Single Amino Acid Residue Replacement Research Articles

Identification of a conserved linear neutralizing epitope recognized by monoclonal antibody 9A9 against serotype A foot-and-mouth disease virus.

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. In recent years, a series of outbreaks of serotype A FMD have occurred in many countries. High-affinity neutralizing antibodies against a conserved epitope have the potential to provide protective immunity against diverse subtypes of FMDV serotype A and to protect against future pandemics. In this study, we produced an A serotype FMDV-specific monoclonal antibody (MAb) against the viral capsid protein VP1, designated 9A9, that potently neutralized FMDV A/JLYS/CHA/2014 with a 50% neutralization titer (NT50) of 4,096. GST-fusion proteins expressing truncated peptides of VP1 were subjected to Western blot analysis using MAb 9A9, and it was found that the peptide (143)RGDLGPLAARL(153) of VP1 was the minimal epitope for MAb 9A9 binding. Western blot analysis also revealed that the epitope peptide could be recognized by positive sera from serotype A FMDV-infected pigs and cattle. Subsequent alanine-scanning mutagenesis analysis revealed that residues Gly(147) and Leu(149) of the 9A9-recognized epitope are crucial for MAb 9A9 binding. Furthermore, under immunological pressure selected by MAb 9A9, a single amino acid residue replacement (L149P) occurred in a viral neutralization-escape mutant, which verified the location of a critical residue of this epitope at Leu(149). Importantly, the epitope (143)RGDLGPLAARL(153) was highly conserved among different topotypes of serotype A FMDV strains in sequence alignment analysis. Thus, the results of this study could have application potential in the development of epitope-based vaccines and a suitable MAb-based diagnostic method for detection of type A FMDV as well as quantitation of antibodies against FMDV serotype A.

Thermostability of Chimeric Cytidine Deaminase Variants Produced by DNA Shuffling

The DNA shuffling technique has been used to generate libraries of evolved enzymes in thermostability. We have shuffled two thermostable cytidine deaminases (CDAs) from Bacillus caldolyticus DSM405 (T53) and B. stearothermophilus IFO12550 (T101). The shuffled CDA library (SH1067 and SH1077 from the first round and SH2426 and SH2429 from the second round) showed various patterns in thermostability. The CDAs of SH1067 and SH1077 were more thermostable than that of T53. SH2426 showed 150% increased half-time than that of T53 at 70 degrees C. The CDA of SH2429 showed about 200% decreased thermostability than that of T53 at 70 degrees C. A single amino acid residue replacement that presented between SH1077 and SH2429 contributed to dramatic changes in specific activity and thermostability. On SDS-PAGE, the purified CDA of SH1077 tetramerized, whereas that of SH2429 denatured and became almost monomeric at 80 degrees C. A simulated three-dimensional structure for the mutant CDA was used to interpret the mutational effect.

Requirement of the N-terminus for dimer formation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate synthase AroG of Escherichia coli.

The first regulatory step in the synthesis of aromatic amino acids is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). In Escherichia coli, the allosteric DAHPS exists as three isozymes, AroG, AroF and AroH, each independently feedback-inhibited by corresponding end product amino acids, phenylalanine, tyrosine and typtophan. Structural biological evidences have suggested that the N-terminus of AroG is involved in the formation of a putative inhibitor-binding site and feedback inhibition signal transmission. Our previous work showed that a single amino acid residue replacement Ile10Ala or deletion of 15 N-terminal amino acids could lead to a dramatic loss of AroG enzymatic activity (Hu et al. 2003). Here we demonstrate that the deletion of N-terminus prevents the enzyme from forming a dimeric structure, indicating that the N-terminus of AroG plays a critical role in the formation of the essential tight dimeric structure.

Mutations that affect the ability of the vnd/NK-2 homeoprotein to regulate gene expression: transgenic alterations and tertiary structure.

The importance in downstream target regulation of tertiary structure and DNA binding specificity of the protein encoded by the vndNK-2 homeobox gene is analyzed. The ectopic expression patterns of WT and four mutant vndNK-2 genes are analyzed together with expression of two downstream target genes, ind and msh, which are down-regulated by vndNK-2. Three mutants are deletions of conserved regions (i.e., tinman motif, acidic motif, and NK-2 box), and the fourth, Y54M vndNK-2, corresponds to a single amino acid residue replacement in the homeodomain. Of the four ectopically expressed mutant genes examined, only the Y54M mutation inactivates the ability of the vndNK-2 homeodomain protein to repress ind and msh. The acidic motif deletion mutant slightly reduced the ability of the protein to repress ind and msh. By contrast, both tinman and NK-2 box deletion mutants behaved as functional vndNK-2 genes in their ability to repress ind and msh. The NMR-determined tertiary structures of the Y54M vndNK-2 homeodomain, both free and bound to DNA, are compared with the WT analog. The only structural difference observed for the mutant homeodomain is in the complex with DNA and involved closer interaction of the methionine-54 with A2, rather than with C3 of the (-) strand of the DNA. This subtle change in the homeodomain-DNA complex resulted in modifications of binding affinities to DNA. These changes resulting from a single amino acid residue replacement constitute the molecular basis for the phenotypic alterations observed on ectopic expression of the Y54M vndNK-2 gene during embryogenesis.

Surface retention of an inactivating lutropin receptor mutant in exoloop 3.

The heptahelical lutropin receptor (LHR) signals primarily via the Gs-adenylyl cyclase pathway and undergoes ligand-mediated receptor desensitization and internalization. A loss-of-function rat LHR mutant was recently described in which a single amino acid residue replacement in exoloop 3, K583E, had no effect on human choriogonadotropin (hCG) binding but essentially abolished signaling. This LHR mutant is a prime candidate for which to study hCG-mediated receptor internalization since it is highly unlikely that an amino acid residue in exoloop 3 , i.e. an extracellular portion of LHR connecting transmembrane helices 6 and 7, could have any direct interaction with Galpha(s), which is located on the cytoplasmic face of the plasma membrane. A method to study endocytosis was adapted that involves concanavalin A binding to the glycoproteins on the cell surface, thus facilitating separation of the plasma membrane fraction from other cellular membrane fractions by sucrose gradient centrifugation. Conditions were used such that a single round of endocytosis could be determined with [125I]hCG. Endocytic rate constants of 0.03 and O min(-1) were obtained for LHR and the mutant, respectively, in transfected human embryonic kidney 293 cells; moreover, internalization of the mutant could not be restored by the addition of 8-Br-cAMP. Thus, the presence of the second messenger cAMP is not sufficient for internalization of ligand-occupied LHR. Rather, it appears that ligand-mediated activation and subsequent internalization of LHR results from an altered conformational state or a conformation-dependent post-ligand binding modification such as phosphorylation.

Major and minor epitopes on the self antigen mouse cytochrome c mapped by site-directed mutagenesis

Variants of rat (mouse) cytochrome c, prepared by site-directed mutagenesis or represented by closely-related cytochromes c from different species, were employed to map the functional boundaries of a number of mouse monoclonal antibodies (mAb) specific for the major antigenic region on the self antigen (Ag) around residue 62 and the minor antigenic region around residue 44. The recombinant mouse cytochromes c tested were, unlike the tissue-derived Ag, trimethylated at position 72, and included the wild-type which was acetylated at the amino terminus, a variant that was unacetylated at the amino terminus, and variants with the following single amino acid residue replacements: V11I (valine to isoleucine at position 11), Q12M, A15S, A44P, F46Y, D50A, T58I and G89E. Of these, only the A44P variant affected the binding of mAb to the region previously localized to the vicinity of residue 44, thus confirming that assignment. Loss of the acetyl group at the amino terminus affected the binding of most of the mAb to the region around residue 62. The other mutations had little, if any, affect on mAb binding. The epitopes of mAb binding the region around residue 62 were shown in this study to have similar functional boundaries. This site on the self Ag, which encompasses at least three discontinuous segments of the polypeptide chain, is comparable in size to epitopes on other protein Ag that have been mapped by X-ray crystallography and is similar to an epitope in the corresponding region of the foreign Ag, horse cytochrome c, that has been mapped by hydrogen-deuterium exchange. In addition to the mAb binding the regions around residues 44 and 62, a third group of mouse cytochrome c-specific mAb known to be broadly reactive with mammalian cytochromes c and that represents a minor portion of the mAb was tested for binding the site-directed mutants of mouse cytochrome c. None of these mAb were affected by the mutations, indicating the presence of at least one more antigenic region on the self Ag in an area not encompassed by these mutations that is structurally highly conserved.

