Makiko Suwa

Structural Basis for a Broad But Selective Ligand Spectrum of a Mouse Olfactory Receptor: Mapping the Odorant-Binding Site

The olfactory receptor (OR) superfamily provides a basis for the remarkable ability to recognize and discriminate a large number of odorants. In mice, the superfamily includes ∼1000 members, and they recognize overlapping sets of odorants with distinct affinities and specificities. To address the molecular basis of odor discrimination by the mammalian OR superfamily, we performed functional analysis on a series of site-directed mutants and performed ligand docking simulation studies to define the odorant-binding site of a mouse OR. Our results indicate that several amino acids in the transmembrane domains formed a ligand-binding pocket. Although other G-protein-coupled receptors (GPCRs) recognize biogenic ligands mainly with ionic or hydrogen bonding interactions, ORs recognize odorants mostly via hydrophobic and van der Waals interactions. This accounts for the broad but selective binding by ORs as well as their relatively low ligand-binding affinities. Furthermore, we succeeded in rational receptor design, inserting point mutations in the odorant-binding site that resulted in predicted changes in ligand specificity and antagonist activity. This ability to rationally design the receptor validated the binding site structure that was deduced with our mutational and ligand docking studies. Such broad and specific sensitivity suggests an evolutionary process during which mutations in the active site led to an enormous number of ORs with a wide range of ligand specificity. The current study reveals the molecular environment of the odorant-binding site, and it further advances the understanding of GPCR pharmacology.

Highly Selective Tuning of a Silkworm Olfactory Receptor to a Key Mulberry Leaf Volatile

BACKGROUND The olfactory system plays an important role in the recognition of leaf volatiles during the search of folivore insects for a suitable plant host. For example, volatiles emitted by mulberry leaves trigger chemotaxis behavior in the silkworms Bombyx mori, and as a consequence, they preferentially reside on and consume mulberry leaves. Here, we aimed to identify natural chemoattractants and their corresponding olfactory receptors (Ors) involved in silkworm behavior to mulberry leaves. RESULTS Chemotaxis behavioral assays for headspace volatiles detected by gas chromatography-mass spectroscopy analysis revealed that among the volatiles that were emitted by mulberry leaves, cis-jasmone was the most potent attractant for silkworms, working at a threshold of 30 pg from [corrected] 20 cm distance. Among a total of 66 Ors identified in the B. mori genome, we found that 23 were expressed in the olfactory organs during larval stages. Functional analysis of all the larvae-expressed Ors in Xenopus oocytes revealed that one Or, termed BmOr-56, showed a high sensitivity to cis-jasmone. In addition, the ligand-receptor activity of BmOr-56 reflected the chemotaxis behavioral response of silkworms. CONCLUSIONS We identified cis-jasmone as a potent attractant in mulberry leaves for silkworms and provide evidence that a highly tuned receptor, BmOr-56, may mediate this behavioral attraction. The current study sheds light on the mechanism of the correlation between olfactory perception in folivore insects and chemotaxis behavior to a natural volatile emitted by green leaves.

Odorant Receptor Map in the Mouse Olfactory Bulb: In Vivo Sensitivity and Specificity of Receptor-Defined Glomeruli

Odorant identity is represented in the olfactory bulb (OB) by the glomerular activity pattern, which reflects a combination of activated odorant receptors (ORs) in the olfactory epithelium. To elucidate this neuronal circuit at the molecular level, we established a functional OR identification strategy based on glomerular activity by combining in vivo Ca(2+) imaging, retrograde dye labeling, and single-cell RT-PCR. Spatial and functional mapping of OR-defined glomeruli revealed that the glomerular positional relationship varied considerably between individual animals, resulting in different OR maps in the OB. Notably, OR-defined glomeruli exhibited different ligand spectra and far higher sensitivity compared to the in vitro pharmacological properties of corresponding ORs. Moreover, we found that the olfactory mucus was an important factor in the regulation of in vivo odorant responsiveness. Our results provide a methodology to examine in vivo glomerular responses at the receptor level and further help address the long-standing issues of olfactory sensitivity and specificity under physiological conditions.

A simple statistical method for discriminating outer membrane proteins with better accuracy

MOTIVATION Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important task both for identifying outer membrane proteins from genomic sequences and for the successful prediction of their secondary and tertiary structures. RESULTS We have systematically analyzed the amino acid composition of globular proteins from different structural classes and outer membrane proteins. We found that the residues, Glu, His, Ile, Cys, Gln, Asn and Ser, show a significant difference between globular and outer membrane proteins. Based on this information, we have devised a statistical method for discriminating outer membrane proteins from other globular and membrane proteins. Our approach correctly picked up the outer membrane proteins with an accuracy of 89% for the training set of 337 proteins. On the other hand, our method has correctly excluded the globular proteins at an accuracy of 79% in a non-redundant dataset of 674 proteins. Furthermore, the present method is able to correctly exclude alpha-helical membrane proteins up to an accuracy of 80%. These accuracy levels are comparable to other methods in the literature, and this is a simple method, which could be used for dissecting outer membrane proteins from genomic sequences. The influence of protein size, structural class and specific residues for discrimination is discussed.

