Glycome project: concept, strategy and preliminary application to Caenorhabditis elegans.

Glycans play a central role as potential mediators between complex cell societies, because all living organisms consist of cells covered with diverse carbohydrate chains reflecting various cell types and states. However, we have no idea how diverse these carbohydrate chains actually are. The main purpose of this article is to persuade life scientists to realize the fundamental importance of taking some action by becoming involved in "glycomics". "Glycome" is a term meaning the whole set of glycans produced by individual organisms, as the third bioinformative macromolecules to be elucidated next to the genome and proteome. Here a basic strategy is presented. The essence of the project includes the following: (a) glycopeptides, but not glycans released from their core proteins, are targeted for linkage to genome databases; (b) Caenorhabditis elegans is used as the first model organism for this project, since its genome project has already been completed; (c) four essential attributes are adopted to characterize each glycopeptide: (i) cosmid identification number (ID), (ii) molecular weight (M(r)), (iii) retention (Rs) of pyridylaminated (PA) oligosaccharides in 2-D mapping, and (iv) dissociation constants (Kd's) of PA-oligosaccharides for a set of lectins. Thus, the obtained ID, M(r), R and Kd's construct the glycome database, which will be open as the previous genome and proteome databases. For the project to proceed the "glyco-catch" method is proposed, where a group of target glycopeptides are captured by means of lectin-affinity chromatography after protease digestion. Already glycopeptides from asialofetuin and ovalbumin were successfully captured by galectin-agarose and Con A-agarose, respectively. Further, to examine the practical validity of the method, we extracted membrane proteins from C. elegans with 1% Triton X-100, and isolated specific glycopeptides by use of the same galectin column. One of the glycopeptides was successfully identified in the C. elegans genome database. Finally, for determination of Kd between glycopeptides and lectins, a recently reinforced frontal affinity chromatography (FAC) is proposed as an alternative to define glycan structures in place of determining every covalent structure.

Direct binding of the signal-transducing adaptor Grb2 facilitates down-regulation of the cyclin-dependent kinase inhibitor p27Kip1.

Ectopic expression of Jab1/CSN5 induces specific down-regulation of the cyclin-dependent kinase (Cdk) inhibitor p27 (p27(Kip1)) in a manner dependent upon transportation from the nucleus to the cytoplasm. Here we show that Grb2 and Grb3-3, the molecules functioning as an adaptor in the signal transduction pathway, specifically and directly bind to p27 in the cytoplasm and participate in the regulation of p27. The interaction requires the C-terminal SH3-domain of Grb2/3-3 and the proline-rich sequence contained in p27 immediately downstream of the Cdk binding domain. In living cells, enforcement of the cytoplasmic localization of p27, either by artificial manipulation of the nuclear/cytoplasmic transport signal sequence or by coexpression of ectopic Jab1/CSN5, markedly enhances the stable interaction between p27 and Grb2. Overexpression of Grb2 accelerates Jab1/CSN5-mediated degradation of p27, while Grb3-3 expression suppresses it. A p27 mutant unable to bind to Grb2 is transported into the cytoplasm in cells ectopically expressing Jab1/CSN5 but is refractory to the subsequent degradation. These findings indicate that Grb2 participates in a negative regulation of p27 and may directly link the signal transduction pathway with the cell cycle regulatory machinery.

Improved salt tolerance of transgenic tobacco expressing apoplastic yeast-derived invertase.

We investigated the salt tolerance of transgenic tobacco, in which yeast invertase is expressed in the apoplastic (Apo-Inv) spaces. Whereas photosynthetic activities in wild-type tobacco in light were inhibited under salt stress, transgenic Apo-Inv tobacco maintained constant photosynthetic activities. The physical appearance of plants under salt stress also indicates that yeast invertase expression in the apoplastic space is beneficial for inducing salt tolerance. Apo-Inv tobacco had a much higher osmotic pressure increase in the cell sap than did wild-type tobacco under this type of stress. The physiological importance of sucrose metabolism under salt stress is discussed.

Time dependence of aggregation in crystallizing lysozyme solutions probed using NMR self-diffusion measurements.

The time dependence of aggregation in supersaturated lysozyme solutions was studied using pulsed-gradient spin-echo NMR diffusion measurements as a function of lysozyme concentration at pH 6.0 and 298 K in the presence of 0.5 M NaCl. The measurements provide estimates of the weight-averaged diffusion coefficient of the monomeric to intermediate molecular weight lysozyme species present in the solution (very large aggregates and crystals are excluded from the average due to the NMR relaxation-weighting effects inherent in the method). The results show that the average molecular weight of the various lysozyme aggregates changed with sigmoidal kinetics and that these kinetics were strongly influenced by the initial lysozyme concentration. The visualization of the time dependence of the protein aggregation afforded by this method provides a deeper understanding of how the crystallizing conditions (especially the initial protein concentration) are related to the resulting crystals.

