Restoration of cellular function of mesenchymal stem cells from a hypophosphatasia patient.

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages and are used for regenerative treatments for a variety of diseases. However, the patient's cells cannot be used to treat genetic diseases. Allogeneic cells can serve as an alternative but long-term survival is uncertain. Our experience of allo-transplantation to a patient with hypophosphatasia, which is caused by mutations of the tissue non-specific alkaline phosphatase (TNSALP) gene resulting in low serum alkaline phosphatase (ALP) activity and skeletal deformity, did not improve these clinical characteristics. Therefore, we sought to use autologous MSCs for the treatment of hypophosphatasia. MSCs derived from the patient's bone marrow had a similar profile when compared with well-reported MSCs. However, the MSCs had extremely low ALP activity and could not produce a mineralized bone matrix even under the osteogenic culture conditions. We therefore transduced a retroviral vector with TNSALP promoter-driven TNSALP gene in the MSCs. In the culture condition, the MSCs had about 7-fold higher ALP activity than did mock-transduced MSCs, and showed mineralization as well as bone-specific markers. Furthermore, the MSCs, but not mock-transduced MSCs, newly formed bone at the frequency of 50% in nude rats. Transplantation of the TNSALP-transduced autologous MSCs might become a new therapy for hypophosphatasia.

Crystallization of the complexes between M315 idiotope and its monoclonal anti-idiotopic antibody Fab fragment.

The Fab fragment of a monoclonal anti-idiotopic antibody against M315 has been isolated and its complexes with Fv and Fab' fragment of M315 have been crystallized by using poly(ethylene glycol) 6000 or ammonium sulfate. X-ray diffraction photographs showed that the crystal of the complex with Fv diffracts better than that with Fab'. The Fv-complexed crystal was shown to be tetragonal I4, with cell dimensions a = 152 A and c = 69 A, and to contain one complex molecule of about 75,000 molecular weight in the crystallographic asymmetric unit.

Structure of [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus refined at 2.3 A resolution. Structural comparisons of bacterial ferredoxins.

The structure of a low-potential ferredoxin isolated from Bacillus thermoproteolyticus has been refined by a restrained least-squares method. The final crystallographic R factor is 0.204 for 2906 reflections with F greater than 3 sigma F in the 6.0 to 2.3 A resolution range. The model contains 81 amino acid residues, one [4Fe-4S] cluster, and 59 water molecules. The root-mean-square deviation from ideal values for bond lengths is 0.018 A, and the mean coordinate error is estimated to be 0.25 A. The present ferredoxin is similar in the topology of the polypeptide backbone to the dicluster-type ferredoxins from Peptococcus aerogenes and Azotobacter vinelandii, but has considerable insertions and deletions of the peptide segments as well as different secondary structures. Although all but the C-terminal C zeta atoms of P. aerogenes ferredoxin superpose on the C alpha atoms of A. vinelandii ferredoxin, only 60% superpose on the C alpha atoms of B. thermoproteolyticus ferredoxin, with a root-mean-square distance of 0.82 A for each pair. The conformations of the peptide segments surrounding the [4Fe-4S] clusters in these three ferredoxins are all conserved. Moreover, the schemes for the NH...S hydrogen bonds in these ferredoxins are nearly identical. The site of the aromatic ring of Tyr27 in B. thermoproteolyticus ferredoxin is close spatially to that of Tyr28 in P. aerogenes ferredoxin with reference to the cluster, but these residues do not correspond in the spatial alignment of their polypeptide backbones. We infer that in monocluster-type ferredoxins, the side-chain at the 27th residue has a crucial effect on the stability of the cluster. Of the four cysteine residues that bind to the second Fe-S cluster in the dicluster-type ferredoxins, two are conserved in the monocluster-type ferredoxins from Desulfovibrio gigas. D. desulfuricans Norway, and Clostridium thermoaceticum. The tertiary structure of B. thermoproteolyticus ferredoxin suggests that in such monocluster-type ferredoxins these two cysteine residues, which in it correspond to Ala21 and Asp53, form a disulfide bridge.

Crystallization of a complex between an elastase-specific inhibitor elafin and porcine pancreatic elastase.

