Genetically fused charged peptides induce rapid crystallization of proteins.

We utilized electrostatic interaction to induce rapid crystallization of streptavidin. Simply mixing streptavidins possessing either a positively or negatively charged peptide at their C-terminus generated diffraction-quality crystals in a few hours. We modified the streptavidin crystals with fluorescent molecules using biotin, demonstrating the concept of protein crystals as functional biomaterials.

Decontamination of outdoor school swimming pools in Fukushima after the nuclear accident in March 2011.

Because of radioactive fallout resulting from the Fukushima Daiichi Nuclear Power Plant (NPP) accident, water discharge from many outdoor swimming pools in Fukushima was suspended out of concern that radiocesium in the pool water would flow into farmlands. The Japan Atomic Energy Agency has reviewed the existing flocculation method for decontaminating pool water and established a practical decontamination method by demonstrating the process at eight pools in Fukushima. In this method, zeolite powder and a flocculant are used for capturing radiocesium present in pool water. The supernatant is discharged if the radiocesium concentration is less than the targeted level. The radioactive residue is collected and stored in a temporary storage space. Radioactivity concentration in water is measured with a NaI(Tl) or Ge detector installed near the pool. The demonstration results showed that the pool water in which the radiocesium concentration was more than a few hundred Bq L was readily purified by the method, and the radiocesium concentration was reduced to less than 100 Bq L. The ambient dose rates around the temporary storage space were slightly elevated; however, the total increase was up to 30% of the background dose rates when the residue was shielded with sandbags.

Crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus.

The crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus KM1 has been solved by multiple isomorphous replacement. The enzyme is an alpha-amylase (family 13) with unique exo-amylolytic activity for glycosyltrehalosides. It cleaves the alpha-1,4 glycosidic bond adjacent to the trehalose moiety to release trehalose and maltooligo saccharide. Unlike most other family 13 glycosidases, the enzyme does not require Ca(2+) for activity, and it contains an N-terminal extension of approximately 100 amino acid residues that is homologous to N-terminal domains found in many glycosidases that recognize branched oligosaccharides. Crystallography revealed the enzyme to exist as a homodimer covalently linked by an intermolecular disulfide bond at residue C298. The existence of the intermolecular disulfide bond was confirmed by biochemical analysis and mutagenesis. The N-terminal extension forms an independent domain connected to the catalytic domain by an extended linker. The functionally essential Ca(2+) binding site found in the B domain of alpha-amylases and many other family 13 glycosidases was found to be replaced by hydrophobic packing interactions. The enzyme also contains a very unusual excursion in the (beta/alpha)(8) barrel structure of the catalytic domain. This excursion originates from the bottom of the (beta/alpha)(8) barrel between helix 6 and strand 7, but folds upward in a distorted alpha-hairpin structure to form a part of the substrate binding cleft wall that is possibly critical for the enzyme's unique substrate selectivity. Participation of an alpha-beta loop in the formation of the substrate binding cleft is a novel feature that is not observed in other known (beta/alpha)(8) enzymes.

Functional analysis of the C-terminal region of recombinant human thrombopoietin. C-terminal region of thrombopoietin is a "shuttle" peptide to help secretion.

Thrombopoietin (TPO) is a cytokine that primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO has a unique C-terminal tail peptide of about 160 amino acids that consists mostly of hydrophilic residues and contains six N-linked sugar chains. In order to investigate the biological function of the C-terminal domain, two series of mutations were performed. One is systematic truncation from the C terminus. Another is elimination of N-glycosylation sites in the C-terminal domain by Asn to Gln mutations. After the mutant proteins were expressed by mammalian cells, it was found that the elimination of the N-linked sugar sites did not affect the biological activity, whereas truncation of the C-terminal domain resulted in elevation of in vitro activity up to 4-fold. The C-terminal peptide itself was found to inhibit the in vitro activity. Moreover, both the C-terminal truncation and the elimination of the N-glycosylation sites decreased the secretion level progressively down to (1)/(10) that of wild type, and the amount of the mutant left in the cell increased. The N-glycosylation in the C-terminal region was found to be important for secretion of TPO. Among six N-glycosylation sites in the C-terminal region, two locations, Asn-213 and Asn-234, were found to be critical for secretion, and two other locations, Asn-319 and Asn-327, did not affect the secretion.

