primer (5'-ATT TGC AGATCT TTA TCA CAA GTC TTC TTC AGA AAT AAG CTT TTG TTC CCA GTG TTT CAG AAC CGG CTC-3') that encodes a c-myc epitope tag and a BglII diagnostic site downstream of the {alpha}-chain. PCR SOEing (splicing by overlap extension) (Horton et al., 1989Go; Warrens et al., 1997Go) was used to fuse the I-Ag7 ß-chain and {alpha}-chain PCR products through an additional amplification step with the ß-chain forward primer and the {alpha}-chain reverse primer. A construct that contains an N-terminal peptide, GAD65(78–97) [KPCNCPKGDVNYAFLHATDL], was generated by PCR amplification of the scI-Ag7 fusion product using a forward primer (5'-ATT GCA GCT AGC AAA CCA TGT AAT TGT CCA AAA GGT GAT GTT AAT TAT GCT TTT TTG CAT GCT ACT GAT CTT AAG GGT GGC GGC GGT TCT TTA GTT CCA-3') that encoded the GAD65(78–97) peptide between NheI and AflII restriction sites upstream of the ß-chain and the {alpha}-chain reverse primer. Both the scI-Ag7 construct and the GAD65(78–97) containing scI-Ag7 construct were digested with NheI and BglII and ligated to NheI–BglII-digested yeast surface display vector pCT302. The ligation mixtures were transformed into DH10B Escherichia coli (Invitrogen, Carlsbad, CA) and transformants were plated on LB plates supplemented with ampicillin at 100 µg/ml. Cassette ligations were used to generate insulin B (9–23) [SHLVEALYLVCGERG] scIAg7 and BDC2.5(A) [GKKVAAPAWARMG] scI-Ag7 constructs. BDC2.5(A) peptide and insulin B (9–23) peptide sense (5'-CTA GCG GTA AAA AGG TTG CTG CAC CAG CTT GGG CTC GTA TGG GTC-3'; 5'-CTA GCT CTC ATT TGG TTG AAG CTT TGT ATT TGG TTT GTG GTG AAA GAG GTC-3') and anti-sense (5'-TTA AGA CCC ATA CGA GCC CAA GCT GGT GCA GCA ACC TTT TTA CCG-3'; 5'-TTA AGA CCT CTT TCA CCA CAA ACC AAA TAC AAA GCT TCA ACC AAA TGA GAG-3') 5'-phosphorylated oligonucleotides with NheI and Afl II restriction site overhangs were mixed in equimolar ratios. Peptide specific forward and reverse primers were incubated at 100°C for 1 min and allowed to anneal at 25°C to generate the peptide-encoded cassettes. The cassettes were ligated to NheI–Afl II-digested GAD65(78–97) scI-Ag7/pCT302 and transformed into DH10B E.coli. Plasmid DNA was sequenced and transformed using lithium acetate (Gietz et al., 1995Go) into the yeast strain EBY100 (Boder and Wittrup, 1997Go).

Flow cytometry

Yeast transformed with various scI-Ag7 constructions was grown in SD-CAA [2% dextrose, 0.67% yeast nitrogen base, 1% casamino acids (Difco, Sparks, MD)] at 30°C for 18–20 h. To induce AGA2 fusion protein expression, yeast was pelleted by centrifugation, suspended to an OD600 of ~1.0 in SG-CAA (2% galactose, 0.67% yeast nitrogen base, 1% casamino acids) and incubated at 20°C. After ~18–20 h, cultures were harvested and ~107 cells/tube were incubated on ice, washed with PBS (10 mM NaH2PO4, 150 mM NaCl, pH 7.3) that contained 0.5% bovine serum albumin (BSA) and incubated for 1 h with 25 ml of antibody staining reagent. The antibodies consisted of 10 µg/ml anti-HA mAb 12CA5 (Boehringer Mannheim, Indianapolis, IN), anti-c-myc mAb 9E10 (1:50), 10 µg/ml anti-I-Ag7 mAb 10.2.16, 10 µg/ml anti-I-Ag7 mAb AG2.42.7 or 10 µg/ml anti-I-Ag7 mAb AG2.27.5. Cells were washed with PBS 0.5%BSA and incubated for 1 h on ice with FITC-labeled F(ab')2 goat anti-mouse IgG (1:50) (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Labeled cells were washed with PBS–0.5% BSA and analyzed on a Coulter Epics XL instrument.