A complex between synthetic elafin, an elastase-specific inhibitor, and porcine pancreatic elastase was crystallized using 2-methyl-2,4-pentanediol as precipitant. The crystals belong to the monoclinic space group P2(1) with cell parameters a = 37.91 A, b = 73.32 A, c = 48.92 A, beta = 105.4 degrees, and one complex molecule in the asymmetric unit. The crystals diffract X-rays to 1.9 A resolution and are suitable for crystallographic structure analysis.

The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization.

The crystal structure of oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) from Bacillus cereus ATCC7064 has been refined to 2.0 A resolution with an R-factor of 19.6% for 43,328 reflections. The final model contains 4646 protein atoms and 221 ordered water molecules with respective root-mean-square deviations of 0.015 A for bond lengths and of 3.166 degrees for bond angles from the ideal values. The structure consists of three domains: the N-terminal domain (residues 1 to 104 and 175 to 480), the subdomain (residues 105 to 174) and the C-terminal domain (residues 481 to 558). The N-terminal domain takes a (beta/alpha)8-barrel structure with additions of an alpha-helix (N alpha6') between the sixth strand Nbeta6 and the sixth helix N alpha6, an alpha-helix (N alpha7') between the seventh strand Nbeta7 and the seventh helix N alpha7 and three alpha-helices (N alpha8', N alpha8" and N alpha8'" between the eighth strand Nbeta8 and the eighth helix N alpha8. The subdomain is composed of an alpha-helix, a three-stranded antiparallel beta-sheet, and long intervening loops. The C-terminal domain has a beta-barrel structure of eight antiparallel beta-strands folded in double Greek key motifs, which is distorted in the sixth strand Cbeta6. Three catalytic residues, Asp199, Glu255 and Asp329, are located at the bottom of a deep cleft formed by the subdomain and a cluster of the two additional alpha-helices N alpha8' and N alpha8" in the (beta/alpha)8-barrel. The refined structure reveals the locations of 21 proline-substitution sites that are expected to be critical to protein thermostabilization from a sequence comparison among three Bacillus oligo-1,6-glucosidases with different thermostability. These sites lie in loops, beta-turns and alpha-helices, in order of frequency, except for Cys515 in the fourth beta-strand Cbeta4 of the C-terminal domain. The residues in beta-turns (Lys121, Glu208, Pro257, Glu290, Pro443, Lys457 and Glu487) are all found at their second positions, and those in alpha-helices (Asn109, Glu175, Thr261 and Ile403) are present at their N1 positions of the first helical turns. Those residues in both secondary structures adopt phi and phi values favorable for proline substitution. Residues preceding the 21 sites are mostly conserved upon proline occurrence at these 21 sites in more thermostable Bacillus oligo-1,6-glucosidases. These structural features with respect to the 21 sites indicate that the sites in beta-turns and alpha-helices have more essential prerequisites for proline substitution to thermostabilize the protein than those in loops. This well supports the previous finding that the replacement at the appropriate positions in beta-turns or alpha-helices is the most effective for protein thermostabilization by proline substitution.

Crystal structures of a mutant maltotetraose-forming exo-amylase cocrystallized with maltopentaose.

The three-dimensional structures of the catalytic residue Glu219-->Gln mutant of Pseudomonas stutzeri maltotetraose-forming exo-alpha-amylase, and its complex with carbohydrate obtained by cocrystallization with maltopentaose were determined. Two crystal forms were obtained for the complexed enzyme, and a bound maltotetraose was found in each. The structures were analyzed at 2.2 A and 1.9 A resolution, respectively for the uncomplexed and complexed mutant. These structures were compared with the wild-type enzyme structure. In the complexed crystals, the maltotetraose was firmly bound, extensively interacting with the amino acid environments in the active cleft. The non-reducing end glucose unit was hydrogen bonded to the side-chain of Asp160 and the main-chain nitrogen of Gly158, which seem to be predominantly required for the recognition of the non-reducing end of the substrate that determines the exo-wise degradation of this enzyme. The reducing end glucose unit of bound maltotetraose showed clear deformation, adopting a half-chair conformation with extensive hydrogen bonds to surrounding polypeptides. The C1-atom of this deformed glucose unit lies very close to Asp193OD1 with a distance of 2.6 A. The catalytic residue Asp294 is firmly hydrogen-bonded to the O2 and O3-hydroxyl groups of the deformed reducing end glucose unit. Upon binding of the carbohydrate, small but significant induced fits were observed in the regions of Asp294, Phe156, Ile157, and Asp160. Possible roles of the three catalytic residues are also discussed.

