Author: Nyamekye Obeng-Adjei
Institution: University of Maryland Eastern Shore
Date: November 2006
Mycoplasma arthritidis-derived Mitogen (MAM) is a superantigen that can dimerize major histocompatibility complex (MHC) antigen, HLA-DR1 molecule. MAM is known to induce chronic arthritis in rodents which bear a resemblance to human rheumatoid arthritis. MAM stimulates T-cell activation by interacting with class II MHC including HLA-DR1, which is one of the important disease risk genes in rheumatoid arthritis. It has been suspected that MAM a plays role in human rheumatoid arthritis. The high affinity of MAM to the peptide/MHC complex is due to interaction between the N-terminal domain of MAM and the antigen-presenting domain of MHC. In this study, we created mutants at the N-terminal domain of MAM to investigate the MAM residues responsible for this interaction. We mutated four amino acids: threonine, arginine, lysine and glutamine at positions 89, 91, 92, and 99, respectively. The four residues, which are at the MAM-MHC interface, were individually converted to alanine. Sedimentation velocity of analytical ultracentrifuge was used to identify the heterogeneous interaction between the mutant and the MHC molecule. The dissociation constants (KD) of the mutant MAM-MHC complexes were measured using sedimentation equilibrium. Of the four mutants, R91A binds to HLA DR1 with the lowest affinity, which is about twenty times lower than that of the wild-type. Other mutants reduce the binding affinity of MAM to HLA DR1 by 7-10 folds. These data provide evidence that substitution of any of these four residues will cause the affinity of MAM to the peptide/HLA-DR1 complex to decrease. Knowing the KD of the mutants will help define MAM residues responsible for the high affinity binding between MAM and the receptor peptide/HLA-DR1 complex. In long term, the knowledge will shed lights on the biological function of MAM and a path-way to the development of a vaccine in the future.
Superantigens (SAg) are functionally related immunoregulatory proteins that stimulate large number of T lymphocytes and are known to be produced by bacteria and viruses. The stimulation of T lymphocytes occurs upon the binding of SAgs to major histocompatibility complex (MHC) class II molecules. This discovery offered hopes for effective T cell -based therapies and has become effective tool for studying interactions within trimolecular complex (Zhao et al., 2004).
Mycoplasma arthritidis-derived mitogen (MAM) functions as a SAg that causes spontaneous onset of chronic arthritis in genetically susceptible strains of rodents and is suspected to play a role in human rheumatoid arthritis (RA). MAM is produced by Mycoplasma arthritidis (Cole, 1991). Using MAM and Human Leukocyte Antigens (HLA), which is a series of proteins on the white blood cells that help the cell differentiate between self and foreign antigens, previous studies uncovered the ability of SAgs to dimerize MHC molecules. MAM was found to preferentially bind to HLA-DR1 with high affinity, similar to murine MHC class II (Etongue-Mayer et al., 2002). Genes in the HLA region remain the most powerful disease risk genes in RA. Several allelic variants of HLA-DRB1 genes have been associated with RA, supporting a role for T-cell receptor-HLA-antigen interactions in the pathologic process (Weyand and Goronzy, 2000)
In this study, the affinities of MAM mutants to the receptor, HLA-DR1, were measured and compared to the affinity of the wild-type MAM to the same receptor. The identification of a particular amino acid sequence vital for the high affinity between MAM and the peptide/HLA-DR1 complex will help in understanding the SAg's biological function and contribute to the manufacturing of a vaccine in the future. The previous crystallographic structure of the demerized MAM/HLA-DR1 complex indicates that the complex is formed through contacts between the N-terminal domain of MAM and the antigen-presenting domain of HLA-DR1 (Zhao et al., 2004). The mutations were targeted at the residues where the contacts occur. The site-directed mutagenesis was used to individually change threonine (Thr89), arginine (Arg91), lysine (Lys92), and glutamine (Gln99) to alanine. Glutathione-S-transferase (GST)-expression system was used for protein expression. Affinity and size exclusion chromatographies were used to purify the mutant proteins. Finally, sedimentation velocity (SV) and sedimentation equilibrium (SE) from an analytical ultracentrifugation were used to calculate the mutant's affinity to the receptor.