Generation of mutated scI-Ag7 yeast libraries

Random and site-directed DNA mutagenesis strategies were used to generate scI-Ag7 libraries. The random mutagenesis strategy used error-prone PCR, followed by yeast homologous recombination to clone into the pCT302 yeast display vector. Briefly, the scI-Ag7 constructs were amplified using a flanking AGA-2-specific upstream primer (5'-GGC AGC CCC ATA AAC ACA CAG TAT-3') and a downstream primer (5'-TAA TAC GAC TCA CTA TAG GG-3') such that ~100 bp upstream of the I-Ag7 insert and ~300 bp downstream of the I-Ag7 insert extended into the display vector (pCT302). Random nucleotide errors were incorporated using Taq polymerase (Invitrogen) in the presence of 2 mM MgCl2 and 0.3 mM MnCl2. The site-directed mutagenesis strategy used PCR SOEing reaction with degenerate PCR primers to mutate the ß56 and ß57 residues of scI-Ag7 ß-chain, followed by yeast homologous recombination (Orr-Weaver and Szostak, 1983Go; Raymond et al., 1999Go). Homologous recombination uses the yeast recombination machinery important in double-stranded gap repair, thereby allowing the direct cloning and transformation of linear DNA products (Suzuki et al., 1983Go). The cloning process involved a PCR SOEing step with a forward primer degenerate at codons ß56 and ß57 (5'-TAC CGC GCG GTG ACC GAG CTC GGG CGG NNS NNS GCC GAG TAC TAC AAT AAG C-3', where N is any nucleotide and S is C or G) and a reverse primer (5'-CCG CCC GAG CTC GGT CAC CGC GCG GTA CTC GCC CAC GTC G-3') complementary to the 18 nucleotides at the 5' end of the forward primer. These primers were used together with the I-Ag7 flanking primers described above to amplify the entire scI-Ag7 ß56ß57 construct. Underlined bases indicate the position of a silent mutation that incorporates a SacI restriction site in order to identify recombinants.

For homologous recombination and transformation, 150 ng of random or site-directed mutagenized PCR product and 150 ng of NheI–BglII-digested 7M–scFv-4F10 pcT302 vector (Griffin et al., 2001Go) were combined and transformed into EBY100 yeast cells by electroporation. Ten to twenty independent transformations were pooled in ~250 ml of SD-CAA and grown at 30°C for 48 h. Plasmids from isolated clones from each mutagenic strategy were rescued and sequenced to verify successful mutagenesis. Approximately 4–7 nucleotide errors per 1000 base pairs were incorporated into each construction using the error-prone mutagenic approach.

Flow cytometric sorting and analysis

Yeast cell libraries generated by random mutagenesis were incubated with 25 µl of anti-I-Ag7 antibody 10.2.16 (10 µg/ml), washed with PBS–0.5% BSA and incubated with FITC-labeled F(ab')2 goat anti-mouse IgG (1:50). After washing, samples were sorted in purification mode (i.e. coincident negative cells rejected) using a Cytomation MoFlo sorter with an event rate of ~50 000 cells/s. A total of 2x107 cells were examined during the first sorting round, collecting ~1% of the population. Collected cells were cultured at 30°C in SD-CAA followed by induction in SG-CAA to induce protein expression. After three additional rounds of sorting with anti-I-Ag7 antibody 10.2.16, the sorted scI-Ag7 libraries were incubated with 25 µl of anti-c-myc antibody 9E10 (1:50), washed with PBS–0.5% BSA, incubated with FITC-labeled F(ab')2 goat anti-mouse IgG and sorted, collecting the top 0.25% of the population. Sorted cells were plated on selective glucose medium to isolate individual clones.

A sequential sorting method that took only a single day was used to select the ß56ß57 site-directed library. The yeast cell library was stained with 12.5 µl of anti-IAg7 antibody 10.2.16 (10 µg/ml) and 12.5 µl of biotin-labeled anti-c-myc antibody 9E10 (1:100), washed with PBS–0.5% BSA and incubated with 12.5 µl of FITC-labeled F(ab')2 goat anti-mouse, {gamma}2b chain specific, IgG2b (1:50) and SA–PE conjugate (1:100). A total of ~2x107 cells were examined during the first sorting round, in purification mode, collecting ~1% of the population. The collected cells were sequentially sorted twice more on the same day, collecting the top ~1% of the population. The cells collected from the third sort were plated onto selective glucose plates and cultured for ~48 h.