Crystallization and preliminary X-ray studies of Bacillus pumilus IPO xylanase.

The xylan-degrading enzyme xylanase, from Bacillus pumilus IPO, has been crystallized. The crystals are monoclinic, space group P21 with a = 40.8 A, b = 66.8 A, c = 34.7 A and beta = 103.0 degrees. The asymmetric unit contains one molecule of Mr 22,500. The crystals diffract to at least 2.5 A resolution, and they are suitable for X-ray crystal structure analysis at high resolution.

Crystallographic study of endogenous alpha-amylase inhibitor from wheat.

Endogenous alpha-amylase inhibitor from wheat has been crystallized by a microdialysis method. There are two forms of monoclinic crystal in a microdialysis cell with a space group of P2(1). The unit cell dimensions are a = 43.5 A, b = 64.8 A, c = 32.2 A, beta = 113 degrees for the rod-like crystal, and a = 42.5 A, b = 65.2 A, c = 32.2 A, beta = 112 degrees for the plate-like crystal. The former is suitable for structure analysis because it gives the sharp diffraction beyond 2.0 A resolution, and the latter tends to form a twin crystal. A heavy-atom derivative has been successfully prepared with the heavy-atom reagent K2PtCl4, and structure analysis is in progress.

Tertiary structure of Bacillus thermoproteolyticus [4Fe-4S] ferredoxin. Evolutionary implications for bacterial ferredoxins.

The structure of a low-potential [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic R-factor of 0.33, with 2.3 A (1 A = 0.1 nm) resolution data. The least-squares refinement based on the Bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 A, apart. The NH ... S hydrogen bonds between polypeptide nitrogen atoms, and both cysteine and inorganic sulfur atoms, are present, as in ferrodoxin from Peptococcus aerogenes. The polypeptide chain of the B. thermoproteolyticus ferredoxin has a fold closely similar to that of 2[4Fe-4S] ferredoxin from P. aerogenes. The structural correspondence indicates strongly that both types of ferredoxin evolved from a common ancestor. The second cluster-binding region in P. aerogenes ferredoxin corresponds to the alpha-helix in B. thermoproteolyticus ferredoxin. The secondary-structure predictions strongly suggest that the alpha-helix is generally present in the monocluster-type ferredoxins. The conformational change to alpha-helix, insertions of a loop and a protrusion, as well as the absence of the second cluster in B. thermoproteolyticus ferredoxin, result in the lack of 2-fold symmetry present in P. aerogenes ferredoxin. So, the track of gene duplication is no longer detectable in the tertiary structure alone. The evolutionary events that may have occurred in the ferredoxins with the [4Fe-4S] cluster are discussed.

Crystallization and preliminary X-ray studies of glutathione synthetase from Escherichia coli B.

The glutathione synthetase from Escherichia coli B has been crystallized from 27% saturated ammonium sulfate solution (pH 5.5). The crystals are hexagonal, space group P6(2)22 or P6(4)22. The cell dimensions are a = b = 88.0 A, c = 164.2 A, and gamma = 120 degrees. The enzyme is a tetramer (Mr = 143,000) with 222 symmetry, and the asymmetric unit contains one subunit molecule (Mr = 35,600). The crystals diffract to at least 2.5 A resolution.

Crystal structure of a maltotetraose-forming exo-amylase from Pseudomonas stutzeri.

The three-dimensional structure of an exo-type alpha-amylase from Pseudomonas stutzeri which degrades starch from its non-reducing end to produce maltotetraose has been determined by X-ray structure analysis. The catalytic domain of this enzyme (G4-2), whose structure was determined, is a product of spontaneous limited proteolysis in culture broth. It has 429 amino acid residues and a molecular mass of 47,200, and crystallizes in ammonium sulfate solution at pH 7.5. The structure was elucidated by the multiple isomorphous replacement method and refined at 2.0 A resolution, resulting in a final R-factor of 0.178 for significant reflections with a root-mean-square deviation from ideality in bond distances of 0.013 A. The polypeptide chain of this molecule folds into three domains; the first with a (beta/alpha)8 barrel structure, the second with an excursed part from the first one, and the third with five-stranded antiparallel beta-sheets. The active cleft is formed on the C-terminal side of the beta-sheets in the (beta/alpha)8 barrel as in the known endo-type alpha-amylases. A histidine side-chain nitrogen ND1 is coordinated to one of the bound calcium ion. The recognition site of the non-reducing end of the amylose that determines exo-wise degradation is presumed to be at one end of this cleft where there is a disordered loop consisting of the 66th to 72nd residues, and a loop carrying an aspartic acid (Asp160). These structural features may be responsible for the binding of the non-reducing end of the substrate amylose.

