1 Department of Neurology, 2 Department of Biochemistry and Molecular Biology and 4 Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, 3 Neuroimmunology Research and 6 Neurology Service, Veterans Affairs Medical Center and 5 Shriners Hospital for Children, Portland, Oregon 97201, USA
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Abstract |
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Keywords: ß11/drug design/immunotherapy/MHC class II/TCR ligand
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Introduction |
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Complexes of MHCantigen have been purified as detergent extracts of lymphocyte membranes (Sharma et al., 1991) and as associated recombinant proteins using baculovirus and bacterial expression systems (Nag et al., 1993Nag et al., 1996; Kozono et al., 1994
; Arimilli et al., 1995
; Rhode et al., 1996
). These two-chain, four-domain molecular complexes, after loading with selected peptide epitopes, have been demonstrated to interact with T cells in an antigen-specific manner (Matsui et al., 1991
; Nag et al., 1992
Nag et al., 1993; Nicolle et al., 1994
; Spack et al., 1995
). However, because of their size, heterodimeric structure and the presence of multiple disulfide bonds, class II MHC molecules present an inherently difficult in vitro folding problem. Furthermore, MHC class II molecules participate in other interactions during peptide presentation and TCR engagement. The
2 domain appears to contribute to ordered oligomerization in T cell activation (Konig et al., 1995
), and the ß2 domain contains a CD4 binding site that co-ligates CD4 when the MHCantigen complex interacts with the TCR
and ß chains (Fleury et al., 1991
; Cammarota et al., 1992
; König et al., 1992
; Huang et al., 1997
). Moreover, the ß1 domain has recently been implicated in CD4 binding, as shown by a lymphocyte binding assay using synthetic peptides (Brogdon et al., 1998
).
To develop a simple and effective agent that could bind selectively to the TCR, we have engineered small (approximately 200 amino acid residues) molecules consisting of the 1 and ß1 domains of the rat MHC class II RT1.B molecule genetically linked into a single polypeptide chain, with and without covalently coupled antigenic peptide (Figure 1
). We describe in this report the design, production and purification of MHC class II-derived `ß1
1' molecules that retain the biochemical properties required for TCR engagement. These findings anticipate the production of large amounts of TCR ligands for structural characterization and immunotherapeutic applications. Molecules with this design would be useful for studying binding specificity in vitro, for exploring primary TCR signalling events independent of co-stimulatory input, and for treating CD4+ T cell-mediated autoimmune disease in an epitope-specific manner.
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Materials and methods |
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Sequence alignment of MHC class II molecules from rat, human and mouse species provided a starting point for our studies. The program Sybyl (Tripos Associates, St Louis, MO) was used to generate graphic images using an O2 workstation (Silicon Graphics, Mountain View, CA) and coordinates deposited in the Brookhaven Protein Data Bank (Brookhaven National Laboratories, Upton, NY). Structure-based homology modeling was based on the refined crystallographic coordinates of human DR1 (Brown et al., 1993; Murthy and Stern, 1997
), murine I-Ek molecules (Fremont et al., 1996
) and scorpion toxins (Zhao et al., 1992
; Housset et al., 1994
; Zinn-Justin et al., 1996
). Amino acid residues in human DR1 (PDB accession numbers 1SEB, 1AQD) were substituted with the rat RT1.B side chains, and the peptide backbone was modeled as a rigid body during structural refinement using local energy minimization.