Purifying the mutant's protein
Affinity and size exclusion chromatography was used to purify mutant proteins. Figure 1 shows an example of SDS-PAGE analysis of one mutant's expression and purification profile from its uninduced sample to purification with size exclusion chromatography. Figure 2 shows a representative profile for size exclusion chromatography of the mutant.
Sedimentation Velocity (SV)
Having expressed and purified the proteins of the mutants, we investigated the ability of the MAM mutants (T89A, R91A, K92A and Q99A) to bind a HLA-DR1 molecule by using analytical ultracentrifugation sedimentation velocity. The sedimentation coefficients of the HLA-DR1 molecule, MAM, and the MAM-DR1 complex were determined to be 2.1S, 3.5S, and 4.2S, respectively (Figure 3a). The mutants T89, R91, and Q99 bind HLA-DR1 with a sedimentation coefficient of 4.2S (Figure 3b). The peaks at sedimentation coefficient 2.1S represent free MAM mutants that are in excess amount compared to HLA-DR1.
Sedimentation Equilibrium ( SE)
Sedimentation equilibrium analysis was used to estimate the dissociation constant (KD) of the MAM proteins and their receptors. By using SEDPHAT, fitting experimental sedimentation equilibrium data generated the dissociation constants for MAM (or MAM mutants) interactions with the HLA-DR1/HA complex (Figure 4a, b and Table 1).
The formations of the mutant MAM-HLA/DR1 complex were verified by comparing the coefficients of the mutant and that of the HLA-DR1 molecule (Figure 3a, b). Clearly, all MAM mutants and MAM wild-type bind the HLA-DR1/HA complex with an sedimentation coefficients of 4.2S.
The dissociation constant indicates the strength of binding between the two molecules in terms of how easy it is to separate the complex. If a high concentration of both macromolecules is required to form the complex, it indicates that the strength of binding is low and vice versa. The high dissociation constant of the R91A mutant (10 M) makes its affinity the lowest of all, followed by mutants T89A (4.2 M), Q99A (3.8 M), K92A (3.2 M), and MAM wild-type (0.5 M) (Table 1).
As shown in Figure 4a, all four residues (Thr89, Arg91, Lys92 and Gln99) are required for high affinity in MAM/HLA complex. The complex affinity decreased after mutation of each of these four amino acids on the peptide chain of the MAM. Thr89, Arg91, Lys92 and Gln99 each decrease by 10,22,7 and 8 times respectively.
Although MAM is known to bind HLA-DR1 with high affinity through the MAM N-terminal domain, until now, there was no direct evidence to demonstrate which residue at the N-terminal is vital for the high affinity binding to MHC. Investigation to this matter leads to the mutation of four residues and to study how these mutants affect the affinity between MAM and the HLA-DR1 complex. Data obtained in the course of the present investigation provides the first direct evidence of decrease in affinity of MAM/HLA-DR1 complex due to mutation of the MAM residues. All of the four alanine mutants showed significant decrease in affinity, with the lowest affinity for the R91A mutant.
The crystal structure of the MAM/HLA-DR1 complex indicated that only about 25% of the surface area of Arg91 is buried upon complex formation (Figure 5) (Zhao et al., 2004). However, mutation of arginine to alanine resulted in the lost of two hydrogen bonds between MAM Arg91 and HLA-DR1 Gln18. This implies that the contribution of Arg91 to the high affinity within the complex comes from the residue's interaction with the Glutamine (Gln18α) on the HLA-DR1 molecule.
Future Studies will include analyzing eleven other residues by mutagenesis to determine their contribution to the MAM/HLA-DR1 interaction, which will help in determine the type of forces, hydrogen bonds or van der Waals contacts, that contribute the most to this interaction.