Isolated clones were examined by flow cytometry on a Coulter Epic flow cytometer. Two million cells were incubated with a total volume of 25 µl of primary antibody, followed by the appropriate secondary reagent, as described above.

Plasmid rescue and sequencing

Plasmids were rescued from yeast using a Zymoprep miniprep kit (Zymo Research, Orange, CA). Plasmid DNA was transformed into E.coli DH10B (Invitrogen) competent cells by electroporation. Transformations were plated on LB plates supplemented with 100 µg/ml ampicillin and cultured overnight at 37°C. Individual colonies were picked and cultured in 2 ml of LB-AMP (100 mg/ml) at 37°C overnight. QIAprep spin miniprep kits (Qiagen, Valencia, CA) were used to isolate plasmid DNA. Sequencing reactions were performed using the scI-Ag7 flanking primers described above and a scI-Ag7 ß-chain specific primer, scI-Ag7 {alpha} LNK (5'-CCA GGA CAG AGG CCC TCA AC-3'), using fluorescence automated sequencing performed by the University of Illinois Biotechnology Center.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Analysis of yeast surface expression of wild-type single-chain I-Ag7

Previous studies have shown that an unstable single-chain T cell receptor (TCR) and antibody Fv fragment could not be expressed on the surface in a yeast display system (Kieke et al., 1999Go; Griffin et al., 2001Go). However, selection of a mutated library of the TCR or scFv for display on the surface yielded mutants with greater thermal stability (Kieke et al., 1999Go; Shusta et al., 1999Go, 2000Go; Griffin et al., 2001Go). To examine a class II protein in the yeast display system, a gene encoding a single-chain scI-Ag7 construct was cloned into the yeast pCT302 display vector as AGA2 polypeptide fusions downstream of an inducible GAL1-10 promoter (Boder and Wittrup, 1997Go; Holler et al., 2000Go) (Figure 1AGo). In addition, the genes encoding three different peptides that bind to I-Ag7 were also cloned at the N-terminus of the I-Ag7 single-chain construction (Figure 1BGo). The three peptides included GAD65(78–97) (sequence KPCNCPKGDVNYAFLHATDL), insulin B (9–23) (sequence SHLVEALYLVCGERG) and BDC2.5(A) (sequence GKKVAAPAWARMG).



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Fig. 1. Schematic diagram of single-chain I-Ag7 fusions. (A) Extracellular domains of the I-Ag7 ß- and I-Ad {alpha}-chains were joined by linker 2 (Gly4–Ser–Gly4) and fused in-frame to the AGA2 protein (not shown), a hemaglutinin (HA) epitope tag (YPYDVPDYA) and linker 1 (Gly4–Ser–Leu–Val–Pro–Arg–Gly–Ser–Gly4–Ser). A c-myc epitope tag (EQKLISEEDL) was fused at the C-terminus, followed by a stop codon. (B) Three different I-Ag7-binding peptides were produced as N-terminal fusions of the scI-Ag7. These included the peptides GAD65(78–97) [KPCNCPKGDVNYAFLHATDL], insulin B (9–23) [SHLVEALYLVCGERG] and BDC2.5(A) [GKKVAAPAWARMG].

 
Analysis of hemagglutinin (HA) and c-myc epitope tags that flank the scI-Ag7 constructions by flow cytometry allowed the characterization of yeast surface expression levels. A positive staining population was observed with the anti-HA antibody in all four constructions (Figure 2Go). The unshaded histograms in this and subsequent figures represent cells that were stained only with the secondary antibodies. The negative populations seen in histograms are yeast cells in a particular stage of the cell cycle that do not express high levels of the fusion protein. This negative population has been observed for all yeast displayed proteins (Boder and Wittrup, 1997Go; Kieke et al., 1997Go). Binding was not observed with the antibody to the C-terminal c-myc tag or with the anti-I-Ag7 monoclonal antibody 10.2.16 (Figure 2Go). Our previous studies of the TCR have shown that the inability to detect the fused protein is due to the fact that proteolytic cleavage has occurred to the carboxy end of Aga-2 and the HA tag (Shusta et al., 1999Go). This cleavage occurs as a result of unstable protein domains associated with the AGA2 fusion. It is possible to generate mutants of the fused proteins that exhibit higher surface levels and greater thermal stability (Kieke et al., 1999Go; Shusta et al., 1999Go, 2000Go; Griffin et al., 2001Go). These stabilized mutants can be isolated from a random library of mutants by selection for yeast with higher surface levels of the fused protein.



