Calcium interactions with D-glucans: crystal structure of alpha, alpha-trehalose-calcium bromide monohydrate

Three-dimensional X-ray diffraction data were used to determine the crystal structure of α,α-trehalose-calcium bromide monohydrate, a model system for investigation of factors involved in the binding of calcium ions to d-glucans of dental plaques. Crystals of C₁₂H₂₂O₁₁ ·CaBr₂·H₂O are orthorhombic, space group C222₁, with a  11.058(1) b  11.537(1), c  15.101(1) Å, and Z  4. Intensity data for 925 independent reflections were measured with an automated diffractometer. A trial structure, obtained by the heavy-atom method, was refined by least-squares to R  0.03. An outstanding feature of the crystal packing is the interaction of trehalose molecules with calcium ions. Each calcium is coordinated to hydroxyl groups from four symmetry-related d-glucose moieties, thereby cross-linking the trehalose molecules. Similar interactions between calcium ions and the d-glucose residues of extracellular d-glucans may be of importance in the agglutination processes involved in dental-plaque formation.     

Catalytic mechanism of a C-C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme

2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the hydrolytic cleavage of the extradiol ring fission product on the phenylpropionate catabolic pathway and is a member of the alpha/beta hydrolase family. The catalytic mechanism of this enzyme has previously been shown to proceed via initial ketonization of the dienol substrate (Henderson, I. M. J., and Bugg, T. D. H. (1997) Biochemistry 36, 12252-12258), followed by stereospecific fragmentation. Despite the implication of an active site serine residue in the alpha/beta hydrolase family, attempts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a (14)C-labeled substrate yielded a stoichiometry of <1% covalent intermediate, which could be accounted for by nonenzymatic processes. In contrast, incorporation of 5-6% of two atoms of (18)O from H(2)(18)O into succinic acid was observed using the natural substrate, consistent with the reversible formation of a gem-diol intermediate. Furthermore, time-dependent incorporation of (18)O from H(2)(18)O into the carbonyl group of a nonhydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presence of MhpC, consistent with enzyme-catalyzed attack of water at the ketone carbonyl. These results favor a catalytic mechanism involving base-catalyzed attack of water, rather than nucleophilic attack of an active site serine. The implication of this work is that the putative active site serine in this enzyme may have an alternative function, for example, as a base.     

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.     

From peptidoglycan to glycoproteins: Common features of lipid-linked oligosaccharide biosynthesis

Abstract The peptidoglycan layer of bacterial cell walls is biosynthesised using a lipid carrier undecaprenyl phosphate to assemble and transport the MurNAc(GlcNAc)-pentapeptide precursor. Similar lipid-linked cycles are involved in the biosynthesis of other bacterial exopolysaccharides and eukaryotic asparagine-linked glycoproteins, the latter involving the structurally related dolichyl phosphate as a lipid carrier. Recent protein sequence data and common inhibitors of the bacterial and eukaryotic systems have revealed functional similarities between the two systems. Biological and physical studies on the lipid carriers themselves have provided clues to their role in oligosaccharide translocation, but have not revealed significant differences in function between undecaprenyl phosphate and dolichyl phosphate. The presence of dolichyl phosphate and a family of saturated isoprenoid lipids in Archaebacteria suggests a possible evolutionary link between the two systems.     

Rotation function studies of human C-reactive protein

Rotation function studies of two tetragonal crystal forms of human C-reactive protein have confirmed the pentameric structure of the molecule. The two crystal forms have space groups P4122 (I) and P4222 (II) with closely similar unit cells and are often twinned together. Investigation of the crystallization conditions indicates that dissociation heterogeneity has been a major limiting factor in the reproducible growth of good single crystals. The orientation of the pentameric molecule is shown to be almost identical in both forms, about the axial direction omega = 57 degrees, phi = 45 degrees, i.e. 57 degrees away from c in the (110) plane.     

Comparison of the three-dimensional structures of human, yeast, and oat ubiquitin

The crystal structure of human ubiquitin has been solved by x-ray diffraction methods and refined by standard procedures to a conventional crystallographic R factor of 0.176 at 1.8-A resolution (Vijay-Kumar, S., Bugg, C.E., and Cook, W.J. (1987) J. Mol. Biol. 194, 525-538). Crystals of yeast and oat ubiquitin have been grown using human ubiquitin crystals as seeds. Diffraction data for yeast and oat ubiquitin have been collected to a resolution of 1.9 and 1.8 A, respectively. Difference Fourier electron-density maps reveal that the structures of yeast and oat ubiquitin are quite similar to human ubiquitin. All the amino acid changes are clustered in two small patches on one surface of the molecule. This surface is probably not involved in conjugation with proteins destined for ATP-dependent proteolysis.     