Cloning the ß 1 1 constructs
dsDNA fragments encoding RT1B 1 and ß1 domains were prepared from cloned RT1.B
- and ß-chain coding sequences (Syha et al., 1989
; Syha-Jedelhauser et al., 1991
) by PCR amplification. Generation of dsDNA encoding ß1
1 molecules with covalently coupled antigenic peptides was an extension of the cloning strategy recently described for production of empty ß1
1 molecules (Burrows et al., 1998
). Production of the covalent ß1
1peptide construct involved adding a 210 bp insertion sequence, what we have termed the `peptidelinker cartridge' DNA fragment, near the 5' end of the fragment encoding ß1
1. The primers used to generate the ß1
1peptidelinker cartridge DNA were the XhoI 5' oligo and the 5'-GAAATCCCGCGGGGAGCCTCCACCTCCAGAGCCT- CGGGGCACTAGTGAGCCTCCACCTCCGAAGTGCACCACTGGGTTCTCATCCTGAGTCCTCTGGCTCTTCTGTGGGGAGTCTCTGCCCTCAGTCC-3' (3'-MBP-7289/linker ligation oligo). The primers used to generate the 559 bp DNA fragment with a 5' overhang for annealing to the ß1
1peptidelinker cartridge cDNA were 5'-GCTCCCCGCGGGATTTCGTGTACCAGTTCAA-3' (5' peptidelinker ligation oligo); and the KpnI 3' oligo. Annealing and extension resulted in the 750 bp full-length ß1
1MBP-7289 construct. PCR was conducted with Taq polymerase (Promega, Madison, WI) and PCR products were isolated by agarose gel electrophoresis and purified using Gene Clean (Bio 101, Inc., La Jolla, CA). ß1
1 and ß1
1MBP-7289 cDNAs were moved into cloning vector pCR2.1 (Invitrogen, Carlsbad, CA) using Invitrogen's TA Cloning® kit. Modifications at the 5' and 3' ends of the ß1
1 and ß1
1MBP-7289 cDNAs were made for directional subcloning into pET21d+ (Novagen, Madison, WI; Studier et al., 1990
). These primers were 5'-CAGGGACCATGGGCAGAGACTCCCCA-3' (NcoI 5' oligo); 5'-GCCTCCTCGAGTTAGTTGACAGCTTGGGTT-3' (XhoI 3' oligo); and, to generate molecules having six histidine residues as a C-terminal extension for streamlining purification, 5'-CTCGAGTTAGTGGTGGTGGTGGTGGTGGTTGACAGCTTGGGTTGAATT-3' (His6-XhoI 3' oligo).
The primers used to generate the MBP-5569linker cartridge were 5'-TATTACCATGGGCAGAGACTCCTCCGGCAAGGATTCGCATCATGCGGCGCGGACGACCCACTACG- GTGGAGGTGGAGGCTCACTAGTGCCCC-3' (5' MBP-5569 oligo); 5'-GGGGCACTAGTGAGCCTCCACCTCCACCGTAGTGGGTCGTCCGCGCCGCATGATGCGAATCCTTGCCGGAGGAGTCTCTGCCCATGGTAATA-3' (3' MBP-5569 oligo). These were gel purified, annealed and then cut with NcoI and XhoI for ligation into ß11MBP-7289 digested with NcoI and XhoI, to produce a plasmid encoding the ß1
1MBP-5569 covalent construct.
The primers used to generate the CM-2linker cartridge were 5'-TATTACCATGGGCAGAGACTCCAAACTGGAA-CTGCAGTCCGCTCTGGAAGAAGCTGAAGCTTCCCTGGAACACGGAGGTGGAGGCTCACTAGTGCCCC-3' (5' CM-2 oligo); 5'-GGGGCACTAGTGAGCCTCCACCTCCGTGTTCCAGGGAAGCTTCAGCTTCTTCCAGAGCGGACTGCAGTTCCAGTTTGGAGTCTCTGCCCATGGTAATA-3' (3' CM-2 oligo). These were gel purified, annealed and then cut with NcoI and XhoI for ligation into ß11MBP-7289 digested with NcoI and XhoI, to produce a plasmid encoding the ß1
1CM-2 covalent construct. All of the constructs have been confirmed by DNA sequencing.
EExpression and in vitro folding of the ß 1 1 constructs
Escherichia coli strain BL21(DE3) cells were transformed with the pET21d+/ß11 and pET21d+/ß1
1/peptide vectors. Bacteria were grown in 1 l cultures to mid-logarithmic phase (OD600 0.60.8) in Luria-Bertani (LB) broth containing carbenicillin (50 µg/ml) at 37°C. Recombinant protein production was induced by addition of 0.5 mM isopropyl ß-D-thiogalactoside (IPTG). After incubation for 3 h, the cells were centrifuged and stored at 80°C before processing. All subsequent manipulations of the cells were at 4°C. The cell pellets were resuspended in ice-cold PBS, pH 7.4, and sonicated for 4x20 s with the cell suspension cooled in a salt/ice/water bath. The cell suspension was then centrifuged, the supernatant fraction was poured off, the cell pellet resuspended and washed three times in PBS and then resuspended in 20 mM ethanolamine/6 M urea, pH 10, for 4 h. After centrifugation, the supernatant containing the solubilized recombinant protein of interest was collected and stored at 4°C until purification. The recombinant proteins of interest were purified and concentrated by FPLC ion-exchange chromatography using Source 30Q anion-exchange media (Pharmacia Biotech, Piscataway, NJ) in an XK26/20 column (Pharmacia Biotech), using a step gradient with 20 mM ethanolamine/6 M urea/1 M NaCl, pH 10. After purification, the protein was dialyzed against 20 mM ethanolamine, pH 10.0, which removed the urea and allowed refolding of the recombinant protein. This step was critical. Basic buffers were required for all of the ß1
1 molecular constructs to fold correctly, after which they could be dialyzed into PBS at 4°C and concentrated by centrifugal ultrafiltration with Centricon-10 membranes (Amicon, Beverly, MA). For purification to homogeneity, a final step was included using size exclusion chromatography on Superdex 75 media (Pharmacia Biotech) in an HR16/50 column (Pharmacia Biotech). The final yield of purified protein varied between 15 and 30 mg/l of bacterial culture.