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Fig. 2. Flow cytometric analysis of yeast transformed with various wild-type scI-Ag7 constructs. Yeast that contain plasmids encoding the wild-type scI-Ag7 (scI-Ag7WT) or the peptide-linked constructs described in Figure 1Go were stained with three different antibodies: anti-HA mAb 12CA5 (10 µg/ml), anti-c-myc mAb 9E10 (10 µg/ml) or anti-I-Ag7 ß-chain specific antibody 10.2.16 (10 µg/ml). Antibody binding was detected with FITC-labeled F(ab')2 goat-anti-mouse IgG (1:50) and surface expression levels were determined by flow cytometry (shaded). Controls with only the secondary reagent FITC-labeled F(ab')2 goat-anti-mouse IgG are also shown (unshaded).

 
Mutagenesis strategy and library construction

In order to use the yeast display system to explore the issue of stability and the I-Ag7 molecule, two DNA mutagenic strategies were used to generate scI-Ag7 libraries. Because residues ß56 and ß57 have been linked to both disease susceptibility and instability of I-Ag7, we targeted these residues using site-directed mutagenesis to examine if substitutions in these residues would allow display (i.e. stabilization) of I-Ag7. Mutagenic primers containing degenerate codons at positions ß56 and ß57 were used to amplify the BDC2.5(A)WT construction and a PCR SOEing method (Horton et al., 1989Go; Warrens et al., 1997Go) was used to generate full-length BDC2.5(A) scI-Ag7 position ß56ß57 mutants.

Residues ß56 and ß57 have also recently been shown to be critical to the formation of the P9 pocket (Corper et al., 2000Go; Latek et al., 2000Go) and in fact I-Ag7 variants that express I-Ad residues Proß56 and Aspß57 present different peptide subsets on antigen-presenting cells (Suri et al., 2002Go). Hence it is reasonable to predict that scI-Ag7 ß56ß57 variants could be defective in the ability to bind and present diabetogenic peptides. Accordingly, it would be desirable to use an alternative strategy for identifying mutations that stabilize the I-Ag7 molecule but reside outside of residues 56 and 57. Toward this possibility, we employed a random mutagenesis approach. The GAD65(78–97)WT and InsB(9–23)WT constructions were amplified under error-prone conditions of PCR using flanking primers. Conditions were adjusted to incorporate ~5–10 random nucleotide errors per single-chain class II MHC coding sequence. Yeast homologous recombination was used to generate the scI-Ag7 mutagenic libraries of >106 transformants.

Selection of single-chain I-Ag7 variants by fluorescence-activated cell sorting

The two randomly mutated I-Ag7 libraries were sorted through four cycles with anti-I-Ag7 antibody 10.2.16 followed by FITC-labeled F(ab')2 goat-anti-mouse IgG to collect the top 1% of the yeast population. In each cycle, yeast cells collected from the previous sort were cultured and protein expression was induced. A significant enrichment in an anti-I-Ag7 antibody-positive population was observed by the fourth sort (data not shown). To ensure that the isolated I-Ag7 variants were not truncated, the randomly mutated I-Ag7 libraries were also sorted using anti-c-myc antibody 9E10 and the top 0.25% of this population was collected, plated on selective media and colonies were isolated.

The ß56ß57 site-directed I-Ag7 library was sorted in a rapid, sequential, one-day selection scheme termed ‘serial’ sorting (Figure 3AGo). This more rapid approach was used because the possible structural diversity in the ß56ß57 library (20x20 = 400 combinations of ß56ß57 structures) is considerably less than the random library and hence it should be more amenable to a sequential sort. The ß56ß57 library was sorted after staining with anti-I-Ag7 antibody 10.2.16 and biotin-labeled anti-c-myc antibody 9E10 followed by FITC-labeled F(ab')2 goat-anti-mouse {gamma}2b-specific and SA–PE secondary reagents. The top 1% of positive yeast cells was collected in each of three sequential sorts and cells were directly plated on selective media. Unlike sorting of the random mutagenic library, the ß56ß57 site-directed library contained a detectable antibody-positive population in the unsorted library and after the first sort these positive cells were highly enriched (Figure 3AGo). These results suggest that diverse amino acid residues at positions 56 and 57 could yield surface expression.