Structure of the [2Fe-2S] ferredoxin I from the blue-green alga Aphanothece sacrum at 2.2 A resolution.

Crystals of a [2Fe-2S] ferredoxin (Fd) I with a relative molecular mass of 10,480 were obtained from the blue-green alga Aphanothece sacrum. Each asymmetric unit of the crystal contains four molecules. An electron density map calculated by the single isomorphous replacement method with the anomalous dispersion at 2.5 A resolution was refined by averaging the four molecules in the asymmetric unit. Positional and isotropic thermal parameters for the non-hydrogen atoms of the four molecules and 158 water molecules were refined to an R-factor (R = sigma[Fo-Fc[/sigma Fo) of 0.23 by the restrained least-squares method. The estimated root-mean-square (r.m.s.) error for the atomic positions is 0.3 A. The r.m.s. deviations of equivalent C alpha atoms of the asymmetric-unit molecules superposed by the least-squares method average 0.35 A. The Fd molecule has a structure like the beta-barrel in the molecule of the [2Fe-2S] Fd from Spirulina platensis. A [2Fe-2S] cluster is bonded covalently to the protein molecule by four Fe-S, in which three of the Fe-S bonds are in a loop segment from position 38 to 47. The hydrophobic core inside the beta-barrel is formed by seven conservative residues: Val15, Val18, Ile24, Leu51, Ile74, Ala79 and Ile87. The molecular surface around Tyr23, Tyr80 and the active center may interact with ferredoxin-NADP+ reductase. One of the two iron atoms of the [2Fe-2S] cluster should be more easily reduced than the other because of differences in the hydrogen-bonding scheme and the hydrophobicity around the atoms.

Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution.

The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution. The final R-factor is 0.185 for 20,307 reflections. The crystal asymmetric unit has one subunit consisting of 345 amino acid residues. The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs. The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases. A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry. Close subunit contacts are also present at two alpha-helices in the second domain. These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer. Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding. These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme.

Preparation of human manganese superoxide dismutase by tri-phase partitioning and preliminary crystallographic data.

Human liver manganese superoxide dismutase has been purified by a short procedure that includes a tri-phase partitioning step to provide materials that can be crystallized from ammonium sulfate. X-ray diffraction studies at 3 A resolution show that the crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with cell dimensions a = b = 81.1 A, c = 242.2 A. Manganese superoxide dismutase levels as determined by enzymatic assay as well as by enzyme-linked immunosorbent assay indicated that considerable variations occur in different livers but the total superoxide dismutase activity (Mn superoxide dismutase plus Cu,Zn superoxide dismutase) seems to be kept at constant values.

Crystallization and preliminary X-ray diffraction studies of an endoglucanase from Aspergillus aculeatus.

Most fungal cellulases are found in multiple forms varying in size and substrate specificity. Aspergillus aculeatus is known to produce nine cellulolytic enzymes including an endoglucanase (FI CM-cellulase, M(r) = 24,002) as the major component. Single crystals of FI CM-cellulase from Aspergillus aculeatus have been prepared by sitting-drop vapour diffusion using ammonium sulphate as a precipitant. The cellulase crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 52.79(2) A, b = 106.40(4) A and c = 33.15(1) A. The crystals contain one enzyme molecule per asymmetric unit. They diffract to at least 2.0 A resolution and are very stable against X-ray irradiation.

Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution.

Glutathione synthetase (gamma-L-glutamyl-L-cysteine: glycine ligase (ADP-forming) EC 6.3.2.3: GSHase) catalyzes the synthesis of glutathione from gamma-L-glutamyl-L-cysteine and Gly in the presence of ATP. The enzyme from Escherichia coli is a tetramer with four identical subunits of 316 amino acid residues. The crystal structure of the enzyme has been determined by isomorphous replacement and refined to a 2.0 A resolution. Two regions, Gly164 to Gly167 and Ile226 to Arg241, are invisible on the electron density map. The refined model of the subunit includes 296 amino acid residues and 107 solvent molecules. The crystallographic R-factor is 18.6% for 17.914 reflections with F > 3 sigma between 6.0 A and 2.0 A. The structure consists of three domains: the N-terminal, central, and C-terminal domains. In the tetrameric molecule, two subunits that are in close contact form a tight dimer, two tight dimers forming a tetramer with two solvent regions. The ATP molecule is located in the cleft between the central and C-terminal domains. The ATP binding site is surrounded by two sets of the structural motif that belong to those respective domains. Each motif consists of an anti-parallel beta-sheet and a glycine-rich loop.

Molecular organization of a high molecular weight multi-protease complex from rat liver.

A latent multifunctional protease with a molecular weight of 722,000 to 760,000 purified from rat liver cytosol has been reported. This paper reports on the structure and subunit composition of the enzyme. Electron microscopy showed that the enzyme was a ring-shaped particle of 160(+/- 7) A diameter and 110(+/- 10) A height with a small hole of 10 to 30 A diameter (1 A = 0.1 nm). Small-angle X-ray scattering analysis indicated that the enzyme had a prolate ellipsoidal structure with an ellipsoid cavity in the center. The maximum dimension of the enzyme was estimated to be 210 A from a pair-distance distribution function. The radius of gyration obtained from a Guinier plot and the Stokes radius based on the ellipsoidal model were 66 A and 76 A, respectively. On two-dimensional gel electrophoresis, the purified enzyme separated into 13 to 15 characteristic components with molecular weights of 22,000 to 33,000 and isoelectric points of 4 to 9. These multiple components were not artifacts produced by limited proteolysis during purification of the enzyme, because the cell-free translation products in a reticulocyte lysate with poly(A)-mRNA of rat liver consisted of multiple components of similar sizes, and because peptide mapping analyses with lysylendopeptidase and V8 protease demonstrated clear differences in the primary structures of these components. The 13 main components were isolated from the purified enzyme by reverse-phase high performance liquid chromatography and shown to be non-identical. A model of the enzyme is proposed on the basis of these observations and previous physicochemical studies. Interestingly, the morphology of this protease is similar to that of the 16 to 22 S ring-shaped particles found in a variety of eukaryotic organisms. The structural similarity between this multi-protease complex and various reported subcellular particles is discussed.

Molecular structural studies of effector functions of a mouse immunoglobulin G that lacks the entire CH1 domain: small-angle X-ray scattering, nanosecond fluorescence depolarization and stable isotope-aided NMR analyses.

Molecular structural studies are reported of a short-chain mouse IgG2a antibody that lacks the entire CH1 domain. We have recently shown that (1) this short-chain antibody comprises two components in which the inter light-chain disulfide bridge does and does not exist, and (2) these two components are different in the constitutive complement-activating activity [Mizutani et al. (1993) J. Immunol. 150, 131-138]. Structures were compared for these two components on the basis of small-angle X-ray scattering, nanosecond fluorescence depolarization and isotope-aided NMR data. It has been discussed how the presence and absence of the inter light-chain disulfide bridges affect the complement-activating activity of the two components of the short-chain antibody.

Overproduction, purification and crystallization of Bacillus cereus oligo-1,6-glucosidase.

The gene coding for oligo-1,6-glucosidase from Bacillus cereus ATCC7064 has been overexpressed in Escherichia coli MV1184 cells under the control of the lac promoter in the genetically engineered plasmid pBCE4-2. Oligo-1,6-glucosidase was purified in large quantities and was crystallized at 25 degrees C by using a hanging drop vapor diffusion method with 53% saturated ammonium sulfate. The crystals have the shape of hexagonal bipyramids and belong to the space group P6(2) or P6(4) with lattice constants of a = b = 106.1 A, c = 120.0 A and gamma = 120 degrees.

Preliminary X-ray crystallographic analysis of tryptophanase from Escherichia coli.

Tryptophanase (L-tryptophan indole-lyase) from Escherichia coli has been crystallized from ammonium sulfate solution using a vapor diffusion method. The crystals are tetragonal and belong to space group P4(1)2(1)2 or its enantiomorph. The cell dimensions of the crystals are a = b = 113.4 A, and c = 232.2 A, with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution, and are suitable for X-ray structural analysis.