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Results |
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Protein expression was tested in a number of different E.coli strains, including a thioredoxin reductase mutant which allows disulfide bond formation in the cytoplasm (Derman, 1993). With such a small molecule, it became apparent early on in our studies that the greatest yield of material could be readily obtained by harvesting protein expressed as insoluble inclusion bodies in E.coli strain BL21(DE3). This allowed us to avoid problems with proteases associated with large scale production of recombinant protein in bacteria. After purification, the protein was dialyzed against 20 mM ethanolamine, pH 10.0, which removed the urea and allowed refolding of the recombinant protein. The same methodology was used to make the empty ß11 and covalent complexes of ß1
1 with MBP-7289, and control peptides Gp-MBP-5569 and CM-2. Both the rat and guinea pig MBP-7289 covalent constructs (rat, Rt-MBP-7289: PQKSQRTQDENPVVHF; guinea pig, Gp-MBP-7289: PQKSQRSQDENPVVHF) have been produced, and the MBP-5569 peptide is the guinea pig sequence (Figure 4
). The final yields of ß1
1 and covalent ß1
1peptide molecules was approximately 1530 mg/l culture.
Biochemical characterization
The presence of the native disulfide bond between cysteines ß15 and ß79 (empty ß11 amino acid numbering) was demonstrated by a gel shift assay in which identical samples with or without the reducing agent ß-mercaptoethanol (ß-ME) were boiled 5 min prior to SDSPAGE. In the absence of ß-ME disulfide bonds are retained and proteins typically move through acrylamide gels faster due to their more compact structure (Burrows et al., 1998
). All of the ß1
1 and Rt-ß1
1peptide molecules produced showed this pattern, indicating the presence of the native conserved disulfide bond (data not shown). These data represent a primary confirmation of the conformational integrity of the molecules. Samples of the ß1
1 and covalent ß1
1peptide molecules from SDSPAGE were transferred to a PVDF membrane for immunoblotting using the rat MHC class II RT1.B specific monoclonal antibody OX-6 as previously described (Burrows et al., 1998
). All of the ß1
1 and ß1
1peptide molecules were recognized by this antibody (data not shown).
Structural analysis
Circular dichroism (CD) reveals that the ß11 molecules have highly ordered secondary structures. The empty ß1
1 molecule contained approximately 30%
-helix, 15% ß-strand, 26% ß-turn and 29% random coil structures. The covalent ß1
1MBP-7289 and ß1
1CM-2 showed similar, although not identical, secondary structural features (Figure 5
). Comparison with the secondary structures of class II molecules determined by X-ray crystallography (Madden et al., 1991
; Brown, et al., 1993; Fremont et al., 1996
) provided strong evidence that the ß1
1 and ß1
1peptide molecules shared the ß-sheet platform/anti-parallel
-helix secondary structure common to all class II antigen binding domains (Table II
). Thermal denaturation studies revealed a high degree of cooperativity and stability of the molecules (Figure 6
). The biological integrity of these molecules has been demonstrated by their ability to detect and inhibit rat encephalitogenic T cells and treat experimental autoimmune encephalomyelitis (Burrows et al., 1998
; Burrows,G.G., manuscript submitted).
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Discussion |
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Our approach used molecular modeling, combining structural information from X-ray crystallographic data with recombinant DNA technology to design and produce single chain TCR ligands based on the natural MHC class II peptide binding/T cell recognition domain. In the native molecule this domain is derived from portions of the and ß polypeptide chains which fold together to form a tertiary structure, most simply described as a ß-sheet platform upon which two anti-parallel helical segments interact to form an antigen-binding groove. A similar structure is formed by a single exon encoding the alpha-1 and alpha-2 domains of MHC class I molecules, with the exception that the peptide-binding groove of MHC class II is open-ended, allowing the engineering of single-exon constructs that encode the peptide binding/T cell recognition domain and an antigenic peptide ligand. Our extension of this work towards designing covalent
1
2peptide MHC class I molecules has been supported by recent structural data demonstrating an MHC class I molecule with a peptide extending from one end of a class I MHC binding site (Collins et al., 1994). While a pharmacological approach to T cell mediated diseases is still in its infancy, development of the ß1
1 molecules described in this report will allow careful evaluation of the specific role played by the ß1
1 domain, independent from the platform
2ß2-Ig-fold domains.