High-level secretion of biologically active recombinant human macrophage inflammatory protein-1alpha by the methylotrophic yeast Pichia pastoris.

The human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) was produced at a high level in Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 promoter. To ensure proper folding and secretion of the recombinant polypeptide, the MIP-1alpha gene had been fused to the Saccharomyces cerevisiae alpha-factor prepropeptide. As was revealed by analysis of the cell culture supernatant of recombinant Pichia pastoris, MIP-1alpha was efficiently secreted. Immunoblot analysis of secreted proteins from recombinant clones using a polyclonal antibody directed against MIP-1alpha revealed an apparent molecular mass of 8 kDa for the recombinant polypeptide. Up to 70 mg of MIP-1alpha was purified from 1 liter of yeast culture supernatant by a single chromatography step. Biological activity of recombinant MIP-1alpha was shown in a chemotaxis assay. Here, the polypeptide specifically induced migration of U937 cells expressing the CCR1 (MIP-1alpha receptor). Also, in competition binding assays the recombinant MIP-1alpha displayed high affinity binding.

Atomic structure of the GCSF-receptor complex showing a new cytokine-receptor recognition scheme.

Granulocyte colony-stimulating factor (GCSF) is the principal growth factor regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes and is used to treat neutropenia. GCSF is a member of the long-chain subtype of the class 1 cytokine superfamily, which includes growth hormone, erythropoietin, interleukin 6 and oncostatin M. Here we have determined the crystal structure of GCSF complexed to the BN-BC domains, the principal ligand-binding region of the GCSF receptor (GCSFR). The two receptor domains form a complex in a 2:2 ratio with the ligand, with a non-crystallographic pseudo-twofold axis through primarily the interdomain region and secondarily the BC domain. This structural view of a gp130-type receptor-ligand complex presents a new molecular basis for cytokine-receptor recognition.

Structural basis of the conversion of T4 lysozyme into a transglycosidase by reengineering the active site.

In contrast to hen egg-white lysozyme, which retains the beta-configuration of the substrate in the product, T4 lysozyme (T4L) is an inverting glycosidase. The substitution Thr-26 --> His, however, converts T4L from an inverting to a retaining enzyme. It is shown here that the Thr-26 --> His mutant is also a transglycosidase. Indeed, the transglycosylation reaction can be more effective than hydrolysis. In contrast, wild-type T4L has no detectable transglycosidase activity. The results support the prior hypothesis that catalysis by the Thr-26 --> His mutant proceeds via a covalent intermediate. Further mutations (Glu-11 --> His, Asp-20 --> Cys) of the T26H mutant lysozyme indicate that the catalytic mechanism of this mutant requires Glu-11 as a general acid but Asp-20 is not essential. The results help provide an overall rationalization for the activity of glycosidases, in which a highly conserved acid group (Glu-11 in T4L, Glu-35 in hen egg-white lysozyme) on the beta-side of the substrate acts as a proton donor, whereas alterations in the placement and chemical identity of residues on the alpha-side of the substrate can lead to catalysis with or without retention of the configuration, to transglycosidase activity, or to the formation of a stable enzyme-substrate adduct.

Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation.

We previously demonstrated by X-ray crystallography and electrospray mass spectrometry that D52E mutant hen lysozyme formed a covalent enzyme-substrate adduct on reaction with N-acetylglucosamine oligomer. This observation indicates that D52E lysozyme may acquire a catalytic pathway via a covalent adduct. To explain this pathway, the formation and hydrolysis reactions of the covalent adduct were investigated. Kinetic analysis indicated that the hydrolysis step was the rate-limiting step, 60-fold slower than the formation reaction. In the formation reaction, the pH dependence was bell-shaped, which was plausibly explained by the functions of the two catalytic pKas of Glu35 and Glu52. On the other hand, the pH dependence in the hydrolysis was sigmoidal with a transition at pH 4. 5, which was identical with the experimentally determined pKa of Glu35 in the covalent adduct, indicating that Glu35 functions as a general base to hydrolyze the adduct. To improve the turnover rate of D52E lysozyme, the mutation of N46D was designed and introduced to D52E lysozyme. This mutation reduced the activation energy in the hydrolysis reaction of the covalent adduct by 1.8 kcal/mol at pH 5.0 and 40 degrees C but did not affect the formation reaction. Our data may provide a useful approach to understanding the precise mechanism of the function of natural glycosidases, which catalyze via a covalent adduct.