Application of reinforced frontal affinity chromatography and advanced processing procedure to the study of the binding property of a Caenorhabditis elegans galectin.

Frontal affinity chromatography is a very useful and simple method to analyze molecular interactions between an analyte and an immobilized ligand by calculating the extent of "retardation" of the elution front. We developed a very simple and efficient data-processing procedure that enables the measurement of very small differences in retardation with precision. This procedure was successfully applied to comparison of the binding properties of recombinant C. elegans galectins for their ligand.

Effects of low-level occupational exposure to styrene on color vision: dose relation with a urinary metabolite.

To investigate the threshold effects of chronic low-level occupational exposure to styrene on color vision, we examined color discrimination in 105 male workers exposed to styrene (mean age 37.7 years; mean length of exposure 6.2 years; mean urinary concentration of mandelic acid 0.21 g/L) and in 117 referents (mean age 37.7 years). We also assessed the effects of styrene by examination of the nature of the relation between disorders of nervous function and age, alcohol consumption, and other variables. A standardized questionnaire was adopted to collect information about work history, occupational or nonoccupational solvent exposure, alcohol consumption, and drug use. Color vision was evaluated by the Lanthony desaturated panel D-15 test. The results of the test were expressed as the color confusion index (CCI). There was a dose-dependent relationship between the urinary concentration of mandelic acid and color vision loss. The CCIs of the subgroups whose urinary mandelic acid levels were 0.1-0.2 and >0.2 g/L were significantly higher than those of each referent group (P<0.05 and P<0.01, respectively), but not in the subgroup whose urinary mandelic acid level was lower than 0.1 g/L. Our study suggests that a low level of styrene, presumably 0.1-0.2 g/L, involves the risk of inducing adverse effects on color vision. After confounding factors were adjusted for, the urinary mandelic acid level had a significant positive relationship with color vision.

Sugar binding properties of the two lectin domains of the tandem repeat-type galectin LEC-1 (N32) of Caenorhabditis elegans. Detailed analysis by an improved frontal affinity chromatography method.

The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14-kDa-type galectins. To elucidate the biological meaning of this unique structure containing two probable sugar binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containing N-linked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2-5-fold stronger binding toward all complex-type sugars compared with Nh. Both Nh and Ch preferred Galbeta1-3GlcNAc to Galbeta1-4GlcNAc. Because the Fucalpha1-2Galbeta1-3GlcNAc (H antigen) structure was found to interact with all immobilized protein columns significantly, the K(d) value of pentasaccharide Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA for each column was determined by analyzing the concentration dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 x 10(-5), 1.3 x 10(-4), and 6.5 x 10(-5) m, respectively. The most significant difference between Nh and Ch was in their affinity for GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA, which contains the blood group A antigen; the K(d) value for immobilized Nh was 4.8 x 10(-5) m, and that for Ch was 8.1 x 10(-4) m. The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.

Reinforcement of frontal affinity chromatography for effective analysis of lectin-oligosaccharide interactions.

Frontal affinity chromatography is a method for quantitative analysis of biomolecular interactions. We reinforced it by incorporating various merits of a contemporary liquid chromatography system. As a model study, the interaction between an immobilized Caenorhabditis elegans galectin (LEC-6) and fluorescently labeled oligosaccharides (pyridylaminated sugars) was analyzed. LEC-6 was coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow (100 microm diameter), and packed into a miniature column (e.g., 10 x 4.0 mm, 0.126 ml). Twelve pyridylaminated oligosaccharides were applied to the column through a 2-ml sample loop, and their elution patterns were monitored by fluorescence. The volume of the elution front (V) determined graphically for each sample was compared with that obtained in the presence of an excess amount of hapten saccharide, lactose (V0); and the dissociation constant, Kd, was calculated according to the literature [K. Kasai, Y. Oda, M. Nishikawa, S. Ishii, J. Chromatogr. 376 (1986) 33]. This system also proved to be useful for an inverse confirmation; that is, application of galectins to an immobilized glycan column (in the present case, asialofetuin was immobilized on Sepharose 4 Fast Flow), and the elution profiles were monitored by fluorescence based on tryptophan. The relative affinity of various galectins for asialofetuin could be easily compared in terms of the extent of retardation. The newly constructed system proved to be extremely versatile. It enabled rapid (analysis time 12 min/cycle) and sensitive (20 nM for pyridylaminated derivatives, and 1 microg/ml for protein) analyses of lectin-carbohydrate interactions. It should become a powerful tool for elucidation of biomolecular interactions, in particular for functional analysis of a large number of proteins that should be the essential issues of post-genome projects.