Preliminary X-ray study of crystals of human C-reactive protein

Two different crystal forms of human C-reactive protein have been grown from solutions of 2-methyl-2,4-pentanediol. Both crystal forms are tetragonal, the space group for form I is P4(1)22 (or P4(3)22), and that for form II is P4(2)22. The unit cell parameters for form I are a = b = 103.0(5) A, c = 308.5(7) A and for form II are a = b = 103.1(2) A, c = 312.7(6) A. The crystals of form II diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.     

Catalytic role for arginine 188 in the C-C hydrolase catalytic mechanism for Escherichia coli MhpC and Burkholderia xenovorans LB400 BphD

The alpha/beta-hydrolase superfamily, comprised mainly of esterase and lipase enzymes, contains a family of bacterial C-C hydrolases, including MhpC and BphD which catalyze the hydrolytic C-C cleavage of meta-ring fission intermediates on the Escherichia coli phenylpropionic acid pathway and Burkholderia xenovorans LB400 biphenyl degradation pathway, respectively. Five active site amino acid residues (Arg-188, Asn-109, Phe-173, Cys-261, and Trp-264) were identified from sequence alignments that are conserved in C-C hydrolases, but not in enzymes of different function. Replacement of Arg-188 in MhpC with Gln and Lys led to 200- and 40-fold decreases, respectively, in k(cat); the same replacements for Arg-190 of BphD led to 400- and 700-fold decreases, respectively, in k(cat). Pre-steady-state kinetic analysis of the R188Q MhpC mutant revealed that the first step of the reaction, keto-enol tautomerization, had become rate-limiting, indicating that Arg-188 has a catalytic role in ketonization of the dienol substrate, which we propose is via substrate destabilization. Mutation of nearby residues Phe-173 and Trp-264 to Gly gave 4-10-fold reductions in k(cat) but 10-20-fold increases in K(m), indicating that these residues are primarily involved in substrate binding. The X-ray structure of a succinate-H263A MhpC complex shows concerted movements in the positions of both Phe-173 and Trp-264 that line the approach to Arg-188. Mutation of Asn-109 to Ala and His yielded 200- and 350-fold reductions, respectively, in k(cat) and pre-steady-state kinetic behavior similar to that of a previous S110A mutant, indicating a role for Asn-109 is positioning the active site loop containing Ser-110. The catalytic role of Arg-188 is rationalized by a hydrogen bond network close to the C-1 carboxylate of the substrate, which positions the substrate and promotes substrate ketonization, probably via destabilization of the bound substrate.     

AANG Prevents Smad3-dependent Diabetic Nephropathy by Restoring Pancreatic β-Cell Development in db/db Mice

Diabetic nephropathy (DN) is a major cause of end-stage kidney disease, where TGF-β1/Smad signaling plays an important role in the disease progression. Our previous studies demonstrated a combination of Traditional Chinese Medicine derived Smad7 agonist Asiatic Acid (AA) and Smad3 inhibitor Naringenin (NG), AANG, effectively suppressed the progression of renal fibrosis in vivo. However, its implication in type-2 diabetic nephropathy (T2DN) is still unexplored. Here, we detected progressive activation of Smad3 but reduction of Smad7 in db/db mice during T2DN development. Therefore, we optimized the dosage and the combination ratio of AANG to achieve a better rebalancing Smad3/Smad7 signaling for treatment of T2DN. Unexpectedly, preventive treatment with combined AANG from week 4 before the development of diabetes and T2DN effectively protected against the onset of T2DN. In contract, these inhibitory effects were lost when db/db mice received the late AANG treatment from 12-24 weeks. Surprisingly, preventive treatment with AANG ameliorated not only T2DN but also the primary disease type-2 diabetes (T2D) with relative normal levels of fasting blood glucose and HbA1c, and largely improving metabolic abnormalities especially on insulin insensitivity and glucose tolerance in db/db mice. Mechanistically, AANG effectively prevented both Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in the diabetic kidney in vivo and advanced glycation end-products (AGE) stimulated tubular epithelial mTEC cells in vitro. More importantly, we uncovered that preventive treatment with AANG effectively protected against diabetic-associated islet injury via restoring the β cell development in db/db mice. Taken together, we discovered that the early treatment with combined AANG can effectively protect against the development of T2D and T2DN via mechanism associated with protection against Smad3-depenedent islet injury.