Materials and methods
The point mutation was performed using temperature cycler and Pfu Turbo® DNA polymerase. The polymerase aids in exact replication of two plasmid strands with high fidelity. Two synthetic oligonucleotide primers containing the desired mutation were inserted into target site of two super coiled double-stranded DNA (dsDNA) vector. The oligonucleotide primers were extended during temperature cycling by DNA polymerase (primers move in opposite direction of the vector). Dpn 1 endonuclease was used to digest the parent template, to provide a mutated MAM plasmid. The plasmid was then transferred into E. coli cell using Bacterial electrocompetent transformation protocol. Detailed protocol can be found in QuikChange® Site-Directed Mutagenesis.
Protein expression and purification
After the mutants were sequenced to verify gene changes at the target site, the plasmids were transformed into expression hosts (BL21 DE3)). The expression was then induced by the addition of IPTG to a growing culture. The induction is to transcribe the interested proteins in the host strain. SDS polyacrylamide gradient gel was run to monitor the protein expression. Following expression, the mutant cells (soluble supernatant) were washed with PBS buffer in a Glutathione 4B column. The fusion protein was then eluted with Glutathione Elution Buffer, which separates GST-MAM proteins from other proteins due to the fusion-protein's high affinity to Glutathione. The eluted fusion protein was digested with PreScission protease, cutting the GST-MAM fusion protein to remove GST. SDS polyacrylamide gradient gel was run again to check the progress of the digestion. The digested samples were loaded to the glutathione affinity column again to remove GST. The flow-through was collected and concentrated to 5 ml. The sample was then loaded to a size exclusion column Superdex S-200 (Pharmacia). Size exclusion chromatography was used to pool fractions with high protein content.
Analytical Ultracentrifugation (AU)
This instrument was used to monitor the sedimentation of proteins to characterize their hydrodynamics. There are two parts to this experiment: sedimentation velocity (SV) and sedimentation equilibrium (SE). The velocity was used to identify the heterogeneous interaction between the SAg and the HLA-DR1. The equilibrium was used to measure the affinity between MAM and MAM mutant and class II MHC molecule HLA-DR1.
Sedimentation-velocity experiments were done at 20 °C in a Beckman Optima XL-I analytical ultracentrifuge at a rotor speed of 55,000 r.p.m. (An50Ti rotor). Double-sector cells were loaded with 400 l of protein samples, in HBS-E buffer (10 mM Hepes, pH7.4, 150 mM NaCl, 3.4 mM EDTA). All the samples were prepared by mixing MAM mutant and HLA-DR1 with a molar ratio of 10:0.5 M. Data were recorded with absorbance detection at wavelengths of 280 nm for all samples. Absorbance fringe displacement profiles were analyzed with the software SEDFIT (http://www.analyticalultracentrifugation.com), using a model for continuous sedimentation coefficient distributions c(s). Distributions were calculated with maximum entropy regularization at a predetermined confidence level of 1 standard deviation. In further analysis, the differential S value distribution was integrated, to determine weight-average sedimentation coefficients.
Sedimentation equilibrium studies were conducted at a temperature of 20°C and at three rotor speeds of 20,000 rpm, 25,000 rpm, and 30,000 rpm. 110 l of proteins were respectively loaded into Epon double-sector centerpieces, at concentrations the same or half-diluted as in the velocity experiments. Equilibrium absorbance profiles were acquired at 280-nm wavelength. The equilibrium sedimentation data were analyzed using the software SEDPHAT (http://www.analyticalultracentrifugation.com). Data analysis was performed by global least-squares analysis of the data from multiple concentrations and multiple rotor speeds, based on the well-known superposition of the Boltzmann distributions of ideal species in the centrifugal field, using conservation of mass constraints.
This research is supported by a grant (AI50628) from the National Institutes of Health (NIH) (to H. Li). N. Obeng-Adjei was supported by the Research Experiences for Undergraduates (REU) program at the Wadsworth Center, provided by grant DBI-9987844 from National Science Foundation. Thanks to the Molecular Genetics Cole at Wadsworth center for DNA sequencing and the Biochemistry core for assistance in analytical ultracentrifugation.
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