Acanthosis nigricans with severe obesity, insulin resistance and hypothyroidism: improvement by diet control.

We report on a 27-year-old man with acanthosis nigricans (AN) associated with severe obesity, insulin resistance and hypothyroidism. A very low-calorie diet treatment decreased his weight and then ameliorated the insulin-resistant state. These effects were followed by remarkable improvement of the AN prior to the correction of the hypothyroidism. This confirms that AN may be mainly attributed to insulin resistance rather than hypothyroidism per se.

Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants.

We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol. Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species. Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli. Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so. It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not. Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response.

Region of FLO1 proteins responsible for sugar recognition.

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.

Structural and thermodynamic responses of mutations at a Ca2+ binding site engineered into human lysozyme.

Structural determinants of Ca2+ binding sites within proteins typically comprise several acidic residues in appropriate juxtaposition. Three residues (Ala-83, Gln-86, and Ala-92) in human lysozyme are characteristically mutated to Lys, Asp, and Asp, respectively, in natural Ca2+ binding lysozymes and alpha-lactalbumins. The effects of these mutations on the stability and Ca2+ binding properties of human lysozyme were investigated using calorimetry and were interpreted with crystal structures. The double mutant, in which Glu-86 and Ala-92 were replaced with Asp, clearly showed Ca2+ binding affinity, whereas neither point mutant showed Ca2+ affinity, indicating that both residues are essential. The further mutation of Ala-83 --> Lys did not affect the Ca2+ binding of the double mutant. The point mutations Ala-83 --> Lys and Glu-86 --> Asp did not affect the stability, whereas the mutation Ala-92 --> Asp was about 1.3 kcal/mol less stable. Structural analyses showed that both Asp-86 and Lys-83 were exposed to solvent. Side chains of Asp-86 and Asp-91 were rotated in opposite directions about chi1 angle, as if to reduce the electrostatic repulsion. The charged amino acids at the Ca2+ binding site did not significantly affect stability of the protein, possibly because of the local conformational change of the side chains.

Structural and functional effect of Trp-62-->Gly and Asp-101-->Gly substitutions on substrate-binding modes of mutant hen egg-white lysozymes.

In order to clarify the structural role of subsite B of hen egg-white lysozyme in hydrolytic activity towards a carbohydrate substrate, we analysed the structures of Trp-62-->Gly and Asp-101-->Gly mutant hen lysozymes, which have no side chain at positions 62 or 101, complexed with a substrate analogue, (N-acetyl-d-glucosamine)3 [(GlcNAc)3], using X-ray crystallography. The overall protein structures in the mutant lysozyme complexes were almost identical to those in the wild type. In the crystals of all the mutant complexes, the (GlcNAc)3 molecule, which is an inhibitor of wild-type lysozyme, had no inhibitory effect, but was hydrolysed as a substrate. One of the products, (GlcNAc)2, the reducing end of which is an alpha-anomer, was bound in an unproductive binding mode, protruding from the active-site cleft, and was able to act as an inhibitor. Hydrolysis of the synthetic substrate by the mutants occurred in a beta-anomer-retaining manner, and so the alpha-anomer product was converted from the beta-anomer product. Thus the interactions of Asp-101 and Trp-62 in subsite B are not essential for the catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.

Structural analysis of mutant hen egg-white lysozyme preferring a minor binding mode.