Species-specific variation of alternative splicing and transcriptional initiation in six eukaryotes

The genome-wide detection of alternative splicing and transcriptional initiation (ASTI) was conducted in six eukaryotes (human, mouse, fruit fly, nematode, cress and rice) whose genome sequencing has been completed or nearly completed. Transcriptional isoforms were collected by mapping a batch of full-length cDNA sequences onto the respective cognate genomic sequences. Isoforms mapped on the same gene locus were compared pair-wise, ASTI patterns were segmented into minimal spans, and then the minimal patterns (ASTI units) were classified into unique types, such as the cassette type or the alternative donor site. All these procedures were performed automatically under the same conditions so that the results obtained from different species could be compared directly. The fraction of loci that underwent ASTI of the total mapped loci was the largest for mammals and fruit fly, and the smallest for plants. Exactly the same trend was observed for the number of unique ASTI types found in each species. The observed fractional representations of the ASTI types were similar between evolutionarily close species, such as human and mouse or cress and rice. On the other hand, the relative orders of abundance in individual ASTI type were considerably different between evolutionarily distant species, such as between mammals and plants. In human and mouse, alternative splicing other than the retained introns tended to occur within the protein coding sequence (CDS) regions rather than within the untranslated regions (UTRs), whereas this tendency was obscure in the other four species. In all the species examined, the difference in alternative exon lengths was most likely in multiples of three, and this tendency was most prominent when the alternative exons were embedded within the CDSs. These observations are generally consistent with the idea that higher organisms utilize the ASTI mechanisms more extensively and in a more complicated manner than lower organisms, and that ASTI actively participates in the enhancement of the functional and structural diversity of products generated from a limited number of genes on a genome.

Neural network‐based prediction of transmembrane β‐strand segments in outer membrane proteins

Prediction of transmembrane β‐strands in outer membrane proteins (OMP) is one of the important problems in computational chemistry and biology. In this work, we propose a method based on neural networks for identifying the membrane‐spanning β‐strands. We introduce the concept of “residue probability” for assigning residues in transmembrane β‐strand segments. The performance of our method is evaluated with single‐residue accuracy, correlation, specificity, and sensitivity. Our predicted segments show a good agreement with experimental observations with an accuracy level of 73% solely from amino acid sequence information. Further, the predictive power of N‐ and C‐terminal residues in each segments, number of segments in each protein, and the influence of cutoff probability for identifying membrane‐spanning β‐strands will be discussed. We have developed a Web server for predicting the transmembrane β‐strands from the amino acid sequence, and the prediction results are available at http://psfs.cbrc.jp/tmbeta‐net/.

Discrimination of outer membrane proteins using support vector machines

MOTIVATION Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important task both for dissecting outer membrane proteins (OMPs) from genomic sequences and for the successful prediction of their secondary and tertiary structures. RESULTS We have developed a method based on support vector machines using amino acid composition and residue pair information. Our approach with amino acid composition has correctly predicted the OMPs with a cross-validated accuracy of 94% in a set of 208 proteins. Further, this method has successfully excluded 633 of 673 globular proteins and 191 of 206 alpha-helical membrane proteins. We obtained an overall accuracy of 92% for correctly picking up the OMPs from a dataset of 1087 proteins belonging to all different types of globular and membrane proteins. Furthermore, residue pair information improved the accuracy from 92 to 94%. This accuracy of discriminating OMPs is higher than that of other methods in the literature, which could be used for dissecting OMPs from genomic sequences. AVAILABILITY Discrimination results are available at http://tmbeta-svm.cbrc.jp.

Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis

Ligation of the chemokine receptor CCR2 on monocytes and macrophages with its ligand CCL2 results in activation of the cascade consisting of phosphatidylinositol-3-OH kinase (PI(3)K), the small G protein Rac and lamellipodium protrusion. We show here that a unique clathrin heavy-chain repeat homology protein, FROUNT, directly bound activated CCR2 and formed clusters at the cell front during chemotaxis. Overexpression of FROUNT amplified the chemokine-elicited PI(3)K–Rac–lamellipodium protrusion cascade and subsequent chemotaxis. Blocking FROUNT function by using a truncated mutant or antisense strategy substantially diminished signaling via CCR2. In a mouse peritonitis model, suppression of endogenous FROUNT markedly prevented macrophage infiltration. Thus, FROUNT links activated CCR2 to the PI(3)K–Rac–lamellipodium protrusion cascade and could be a therapeutic target in chronic inflammatory immune diseases associated with macrophage infiltration.

Software Note: TMBETADISC-RBF: Discrimination of β-barrel membrane proteins using RBF networks and PSSM profiles

Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying OMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. We have developed a method based on radial basis function networks and position specific scoring matrix (PSSM) profiles generated by PSI-BLAST and non-redundant protein database. Our approach with PSSM profiles has correctly predicted the OMPs with a cross-validated accuracy of 96.4% in a set of 1251 proteins, which contain 206 OMPs, 667 globular proteins and 378 alpha-helical inner membrane proteins. Furthermore, we applied our method on a dataset containing 114 OMPs, 187 TMH proteins and 195 globular proteins obtained with less than 20% sequence identity and obtained the cross-validated accuracy of 95%. This accuracy of discriminating OMPs is higher than other methods in the literature and our method could be used as an effective tool for dissecting OMPs from genomic sequences. We have developed a prediction server, TMBETADISC-RBF, which is available at http://rbf.bioinfo.tw/~sachen/OMP.html.

HUNT: launch of a full-length cDNA database from the Helix Research Institute

The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT.