Hiroki Akiba

Selective detection of phosphotyrosine in the presence of various phosphate-containing biomolecules with the aid of a terbium(III) complex.

Protein kinases control many cellular processes by specifically phosphorylating serine (Ser), threonine (Thr), or tyrosine (Tyr) residues in their target proteins. 2] Among these, the phosphorylation of Tyr is a key step in numerous types of cellular regulation. 3] It is well-known that excessive phosphorylation of receptor and nonreceptor tyrosine kinases occurs in some tumor and cancer cell lines. However, phosphorylated Tyr accounts for less than 1 % of total phosphorylated amino acids, and many of the roles of phosphotyrosine (pTyr) are not yet clearly known. Accordingly, precise and selective detection of pTyr in vitro and in vivo should allow for deeper understanding of cellular events and their disorders. Simple and stable probes that respond only to pTyr are thus required. Most of the chemical probes used to label phosphoproteins that have been developed over the decades merely work as affinity tags for phosphate groups and hardly discriminate between pTyr and phosphoserine or phosphothreonine (pSer/pThr). 5] In this study, pTyr is selectively detected through the emission from a Tb complex. On interaction of pTyr with the complex, the ACHTUNGTRENNUNGluminescence from the complex is enormously increased, due to efficient energy transfer from the pTyr benzene ring to the Tb. In contrast, the other two phosphorylated amino acids (pSer/pThr) are almost silent in emission, although they are also bound by the complex. Clear-cut detection of pTyr is accomplished. Luminescence from lanthanide ions shows various features such as long lifetimes, large Stokes’ shifts, and sharply-spiked emission bands. However, the emission intensity produced by the direct excitation of these ions themselves is relatively small because the f–f transitions are Laporte-forbidden. In order to obtain strong emission, a sensitizing chromophore (“antenna”) must be placed close to the ion. Once the antenna absorbs light energy, the ion is excited through an energy transfer process and its luminescence is notably enhanced. Because of this characteristic sensitizing process, both lanthanide ions and their complexes have been used as sensing probes. In the case of pTyr detection, Niedbalski et al. reported that luminescence from the Tb ion is promoted by interaction with pTyr but little affected by other amino acids, including pSer/pThr. However, the Tb ion also bound other molecules—guanosine5’-monophosphate (GMP), for example—and emitted notable luminescence (see below). Parker et al. have also developed lanthanide complexes that selectively bind pTyr and further carry antenna moieties in their ligands for intramolecular energy transfer. Considerable selectivity for pTyr detection was accomplished through sophisticated design of the ligand. In terms of the difference in spectral change, mainly based on affinity preference, however, completely clear-cut discrimination was not very easy. Here we have employed an entirely different strategy for ACHTUNGTRENNUNGselective detection of pTyr. As depicted in Scheme 1, the Tb

Binuclear Terbium(III) Complex as a Probe for Tyrosine Phosphorylation

By using the luminescence from binuclear complexes of Tb(III) (Tb(2)-L(1) and Tb(2)-L(2)), phosphorylated Tyr residue in peptides was selectively detected in neutral aqueous solutions. Neither the non-phosphorylated Tyr, pSer, pThr, nor the other phosphate-containing biomolecules tested affected the luminescence intensity to any notable extent. Upon the binding of the pTyr to these Tb(III) complexes, the luminescence from the metal ion was notably promoted, as the light energy absorbed by the benzene ring of pTyr is efficiently transferred to the Tb(III) center. The binding activity of the binuclear Tb(III) complexes towards pTyr is two orders of magnitude larger than that of the corresponding mononuclear complex. These binuclear complexes were successfully used for real-time monitoring of enzymatic phosphorylation of a peptide by a tyrosine kinase.

Click Conjugation of a Binuclear Terbium(III) Complex for Real-Time Detection of Tyrosine Phosphorylation

Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis-Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery.

Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity.

Use of SpyTag/SpyCatcher to construct bispecific antibodies that target two epitopes of a single antigen

Bispecific antibody targeting of two different antigens is promising, but when fragment-based antibodies are used, homogeneous production is difficult. To overcome this difficulty, we developed a method using the SpyTag/SpyCatcher system in which a covalent bond is formed between the two polypeptides. Using this method, we constructed a bispecific antibody that simultaneously interacted with two different epitopes of roundabout homologue 1 (ROBO1), a membrane protein associated with cancer progression. A bispecific tetravalent antibody with an additional functional moiety was also constructed by using a dimeric biotin-binding protein. An interaction analysis of ROBO1-expressing cells and the recombinant antigen demonstrated the improved binding ability of the bispecific antibodies through spontaneous binding of the two antibody fragments to their respective epitopes. In addition, multivalency delayed dissociation, which is advantageous in therapy and diagnosis.

Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III) Complexes

Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

Roles of the disulfide bond between the variable and the constant domains of rabbit immunoglobulin kappa chains in thermal stability and affinity

Rabbit antibodies show unique structural characteristics in that kappa chains have an inter-domain disulfide bond between the variable and constant domains. Here we characterized this disulfide bond from physicochemical viewpoints both in stability and affinity. It was revealed that the disulfide bond contributed to the thermal stability of the antibody, but the affinity and mechanism of antigen recognition was not altered by the mutation. The present result expands the understanding of how rabbit antibodies with kappa light chains gain affinity under characteristic mechanism to gain thermal stability, and would give suggestions for the methods to artificially stabilize antibody molecules.

Structural behavior of keratin-associated protein 8.1 in human hair as revealed by a monoclonal antibody

Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the proteins roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species.

Tyrosine Sulfation Restricts the Conformational Ensemble of a Flexible Peptide, Strengthening the Binding Affinity for an Antibody

Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated with the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or nuclear magnetic resonance techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide and its interaction with an antibody. Molecular dynamics simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein-protein interactions consists of the limitation of conformational dynamics in the unbound state, thus reducing the loss of entropy upon binding and boosting the affinity for its partner.

Thermodynamic analyses of amino acid residues at the interface of an antibody B2212A and its antigen roundabout homolog 1

Artificial affinity maturation of antibodies is promising but often shows difficulties because the roles of each amino acid residue are not well known. To elucidate their roles in affinity against the antigen and thermal stability, interface residues in single-chain Fv of an antibody B2212A with its antigen roundabout homolog 1 were mutated and analyzed. Some amino acids played important roles in the affinity while others contributed to thermal stability.