ß11/
2ß2-Ig-fold interface
Modeling studies have highlighted a number of interesting features regarding the interface between the ß11 and
2ß2-Ig-fold domains. The
1 and ß1 domains showed an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core. The ß1
1 domain may have the ability to move as a single entity independent from the
2ß2-Ig-fold `platform'. Besides evidence of a high degree of mobility in the side-chains that make up the linker regions between these two domains (Jardetzky et al., 1994
; Fremont et al., 1996
), crystals of MHC class II I-Ek contained a number of water molecules within this interface (Fremont et al., 1996
; Murthy and Stern, 1997
). The interface between the ß1
1 and
2ß2-Ig-fold domains appears to be dominated by polar interactions, with hydrophobic residues potentially serving as a `lubricant' in a highly flexible `ball and socket' type interface (Burrows,G.G., manuscript in preparation). Flexibility at this interface may be required for freedom of movement within the
1 and ß1 domains for binding/exchange of peptide antigen. Alternatively or in combination, this interaction surface may play a potential role in communicating information about the MHC class IIpeptide molecules interaction with TCRs back to the APC.
Conserved ß1-domain disulfide bond
The ß1-domain of MHC class II molecules contains a disulfide bond that covalently couples the C-terminal end of its -helical segment to the first strand of the anti-parallel ß-sheet platform contributed by the ß1 domain. This structure is conserved among MHC class II molecules from rat, human and mouse, and appears to serve a critical function, acting as a `linchpin' that allows primary sequence diversity in the molecule while maintaining its tertiary structure. Critical analysis of the primary sequence using a helical wheel diagram of amino acid residues within two helical turns (7.2 residues) of cysteine 79 as well as analysis of the ß-sheet platform around cysteine 15 reveal a number of interesting features of the molecule, the most significant being very high diversity along the peptide-binding groove face of the helix and ß-sheet platform. Interestingly, a face of the helix composed of residues L68, E69, R72, A73, D76, R80 and Y83 is conserved among all rat, human and mouse class II and may serve an as yet undefined function, such as interaction with other components of the TCR complex. Site-directed mutagenesis studies using the novel ß1
1 molecules described in this report will allow careful dissection of the specific role played by each of these residues.
Circular dichroism spectra
The circular dichroic spectra indicate that the ß11 molecules are highly ordered and that the secondary structural content is consistent with the known features of the peptide binding/T cell recognition domains (
1 and ß1 domains). Thermal stability measurements indicate that the ß1
1 molecules are very stable and unfold cooperatively. Both the covalent ß1
1MBP-7289 and ß1
1CM-2 molecules show a greater thermal stability than the empty ß1
1 molecule, while the ß1
1CM-2 covalent construct with a six-histidine tag on the C-terminal is less stable than the empty ß1
1. An explanation for this observation may be that the histidine tag helps destabilize the hydrogen-bonded ß-sheet platform above 60°C. Evidence that the CM-2 peptide is constrained within the binding groove at room temperature is provided by the CD spectra, which show no gross increase in
-helical content compared with the other ß1
1 molecules.
From a drug engineering and design perspective this prototypic molecule represents a major breakthrough. Development of the ß11 molecules described in this report separates for the first time the peptide binding ß1
1 domains from the platform
2ß2 Ig-fold domains, allowing studies of their biochemical and biological properties independently, both from each other and from the vast network of information exchange that occurs at the cell surface interface between APC and T cell during MHCpeptide engagement with the T cell receptor. Because of their simplicity, biochemical stability, biological properties and structural similarity with human class II homologues, the rat ß1
1 constructs described here raises the possibility of using this construct as a template for engineering human homologues for treatment of autoimmune diseases. Development of ligands that bind T cell receptors in an antigen-specific manner will have a number of practical applications, and have been the subject of intense research by a number of different laboratories over the last decade (Matsui et al., 1991
; Sharma et al., 1991
; Nag et al., 1993 Nag et al., 1996; Kozono et al., 1994
; Rhode et al., 1996
). Practical applications include the ability to target, label and/or purify antigen-specific T cells, activate T cells in combination with other molecules and to treat conditions mediated by antigen-specific T cells (Burrows et al., 1998
).
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Acknowledgments |
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Notes |
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References |
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Received February 2, 1999; revised April 9, 1999; accepted April 26, 1999.