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Fig. 3. Flow cytometry histograms of I-Ag7 ß56ß57 site-directed library and representative I-Ag7 variants selected from random or site-directed libraries. (A) The I-Ag7 ß56ß57 site-directed library was sorted with anti-IAg7 antibody 10.2.16 and biotin-labeled anti-c-myc antibody 9E10, followed by FITC-labeled F(ab')2 goat anti-mouse, {gamma}2b chain specific and SA–PE. Two-channel sorting of the initial yeast population (shown as FITC vs PE) was performed to isolate the top 1% fluorescent cells. The cells stained for the first (1), second (2) and third (3) sorts are shown with their reactivities for anti-IAg7 antibody 10.2.16 (FITC) and the anti-c-myc antibody (PE). (B) Mut1 from the random mutagenesis library and ß56ß57Mut3 from the ß56ß57 site-directed library were stained with anti-HA mAb 12CA5 (10 µg/ml), anti-c-myc mAb 9E10 (10 µg/ml) or anti-I-Ag7 ß-chain specific mAb 10.2.16 (10 µg/ml) followed by FITC-labeled F(ab')2 goat-anti-mouse IgG (1:50) and analyzed by flow cytometry. Binding in the presence (shaded) or absence (unshaded) of the primary antibody is shown.

 
Antibody analyses of isolated single-chain I-Ag7 variants

Twenty colonies isolated from the GAD65(78–97) random library, 20 colonies from the insulin B(9–23) random library and nine colonies from the BDC2.5(A) scI-Ag7 ß56ß57 site-directed library were screened for binding to the anti-HA, anti-c-myc and anti-I-Ag7 antibodies. Those clones that exhibited binding to the anti-I-Ag7 antibody above the wild-type levels were subjected to further flow cytometry and sequencing analysis. Representative histograms of two of the clones are shown in Figure 3BGo. In contrast to the wild-type constructions (Figure 2Go), the mutants showed positive populations with both the anti-c-myc and anti-I-Ag7 antibodies. The mean fluorescence units of antibody binding for clones from the random libraries are shown in Figure 4AGo. Mean fluorescence units of antibody binding for clones from the ß56ß57 site-directed library are shown in Figure 4BGo. All of the mutant clones exhibited higher surface levels as detected with the anti-I-Ag7 reactive antibody 10.2.16 and most showed higher surface levels with anti-c-myc antibody 9E10. The correlation between the levels of binding by the two antibodies was consistent with clones from the site-directed library but less consistent with clones from the error-prone library. The latter may reflect the possibility that multiple mutations (see below) could affect the binding affinities of these antibodies, through direct or indirect effects on their epitopes.



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Fig. 4. Antibody binding analysis of random or site-directed I-Ag7 variants. Total mean fluorescence units of histograms as shown in Figure 3Go (shaded) were determined for each of the variants isolated from the random libraries (A) or from the ß56ß57 site-directed libraries (B). Binding levels of anti-I-Ag7 antibody 10.2.16 (white) and anti-c-myc mAb 9E10 (black) for the yeast-displaying variants are compared to EBY100 control yeast (not transformed) and several wild-type scI-Ag7 yeast.

