Masato Kobori

Structural classification of CDR-H3 revisited: a lesson in antibody modeling.

Among the six complementarity‐determining regions (CDRs) in the variable domains of an antibody, the third CDR of the heavy chain (CDR‐H3), which lies in the center of the antigen‐binding site, plays a particularly important role in antigen recognition. CDR‐H3 shows significant variability in its length, sequence, and structure. Although difficult, model building of this segment is the most critical step in antibody modeling. Since our first proposal of the “H3‐rules,” which classify CDR‐H3 structure based on amino acid sequence, the number of experimentally determined antibody structures has increased. Here, we revise these H3‐rules and propose an improved classification scheme for CDR‐H3 structure modeling. In addition, we determine the common features of CDR‐H3 in antibody drugs as well as discuss the concept of “antibody druggability,” which can be applied as an indicator of antibody evaluation during drug discovery. Proteins 2008.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia

The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3′ UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.

Sepantronium Bromide (YM155) induces disruption of the ILF3/p54nrb complex, which is required for survivin expression

YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin[1]. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.

Systematic classification of CDR‐L3 in antibodies: Implications of the light chain subtypes and the VL–VH interface

Antibody modeling is widely used for the analysis of antibody–antigen interactions and for the design of potent antibody drugs. The antibody combining site is composed of six complementarity determining regions (CDRs). The CDRs, except for CDR‐H3, which is the most diverse CDR, form limited numbers of canonical structures, which can be identified from the amino acid sequences. A method to classify the CDR‐H3 structure from its amino acid sequence was previously proposed. However, since those CDR structures were classified, many more antibody crystal structures have been determined. We performed systematic analyses of the CDR‐L3 structures and found novel canonical structures, and we also classified a previously identified canonical structure into two subtypes. In addition, two differently defined canonical structures in the κ and λ subtypes were classified into the same canonical structure. We also identified a key residue in CDR‐L3, which determines the conformation of CDR‐H3. Several analyses of CDR‐L3 loops longer than nine residues were performed. These new findings should be useful for structural modeling and are eventually expected to accelerate the design of antibody drugs. Proteins 2009.

Large scale isolation of osteoclast‐specific genes by an improved method involving the preparation of a subtracted cDNA library

Osteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast‐specific biological functions remain elusive because only a limited number of osteoclast‐specific genes have been identified. To circumvent this, we isolated a large number of osteoclast‐specific genes by preparing a subtracted cDNA library of high quality.

Use of amino acid composition to predict epitope residues of individual antibodies

We identified specific amino acid propensities at the interfaces of antigen-antibody interactions in non-redundant qualified antigen-antibody complex structures from Protein Data Bank. Propensities were expressed by the frequency of each of the 20 x 20 standard amino acid pairs that appeared at the interfaces of the complexes and were named the antibody-specific epitope propensity (ASEP) index. Using this index, we developed a novel method of predicting epitope residues for individual antibodies by narrowing down candidate epitope residues which was predicted by the conventional method. The 74 benchmarked antigens were used in ASEP prediction. The efficiency of this method was assessed using the leave-one-out approach. On elimination of residues with ASEP indices in the lowest 10% of all measured, true positives were enriched for 49 antigens. On subsequent elimination of residues with ASEP indices in the lowest 50%, true positives were enriched for 40 of the 74 antigens assessed. The ASEP index is the first benchmark proposed to predict epitope residues for an individual antibody. Used in combination with mutation experiments, this index has the potential to markedly increase the success ratio of epitope analysis.

Chemocavity: specific concavity in protein reserved for the binding of biologically functional small molecules.

The idea that there should be a specific site on a protein for a particular functional small molecule is widespread. It is, however, usually not so easy to understand what characteristics of the site determine the binding ability of the functional small molecule. We have focused on the concurrence rate of the 20 standard amino acids at such binding sites. In order to correlate the concurrence rate and the specific binding site, we have analyzed high-quality X-ray structures of complexes between proteins and small molecules. A novel index characterizing the binding site based on the concurrency rate has been introduced. Using this index we have identified that there is a specific concavity designated as a chemocavity where a specific group of small molecules, i.e., canonical molecular group, is highly inclined to be bound. This study has demonstrated that a chemocavity is reserved for a specific canonical molecular group, and the prevalent idea has been confirmed.

Combination of MS Protein Identification and Bioassay of Chromatographic Fractions to Identify Biologically Active Substances from Complex Protein Sources

Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.