Trp62 in hen egg-white lysozyme has general features observed in protein-carbohydrate interactions, a stacking interaction toward nonpolar surface of the substrate sugar residue B and a hydrogen bonding network with the residue C. Our previous report (I. Kumagai, K. Maenaka, F. Sunada, S. Takeda, K. Miura, Eur. J. Biochem. 212 (1993) 151-156.) showed that the substitution of Trp62 changed the substrate binding modes; especially, the Trp62His mutant exhibited the drastic change of the binding mode and preferred to a minor binding mode of the wild-type enzyme. In order to clarify the relationship between functional and structural changes of the Trp62His mutant, we analyzed the structure of the Trp62His mutant hen lysozyme complexed with the substrate analogue, (GlcNAc)3, by X-ray crystallography. The overall protein structure in the mutant lysozyme complex was almost identical to that in the wild-type. His62 shared almost the same plane as the indole ring of Trp62 of the wild-type. Although the (GlcNAc)3 molecule which is an inhibitor against the wild-type lysozyme was cocrystallized, the Trp62His mutant did not put it in the sites A-B-C but hydrolyzed it as a substrate. One of the products, (GlcNAc)2, whose reducing end is alpha-anomer, was bound in another binding mode sticking out from the active-site cleft. The hydrolytic activity against the synthetic substrate showed that the mutant was a beta-anomer retaining enzyme, so the alpha-anomer product was converted from the beta-anomer product. Therefore, the Trp62His mutant showed the remarkable change of the substrate binding modes not by alteration of the catalytic system but possibly by subtle rearrangement of general features of protein-carbohydrate interactions between His62 and the sugar residues B and C.

A covalent enzyme-substrate adduct in a mutant hen egg white lysozyme (D52E).

A mutant hen egg white lysozyme, D52E, was designed to correspond to the structure of the mutant T4 lysozyme T26E (Kuroki, R., Weaver, L. H., and Matthews B. W. (1993) Science 262, 2030-2033) to investigate the role of the catalytic residue on the alpha-side of the saccharide in these enzymes. The D52E mutant forms a covalent enzyme-substrate adduct, which was detected by electron ion spray mass spectrometry. X-ray crystallographic analysis showed that the covalent adduct was formed between Glu-52 and the C-1 carbon of the N-acetylglucosamine residue in subsite D of the saccharide binding site. It suggests that the catalytic mechanism of D52E mutant lysozyme proceeds through a covalent enzyme-substrate intermediate indicating a different catalytic mechanism from the wild type hen egg white lysozyme. It was confirmed that the substitution of Asp-52 with Glu is structurally and functionally equivalent to the substitution of Thr-26 with Glu in T4 lysozyme. Although the position of the catalytic residue on the beta-side of the saccharide is quite conserved among hen egg white lysozyme, goose egg white lysozyme, and T4 phage lysozyme, the adaptability of the side chain on the alpha-side of the saccharide is considered to be responsible for the functional variation in their glycosidase and transglycosidase activities.

[Coagulase typing of Staphylococcus aureus in the geriatric wards after introduction of preventive measures of hospital infection].

In the early 1980's methicillin-resistant Staphylococcus aureus (MRSA) was reported as a major pathogenic organism of geriatric hospital infection in Japan. At the same time in our geriatric wards, including 190 beds, MRSA infection was prevalent. In the early 1980's in our geriatric wards minocycline was one of the most sensitive antibiotics to MRSA isolated in our wards and used frequently against MRSA pneumonias and bacteremia. In the late 1980's resistant strains of MRSA to minocycline rapidly increased because vancomycin was not allowed to introduced for treatment of MRSA before 1991 in Japan. At the same period the predominant coagulase type changed from type II to type VII. To decrease minocycline-resistant strains to MRSA after 1987, use of minocycline was limited. Moreover since Oct. 1991 to decrease nosocomial infections some active preventive measures against hospital infection, including limited use of 2nd and 3rd cephems, were taken. In this study changing patterns of coagulase type of Staphylococcus aureus were discussed. At least 4 years was needed to find out that the predominant coagulase type changed from type VII to type II again in 1991. In this study about 22 antimicrobial agents MICs of 313 strains of Staphylococcus aureus isolated between March 1992 and June 1993 were determined and compared with the data of MICs before introduction of preventive measures. The pattern of susceptibility to MINO was in part improved. Thus the some sensitive strains of S. aureus were observed again in our geriatric wards. Interestingly indeed it took approximately 5 years to find out the emergence of sensitive strains to MINO since limitation of use of MINO in 1987.