 
Anti-I-Ag7 antibody 10.2.16 is also cross-reactive with the I-Ak allele (Oi et al., 1978Go) and therefore it is less specific for the allelic substitutions characteristic of I-Ag7. To examine further the conformation and epitopes of I-Ag7 mutants, we examined the yeast clones with two other monoclonal antibodies, AG2.42.7 and AG2.275, that bind to I-Ag7 (Suri et al., 2002Go). Antibody AG2.42.7 is specific for the I-Ag7 allele, whereas AG2.275 is cross-reactive with I-Ag7, I-Ak, I-Az, I-Au and I-As alleles (P.Allen, personal communication). All three I-Ag7-specific antibodies bound to Ltk cells co-transfected with the I-Ag7 ß- and I-Ad {alpha}-chain genes (Figure 5AGo) but not to untransfected cells (data not shown). Flow histograms of two yeast displayed variants (Mut2 and ß56ß57Mut5) are shown in Figure 5BGo. All three antibodies, 10.2.16, AG2.42.7 and AG2.275, bound to Mut2 (shaded), compared with the control that included only secondary staining reagents (unshaded). In contrast, only antibodies 10.2.16 and AG2.275 and not the I-Ag7-specific antibody AG2.42.7 bound to ß56ß57Mut5. Other yeast displayed I-Ag7 variants were also analyzed by flow cytometry with antibodies 10.2.16, AG2.42.7 and AG2.275 and mean fluorescent units were determined as a measure of binding (random mutants in Figure 5CGo, left panel; ß56ß57 site-directed mutants in Figure 5CGo, right panel). The random mutated I-Ag7 variants Mut2, Mut4, InsB(9–23)Mut5 and InsB(9–23)Mut6 bound all three antibodies, whereas the variant Mut1 bound only antibodies 10.2.16 and AG2.275 (Figure 5CGo, left panel). Like ß56ß57Mut 5, none of the other ß56ß57 variants were recognized by the I-Ag7-specific antibody AG2.42.7. However, all of the ß56ß57 variants were recognized by antibodies 10.2.16 and AG2.275, indicating that properly folded I-Ag7 was present on the yeast surface. Thus, the epitope for antibody AG2.42.7 appears to include the wild type I-Ag7 residues (His and Ser) at positions 56 and/or 57.



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Fig. 5. Flow cytometric analysis of binding by various ß-chain specific monoclonal antibodies. (A) Ltk cells, transfected with genes encoding I-Ag7 ß- and I-Ad {alpha}-chains, were analyzed with anti-I-Ag7 antibodies 10.2.16 (hatched), AG2.42.7 (black) and AG2.275 (gray). Ltk cells stained only with FITC-labeled F(ab')2 goat-anti-mouse IgG are also shown (NEG; unshaded). Untransfected cells were negative for all antibodies (data not shown). (B) Representative flow cytometry histograms of two I-Ag7 variants selected from random or site-directed libraries. Mut2 and ß56ß57Mut5 were stained with antibody 10.2.16 that is specific for I-Ag7 and I-Ak, antibody AG2.42.7 that is specific for I-Ag7, and antibody AG2.275 that cross-reacts with various class II alleles including I-Ag7, I-Ak, I-Az and I-Au. (C) Total mean fluorescence units of histograms as shown in (B) (shaded) were determined for each of the variants isolated from the random libraries (left panel) or from the ß56ß57 site-directed libraries (right panel). Binding levels of antibody 10.2.16 (gray), antibody AG2.42.7 (black) and antibody AG2.275 (hatched) are shown.

 
Sequencing of single-chain I-Ag7 variants

Plasmids from each of the yeast displayed I-Ag7 variants were rescued and sequenced (Figure 6Go). Each of the variants derived from the randomly mutated libraries contained multiple (3–8) amino acid substitutions. Furthermore, several of the variants lacked the region that encodes the N-terminal linked peptide. The oligonucleotide region appears to have been eliminated during the homologous recombination step of library construction (a crossover event occurred between a GT nucleotide rich region of linker 1 and a similar region upstream of the tethered peptide; data not shown). Variants InsB(9–23)Mut5 and InsB(9–23)Mut6 retained the N-terminal-linked insulin peptide, with a single amino acid substitution (serine to proline at peptide position 1). The presence of multiple mutations in each variant makes predictions of the molecular basis of stabilization difficult, but examination of their location within the I-Ag7 structure may shed some light (see Discussion).



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Fig. 6. Sequence analysis of random and site-directed I-Ag7 variants. Only positions where mutations occurred are shown. Asterisk indicates residue is same as wild-type I-Ag7.

 
Each of the variants isolated from the ß56ß57 site-directed library contained substitutions that differed from the wild-type histidine and serine residues. Only one of the sequences (Glu56–Leu57) was isolated multiple times. Position 56 exhibited preferences for either a charged residue (negative or positive) or a glycine residue. Position 57 appeared to be preferentially selective for large hydrophobic residues. The consensus motif that appeared most often, and that was associated with surface display at the highest level, was the glutamic acid at position 56 and the leucine at position 57. The possible molecular basis of these and the other amino acids identified at positions 56 and 57 is discussed below. The identification of multiple different variants suggests that it is possible to generate variability in class II structure and stability at these two residues.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Both class I and II MHC products are non-covalently linked dimers that associate to form a peptide binding groove flanked by two {alpha}-helices and a ß-sheet floor. Class I MHC are encoded by a polymorphic heavy chain ({alpha}1, {alpha}2 and {alpha}3 domains) and an invariant light chain called ß2-microglobulin. The {alpha}1 and {alpha}2 domains form the {alpha}-helices and ß-sheet floor whereas the {alpha}3 and ß2m domains associate to provide structural support. Class II MHC products are encoded by two polymorphic subunits, {alpha} and ß. It has been known for some time that many class I products appear to be more stable than their class II counterparts. This is perhaps best exemplified by the fact that crystal structures and streptavidin/class I tetramers for class I molecules were amenable to analysis well before class II molecules (Brown et al., 1993Go; Stern et al., 1994Go; Hackett and Sharma, 2002Go). Some studies using covalently linked peptides have resolved some of the stability and expression problems (Crawford et al., 1998Go; Cunliffe et al., 2002Go), but difficulties remain with some class II products and peptides. This has been particularly the case with class II molecules refolded from E.coli for tetramer applications (Ferlin et al., 2000Go). Nevertheless, it has been proposed that class II molecules, particularly those associated with autoimmune diseases, exhibit an inherent instability that leads to unstable peptide–class II complexes and the predisposition to incomplete tolerance induction against several self-peptide–class II complexes (Ridgway and Fathman, 1999Go).

To examine further the issues that involve class II MHC stability, we describe here a method for directed evolution of class II proteins yielding class II variants that can be displayed on the surface of yeast. Because our previous studies have shown a direct relationship between surface display and protein stability (Shusta et al., 1999Go, 2000Go), we predict that these mutations have produced stabilized versions of the class II MHC product I-Ag7. In addition, we show that mutations in 56ß and 57ß, the two residues most directly associated with autoimmune diseases such as type I diabetes, could directly increase the display of the properly folded I-Ag7 molecule.

Consistent with a previous study (Suri et al., 2002Go), the histidine at position 56ß appears to be critical as an epitope for the anti-I-Ag7-specific antibody AG2.42.7. This conclusion is based on the finding that variant InsB(9–23)Mut5 contained the Ser57ßLeu mutation, as in variants ß56ß57Mut3, Mut5 and Mut6, but only InsB(9–23)Mut5 retained the ability to bind to AG2.42.7. Thus the substitutions in ß56ß57Mut3, Mut5 and Mut6 of glutamic acid, glycine and arginine residues, respectively, for the histidine at position 56 must be responsible for the loss of antibody binding. As histidine is exposed in I-Ag7 and pointing upward (Figure 7Go), this residue appears to be positioned for interaction with the antibody. It is interesting that Mut1 from the random library also did not bind to antibody AG2.42.7. As shown in Figure 7Go, this variant contains the ß-chain mutation Leu38ßMet at the same end of the molecule as residues 56ß and 57ß. It is reasonable to predict that this non-conservative substitution could affect the structure of the I-Ag7 molecule at the position of the AG2.42.7 epitope.



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Fig. 7. Selected residues of wild type I-Ag7 that were mutated in surface-displayed variants. Structure from C is shown (PDB code 1ES0).

 
The non-diabetes associated allele I-Ad contains an aspartic acid at position 57 in the ß-chain and one of the predominant stabilizing mutations was a glutamic acid at position 56. Structural and biochemical studies of I-Ag7 in comparison with I-Ad have suggested that these two residues are directly involved in the reduced stability and weaker peptide binding ability of I-Ag7 (Carrasco-Marin et al., 1996Go, 1999Go; Hausmann et al., 1999Go; Corper et al., 2000Go; Latek et al., 2000Go; Munz et al., 2002Go). It is possible that the longer side chain of Glu56ß could form a salt bridge with Arg{alpha}76 (as Asp57ß does in I-Ad), but this rationale could not be applied to those variants (ß56ß57Mut2 and Mut6) with lysine or arginine at position 56. Hence it appears that there are multiple solutions to stabilizing this region of the I-Ag7 molecule. It is likely that these mutations have affected not only the binding to antibody AG2.42.7, but also the peptides that normally associate with I-Ag7 and perhaps the interactions of the molecule with T cell receptors. It remains to be seen if it is possible to engineer I-Ag7 variants in positions 56 and 57 that would bind to both autoimmune peptides and the T cell receptors that are associated with disease pathogenesis. In this regard, it is impossible to know if the ß56ß57 variants have endogenous peptides bound to the yeast displayed proteins. Previous studies have shown that MHC proteins can readily bind to peptides from the media or cells and thereby stabilize the MHC complex. Alternatively, it is possible that the mutations may have stabilized the molecule in a conformation that does not require a bound peptide.