Pairing of oligosaccharides in the Fc region of immunoglobulin G.

The Fc portion of immunoglobulin G (IgG) expresses paired oligosaccharides with microheterogeneities, which are associated with efficiencies of effector functions and with pathological states. A comparison of electrospray ionization mass spectrometry data obtained using a variety of Fc fragments derived from human and mouse IgG that do and do not retain the inter-chain disulfide bridge(s) revealed that (1) the Fc portion can be asymmetric as well as symmetric with respect to glycosylation and (2) the ratios of the individual glycoforms are different from what is expected from the random pairing.

Lethal effect of the expression of a killer gene SMK1 in Saccharomyces cerevisiae.

Expression of the SMK1 gene which encodes the yeast killer toxin SMKT is lethal in Saccharomyces cerevisiae. Effects of deletion and site-directed mutagenesis of SMK1 on the lethality and the secretion of the gene products were examined. Deletion of the interstitial gamma peptide or the C-terminal loop from Ala208 to the C-terminal Asp222 had no effect on the lethality. Those SMK1 products that lacked either the gamma peptide or the C-terminal loop were expressed in the cells but were not secreted into the culture medium, suggesting that these peptides may have a role in secretion or in protein stability. On the other hand, deletion of the signal sequence resulted in complete loss of the lethal activity. Entering the secretory pathway may be critical for the lethality. Further, deletion of the region from the C-terminus to Leu207 resulted in loss of the lethal activity. Leu207 is located at the C-terminus of the central strand of the beta-sheet structure of SMKT and its side chain is thrust into a hydrophobic environment between the beta-sheet and the alpha-helices. The result obtained upon substitutions of Ala, Ser or Glu for Leu207 suggested that the side chain of Leu207 stabilizes the hydrophobic environment that contributes to the overall structure of the SMK1 product.

A novel NMR method for determining the interfaces of large protein-protein complexes.

Identification of the interfaces of large (Mr > 50,000) protein-protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein-protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.

Cdk2-dependent and -independent pathways in E2F-mediated S phase induction.

The transcription factor E2F plays an important role in G(1) to S phase transition in the higher eukaryotic cell cycle. Although a number of E2F-inducible genes have been identified, the biochemical cascades from E2F to the S phase entry remain to be investigated. In this study, we generated stably transfected mouse NIH3T3 cells that express exogenous human E2F-1 under the control of a heavy metal-inducible metallothionein promoter and analyzed the molecular mechanism of the E2F-1-mediated initiation of chromosomal DNA replication. Ectopic E2F-1 expression in cells arrested in G(0)/G(1) by serum deprivation enabled them to progress through G(1) and to enter S phase. During the G(1) progression, mouse cyclin E, but little of cyclin D1, was induced to express, which subsequently activated Cdk2. Experiments using the Cdk inhibitory proteins p27, p18, and p19 proved that the activity of Cdk2, but not of Cdk4, was required for S phase entry mediated by E2F-1. Minichromosome maintenance proteins (MCM) 4 and 7, the components of the DNA-replication initiation complex (RC), were constitutively expressed during the cell cycle, although the MCM genes are well known E2F-inducible genes. However, tight association of these two proteins with chromatin depended upon ectopic E2F-1 expression. In contrast, the Cdc45 protein, another RC component, which turned out to be a transcriptional target of E2Fs, was induced to express and subsequently bound to chromatin in response to E2F-1. Experiments utilizing a chemical Cdk-specific inhibitor, butyrolactone I, revealed that Cdk2 activity was required only for chromatin binding of the Cdc45 proteins, and not for the expression of Cdc45 or chromatin binding of MCM4 and -7. These results indicate that at least two separate pathways function downstream of E2F to initiate S phase; one depends upon the activity of Cdk2 and the other does not.

An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein.

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analysis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associate under acidic conditions, but become dissociated and denatured under neutral and basic conditions. In order to understand the unique pH-dependent denaturation mechanism of this protein, a pH titration was performed by utilizing ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subunits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-dependent dissociation occurs in the liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of this protein. In conclusion, the pH titration of a protein by ESI-MS is particularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules.

Structural basis of the interaction between IgG and Fcgamma receptors.

