Organization of glycosaminoglycan sulfation in the biosynthesis of proteochondroitin sulfate and proteodermatan sulfate

Although the intermediates for sulfation of proteochondroitin and proteodermatan have been known for several decades, organizational aspects of this formation have not been clearly defined. Work in several laboratories, including our own, have indicated a pattern which strongly suggests that sulfation ordinarily takes place together with glycosaminoglycan polymerization in the same Golgi sites, and with close relationship to aspects of polymer elongation, polymer modification and polymer termination. the organization of sulfation together with polymerization may be a major factor controlling the location, type, and degree of sulfation, which in turn may direct specific functions of these proteoglycans.     

Evidence for diabetes-induced alterations in the sulfation of heparin sulfate intestinal epithelial cells

Two compounds with high affinity for the "peripheral type" benzodiazepine binding sites, PK 11195 (an isoquinoline derivative) and RO5-4864 (a benzodiazepine derivative) can modify the sensitivity of DBA/2J mice to audiogenic seizures. RO5-4864 (1-15 mg/kg) facilitates in a dose-dependent manner the audiogenic seizures and PK 11195 (2-5 mg/kg) antagonizes the RO5-4864 effects. At these doses PK 11195 alone does not modify the sensitivity to audiogenic seizures, but at doses between 20-80 mg/kg it protects DBA/2J mice against audiogenic seizures. By contrast PK 11195 is inactive against the facilitation of audiogenic seizures by ethyl-beta-carboline-3-carboxylate (a brain benzodiazepine receptor inverse agonist) and against the seizure elicited in absence of noise stimuli by RO5-4864 at doses between 20-40 mg/kg. These results suggest that facilitation by RO5-4864 of the audiogenic seizures and its antagonism by PK 11195 are mediated by the peripheral type benzodiazepine binding sites and agree with the thermodynamic analysis of the binding data which suggested that RO5-4864 might be an agonist and PK 11195 an antagonist. The good correlation between pharmacological effects and the occupancy degree of the binding sites as measured by the displacement of the "in vivo" [3H]-PK 11195 binding give an additional support to binding sites mediated effects.     

A convenient method for the stereoselective conversion of aryl peptidyl ketones into the corresponding aryl aminomethin derivatives, a novel class of modified peptides

In this paper we describe a reductive amination procedure that can be employed in the preparation of a novel class of pseudopeptides in which a specific amide bond is replaced by a CH(Ar)NH group. The developed methodology, performed using NaBH(3)CN and TiCl(4), is characterized by the formation of diastereomeric intermediates in a relative 1:1 ratio. It provides aryl aminomethin pseudopeptides in moderate but satisfactory yields and with definite stereochemistry on the asymmetric centres next to the modified peptide bond.     

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.     

A method for determining tissue sulfate

A method for the determination of the inorganic sulfate present in rat liver homogenates has been developed. In order to determine sulfate, a protein-free extract is required. The classical protein precipitation methods of preparing protein-free extracts gave 2.5–40% recovery of added ³⁵SO₄^2?. Separation of the protein by ultrafiltration gave only 29% recovery when 0.15 m KCl was the homogenizing medium. A homogenization medium containing 0.154 m NH₄OH and 20 g EDTA per liter gave 102 ± 11% recovery of added ³⁵SO₄^2? when the protein was separated by ultrafiltration.     

Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase

Nucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000-fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.     

Selective effects of sodium chlorate treatment on the sulfation of heparan sulfate

We have analyzed the effect of sodium chlorate treatment of Madin-Darby canine kidney cells on the structure of heparan sulfate (HS), to assess how the various sulfation reactions during HS biosynthesis are affected by decreased availability of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate. Metabolically [(3)H]glucosamine-labeled HS was isolated from chlorate-treated and untreated Madin-Darby canine kidney cells and subjected to low pH nitrous acid cleavage. Saccharides representing (i) the N-sulfated domains, (ii) the domains of alternating N-acetylated and N-sulfated disaccharide units, and (iii) the N-acetylated domains were recovered and subjected to compositional disaccharide analysis. Upon treatment with 50 mM chlorate, overall O-sulfation of HS was inhibited by approximately 70%, whereas N-sulfation remained essentially unchanged. Low chlorate concentrations (5 or 20 mM) selectively reduced the 6-O-sulfation of HS, whereas treatment with 50 mM chlorate reduced both 2-O- and 6-O-sulfation. Analysis of saccharides representing the different domain types indicated that 6-O-sulfation was preferentially inhibited in the alternating domains. These data suggest that reduced 3'-phosphoadenosine 5'-phosphosulfate availability has distinct effects on the N- and O-sulfation of HS and that O-sulfation is affected in a domain-specific fashion.     