Our results with the random I-Ag7 libraries provide some evidence that it is possible to engineer residues in I-Ag7 other than ß56 and ß57 that affect stability. Mutants such as these may still have the potential to bind an array of I-Ag7 peptides. In this regard, most mutants bound to all three I-Ag7-reactive antibodies, including antibody AG2.42.7 that is I-Ag7-allele specific (Suri et al., 2002Go). Because these mutants contained between three and eight amino acid substitutions, it is difficult to correlate stabilization with a particular residue. However, several of the mutations could have such an effect, based on their position in the structure and/or the nature of the side-chain substitution (Figure 7Go). For example, hydrophobic surface-exposed residues mutated to charged (Trp153ßArg, Val128{alpha}Asp) or polar residues (Trp43{alpha}Ser). Several mutations lie at interfaces and may act by stabilizing domain to domain interactions. These include Thr120ßSer and Phe92{alpha}Leu that are located at the ß2:{alpha}2 interface and Trp153ßArg which is located near the ß2:{alpha}1 interface.

There are currently no antibody or high-affinity T cell receptor probes for specific peptide–I-Ag7 complexes. However, we believe that it will be possible to engineer such probes, as we have done for class I complexes (Holler et al., 2000Go, 2002Go). This would allow the selection of stabilized variants that have the appropriate peptide bound within the class II binding site. It may also be possible to engineer residues on the class II helices that increase the binding energy of the interaction with T cell receptors. Presumably the basal energies of these interactions have already been selected during thymic development and they represent a significant fraction of the total free energy (Manning and Kranz, 1999Go).

In addition to providing some insight into the stability of the I-Ag7 molecule, the present study shows that it is possible to display and evolve the class II MHC molecule, as has been done for antibodies, another highly diverse class of proteins. It is reasonable to predict that stabilized peptide–class II complexes would find a variety of uses. Although it is commonly accepted that T cells are the predominant mediators of disease, little is known about the autoreactive T cell subsets that initiate and carry out observed disease pathology. Stabilized peptide–class II complexes, perhaps in multivalent form, could serve as reagents to characterize and quantify these T cell subsets (Howard et al., 1999Go; Hackett and Sharma, 2002Go). The peptide–class II complexes could thus provide a new class of novel diagnostics that might be capable of monitoring disease progression. Furthermore, a stable peptide–class II product that contained an antagonist peptide ligand could be used as an agent to control T cell activity (Casares et al., 2002Go). Finally, the ability to engineer class II molecules may provide additional opportunities to study the structure and function of class II alleles that have proven to be problematic in expression or purification (Ferlin et al., 2000Go). We have shown that T cell receptor variants that are expressed at higher levels on the yeast surface are expressed at significantly higher levels in a secretion system (Shusta et al., 1999Go). Hence it may now be possible to produce selected class II products in secretion systems that have been heretofore unsuccessful.


    Notes
 
3 To whom correspondence should be addressed. E-mail: d-kranz{at}uiuc.edu Back


    Acknowledgments
 
We thank Barbara Pilas and Ben Montez of the University of Illinois Flow Cytometry Facility for flow cytometry assistance and sorting analysis. We also thank Andrea Sant for the Ltk cells transfected with the I-Ag7 ß- and I-Ad {alpha}-chain genes, Nicolas Glaichenhaus for plasmids that encode the I-Ag7 ß- and I-Ad {alpha}-chain genes and Paul Allen and David Donermeyer for providing monoclonal antibodies AG2.42.7 and AG2.27.5. This work was supported by grants from the Juvenile Diabetes Foundation (to J.A.B.) and NIH (Grant AI35990 to D.M.K.).


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Received June 30, 2002; revised November 12, 2002; accepted December 17, 2002.





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