The binding of multivalent antigen-antibody complexes to receptors for the Fc portion of IgG (FcgammaR) induces the clustering of the FcgammaR and triggers cell activation leading to defence reactions against pathogens. The Fc portion of IgG consists of two identical polypeptide chains which are related to each other by a 2-fold axis and are folded in two structural domains, the C(H)2 domain, near the flexible hinge region of the IgG molecule, and the C(H)3 domain. We studied the interaction in solution between the Fc fragment of mouse IgG2b and the extracellular region of mouse FcgammaRII. We find that one Fc molecule binds one FcgammaRII molecule only. Using NMR spectroscopy, we show that FcgammaRII binds to a negatively charged area of the C(H)2 domain, corresponding to the lower hinge region, and that the binding of FcgammaRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. We therefore propose a model that explains why IgG molecules are unable to trigger FcgammaR-mediated cellular responses spontaneously in the absence of crosslinking by multivalent antigens.

1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor.

In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-13C6, 2H7]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with 13C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-13C6, 2H7]glucose.

The pH-dependent structural variation of complementarity-determining region H3 in the crystal structures of the Fv fragment from an anti-dansyl monoclonal antibody.

The Fv fragment from an anti-dansyl antibody was optimally crystallized into two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high resolution at 112 K (at 1.45 A for the crystal at pH 5.25 and at 1.55 A for that at pH 6.75). In the two crystal structures, marked differences were identified in the first half of CDRH3 s having an amino acid sequence of Ile95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro1 00H-Trp100aH-Phe100bH-Ala101H- Tyr102H. NMR pH titration experiments revealed the p Kavalues of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (p Ka=6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structures including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His93L and His55H were almost invariant between the two crystal structures, those around His98Hs showed great difference in spite of the small conformational difference of His98H between the two crystal structures. These spectroscopic and crystallographic findings suggested that the change in the protonation state in His98H was responsible for the structural differences between pH 5.25 and 6.75. In addition, the most plausible binding site of the dansyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 and the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue for the binding mode of the dansyl group in the site.

Strategies for diagnosing and alleviating artifactual attenuation associated with large gradient pulses in PGSE NMR diffusion measurements.

The generation of phase-based artifacts resulting from mismatch in the effective areas (i.e., the time integrals) of sequential gradient pulses is discussed in the context of large gradient pulsed-gradient spin-echo (PGSE) NMR diffusion measurements. Such effects result in artifactual attenuation and distortion in the spectra which, in the first instance, are similar to (and commonly mistaken for) the effects of eddy currents. Small degrees of mismatch cause "unphysical" concave downward curvature in PGSE attenuation plots of freely diffusing species. However, larger mismatches can result in artifactual diffraction peaks in the plots which could easily be confused for true restricted diffusion effects. Although "rectangular" gradient pulses are preferable from a theoretical viewpoint, we found that shaped gradient (e.g., half-sine) pulses, which due to their slower rise and fall times were more tractable for the current amplifier, were more sequentially reproducible. As well as generating fewer phase-based artifacts such shaped pulses also decrease the likelihood of vibration problems.

A multinuclear NMR study of the active site of an endoglucanase from a strain of Bacillus. Use of Trp residues as structural probes.

In the hydrolytic reaction catalyzed by an endoglucanase from a Bacillus strain (endoglucanase K), 2 of 12 Trp residues, Trp174 and Trp243, are responsible for binding of the substrate and/or for the catalysis (Kawaminami, S., Ozaki, K., Sumitomo, N., Hayashi, Y., Ito, S., Shimada, I., and Arata, Y. (1994) J. Biol. Chem. 269, 28752-28756). Here we report results of a stable isotope-aided NMR analysis of the active site of endoglucanase K, using Trp174 and Trp243 as structural probes. Hydrogen-deuterium exchange experiments performed for the NH protons of main and side chains of Trp residues revealed that Trp174 and Trp243 are located in the hydrophilic and hydrophobic microenvironments in the active site, respectively. We also carried out pH titration experiments for indole C2 proton resonances of Trp residues and measured the pH dependence of specific activities for wild-type endoglucanase K and its mutants in which Glu or Asp residues are replaced with their respective amide forms. On the basis of the results obtained from the present study, we conclude that (a) Glu130 and Asp191, which are in spatial proximity to Trp174 and Trp243 in the active site, play a crucial role in the enzymatic activity; (b) Glu130 and Asp191 interact with each other in the active site, leading to an increase in the pKa values to 5.5 for both amino acid residues; and (c) the pKa values of Glu130 and Asp191 would lead to an unusually narrow pH-activity profile of the endoglucanase K.