Mutagenesis of the ‘determinant loop’ region of human choriogonadotropin β

The hormone-specific β subunits of the four human glycoprotein hormones are homologous, and mapping studies are underway in many laboratories to delineate the amino acid residues responsible for receptor binding and activation. Results on the human choriogonadotropin β (hCGβ) subunit, obtained using synthetic peptides, chemically modified derivatives, and mutant forms prepared via site-directed mutagenesis, have suggested that amino acid residues enclosed by the purported disulfide loop between Cys-93 and Cys-100 may contribute to receptor binding and perhaps specificity. Indeed, the 93–100 amino acid sequence is referred to as a determinant loop. We have used site-directed mutagenesis to prepare single amino acid residue replacements at positions not previously investigated in full length β subunits; these include Arg-95, Ser-96, Thr-97, and Thr-98. In addition, Leu-92 was studied in an effort to determine whether changes immediately adjacent to the determinant loop alter receptor binding. The wild-type and mutant cDNAs for hCGβ were subcloned into a P rsv expression vector and transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine α. The concentrations of total expressed hCGβ in heterodimer form with the bovine α subunit were determined by radioimmunoassays. The mutant gonadotropins were assayed in vitro using a competitive binding assay with [ 125I]hCG and progesterone production, both in the transformed murine Leydig cell line, MA-10. Mutant β subunits containing the replacements Lys-92, Ser-95, Asp-96, and Tyr-97 exhibited normal α subunit binding. The resulting heterodimers with β replacements at positions 92 and 97 gave comparable potency to control gonadotropin containing hCGβ wild-type, and mutant gonadotropins containing replacements at positions 95 and 96 yielded reduced potency in the binding assay. Interestingly, the potencies of these latter two mutant gonadotropins were comparable to the wild-type gonadotropin in the steroidogenic assay, although the maximal Steroidogenesis was less in the hormone with the Arg-95 → Ser replacement. Substitution of Thr-98 with Asp and with Arg resulted in mutant β subunits with reduced subunit assembly; however, both heterodimers were active in the in vitro bioassays. Accurate potencies could not be obtained with the gonadotropin containing hCGβ(Arg-98), but the activity associated with hCGβ(Asp-98) appeared less than with wild-type gonadotropin. A comparison of these results with other data obtained on the determinant loop region leads to the following conclusions. Arg-94, Arg-95, Ser-96, Thr-98, and Asp-99 contribute to CG/lutropin receptor binding, although neither is absolutely critical since certain substitutions can be tolerated without a total loss of activity. The determinant loop region also appears to participate in some manner to α subunit binding, perhaps involving Thr-98. It remains to be determined if this region serves as a crucial determinant for β chain folding or represents a direct contact site for the α subunit. The question of whether the determinant loop region is important in receptor specificity is still open, but available data argue strongly that several residues in this disulfide loop contribute to CG/lutropin receptor binding.

Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants

We have analyzed the DNA binding properties of Tet-repressor mutants with single amino acid residue replacements at eight positions within the alpha-helix-turn-alpha-helix DNA-binding motif. A saturation mutagenesis of Gln38, Pro39, Thr40, Tyr42, Trp43 and His44 in the second alpha-helix was performed; in addition, several substitutions of Thr27 and Arg28 in the first alpha-helix were constructed. The abilities of these mutant repressors to bind a set of 16 operator variants were determined and revealed 23 new binding specificities. All repressor mutants with DNA-binding activity were inducible by tetracycline, while mutants lacking binding activity were trans-dominant over the wild-type. All mutant proteins were present at the same intracellular steady-state concentrations as the wild-type. These results suggest the structural integrity of the mutant repressors. On the basis of the new recognition specificities, five contacts between a repressor monomer and each operator half-site and the chemical nature of these repressor-operator interactions are proposed. We suggest that Arg28 contacts guanine of the G.C base-pair at operator position 2 with two H-bonds, Gln38 binds adenine of the A.T base-pair at position 3 with two H-bonds, and the methyl group of Thr40 participates in a van der Waals' contact with cytosine of the G.C base-pair at position 6 of tet operator. A previously unrecognized type of interaction is proposed for Pro39, which inserts its side-chain between the methyl groups of the thymines of T.A and A.T base-pairs at positions 4 and 5. Computer modeling of these proposed contacts reveals that they are possible using the canonical structures of the helix-turn-helix motif and B-DNA. These contacts suggest an inverse orientation of the Tet repressor helix-turn-helix with respect to the operator center as compared with non-inducible repressor-operator complexes, and are supported by similar contacts of other repressor-operator complexes.

A single amino acid residue replacement in the beta subunit of human chorionic gonadotrophin results in the loss of biological activity.

The heterodimer, human chorionic gonadotrophin (hCG), contains an alpha subunit that is common to the glycoprotein hormones and a hormone-specific beta subunit. A comparison of all known beta amino acid sequences shows that an aspartic acid at position 99 (with the numbering scheme for hCG-beta) is one of the seven non-Cys invariant residues. Using site-directed mutagenesis we have replaced hCG-beta Asp99 with Arg. Chinese hamster ovary cells, containing a stably integrated gene for bovine alpha subunit, were transiently transfected with plasmids containing wild-type and mutant hCG-beta cDNAs. The Arg99 beta mutant associated with the alpha subunit, but the resulting heterodimer failed to enhance intracellular cyclic AMP production in a gonadotrophin-responsive transformed murine Leydig cell line. Thus, a single amino acid residue replacement in this glycosylated heterodimer containing 237 amino acid residues is sufficient to abolish activity.