Membrane hemifusion is a stable intermediate of exocytosis

Membrane fusion during exocytosis requires that two initially distinct bilayers pass through a hemifused intermediate in which the proximal monolayers are shared. Passage through this intermediate is an essential step in the process of secretion, but is difficult to observe directly in vivo. Here we study membrane fusion in the sea urchin egg, in which thousands of homogeneous cortical granules are associated with the plasma membrane prior to fertilization. Using fluorescence redistribution after photobleaching, we find that these granules are stably hemifused to the plasma membrane, sharing a cytoplasmic-facing monolayer. Furthermore, we find that the proteins implicated in the fusion process-the vesicle-associated proteins VAMP/synaptobrevin, synaptotagmin, and Rab3-are each immobile within the granule membrane. Thus, these secretory granules are tethered to their target plasma membrane by a static, catalytic fusion complex that maintains a hemifused membrane intermediate.     

Quantification of protein-bound sodium dodecyl sulfate by Rhodamine B: a method for identification of kinetically stable proteins

Sodium dodecyl sulfate (SDS) bound to proteins in solution could be estimated by passing through Extracti-Gel that removes free SDS followed by specific interaction of the fluorophore Rhodamine B with protein-bound SDS. The resulting fluorescence intensity is compared with a calibration curve. Whereas globular proteins respond to binding of 1.4 mg SDS/mg protein under native conditions, “kinetically stable” proteins that are otherwise resistant to denaturation due to structural integrity show a low level of SDS binding. Analysis of the circular dichroism spectrum shows that in spite of the low level of SDS binding to kinetically stable proteins under nondenaturing conditions, the detergent generates considerable secondary structure in these proteins. Because the low level of SDS binding is a general feature of kinetically stable proteins, the protocol may fulfill one of the criteria to classify a protein as kinetically stable.     

Regioselectivity in the sulfation of dermatan sulfate and methyl 4,6-O-benzylidene-alpha-D-idopyranoside.

The sulfation of dermatan sulfate by SO3-trimethylamine in N,N-dimethylformamide led to substitution initially at HO-6 of residues of 2-acetamido-2-deoxy-beta-D-galactopyranosyl 4-sulfate (1), to produce the 4,6-disulfate (6). When this step reached a level of greater than 50%, sulfation occurred with equal facility at HO-2 and HO-3 of residues of alpha-L-idopyranosyluronic acid (2), giving rise to a mixture of 2-,3-, and 2,3-disulfates. An analogous substitution pattern was observed for HO-2 and -3 of a simpler idopyranose unit, in the sulfation of methyl 4,6-O-benzylidene-alpha-D-idopyranoside (12). This lack of regioselectivity in the reaction of 2 (and 12) contrasts markedly with the high affinity of the reagent for HO-3 of residues of alpha-L-idopyranosyluronic acid present in a modified form of heparin. It is attributed to a difference between the two polymers in the relative orientation of their neighboring amino sugar residues, whereby there is an unobstructed access of the reagent in one instance, and hindrance of HO-2 selectively in the other. Enzymolysis by chondroitinase ABC was found to yield unsaturated disaccharide containing residues of 4,6-disulfate, as well as larger fragments containing unsaturated glycosyl groups derived from L-idopyranosyluronic acid 2-sulfate, evidence of a relatively broad enzyme specificity. The presence of extra sulfate groups in dermatan sulfate did not enhance its weak antithrombotic activity, as measured by anti Xa assay, in disagreement with earlier reports.     

Heparan sulfate biosynthesis: a theoretical study of the initial sulfation step by N-deacetylase/N-sulfotransferase

Heparan sulfate N-deacetylase/N-sulfotransferase (NDST) catalyzes the deacetylation and sulfation of N-acetyl-D-glucosamine residues of heparan sulfate, a key step in its biosynthesis. Recent crystallographic and mutational studies have identified several potentially catalytic residues of the sulfotransferase domain of this enzyme (, J. Biol. Chem. 274:10673-10676). We have used the x-ray crystal structure of heparan sulfate N-sulfotransferase with 3'-phosphoadenosine 5'-phosphate to build a solution model with cofactor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and a model heparan sulfate ligand bound, and subsequently performed a 2-ns dynamics solution simulation. The simulation results confirm the importance of residues Glu(642), Lys(614), and Lys(833), with the possible involvement of Thr(617) and Thr(618), in binding PAPS. Additionally, Lys(676) is found in close proximity to the reaction site in our solvated structure. This study illustrates for the first time the possible involvement of water in the catalysis. Three water molecules were found in the binding site, where they are coordinated to PAPS, heparan sulfate, and the catalytic residues.     

Activation of initiator caspases through a stable dimeric intermediate

Structural and biochemical studies have revealed that procaspases form dimers prior to proteolytic activation. How the two procaspases interact in the dimer is unclear. To study the mechanisms of dimer-dependent caspase activation we used a heterodimeric system so that two caspase molecules can be specifically brought together. Surprisingly, only one caspase partner in the dimer needs to be enzymatically active for caspase processing and activation to occur. Caspase activation is inefficient in the dimer in the absence of intramolecular processing, suggesting that caspase activation is initiated via intramolecular processing. Homodimerization of caspase-8 or caspase-9 leads to the formation of a stable dimeric complex. However, heterodimerization between caspase-8 and caspases-3, -9, or -10 failed to induce stable dimer formation or caspase activation. Our data suggest that the formation of a stable dimeric intermediate initiates caspase activation.