Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Design of new reaction conditions for characterization of a mutant thermophilic α-l-fucosidase

Glycosynthases are mutant glycosidases, which in the presence of activated glycosides and suitable reaction conditions, synthesize oligosaccharides without hydrolysing them. This feature makes these catalysts promising tools for the large scale synthesis of carbohydrates. However, despite the popularity of the glycosynthetic approach, the number of enzymes effecting glycosynthetic reactions is still limited. We report here on the design of novel reaction conditions for a thermophilic α-l-fucosidase mutant, which might provide a route for the production of novel glycosynthases.     

Nonradioactive immunoquantification of α-l-fucosidase protein in human colon tissues

α-l-Fucosidase is a glycosidase involved in the degradation of fucoglycoconjugates and has a diagnostic significance because it has been described to be altered in several known diseases. However, in vitro studies on enzymatic activities may not reflect the real protein levels in tissues. This paper describes a simple method to quantify α-l-fucosidase protein levels in human crude extracts, combinding the slot-blot technique and a nonradioactive immunoassay. Taking advantage of the similarities in different mammalian fucosidases, a polyclonal antiserum was raised against commercial purified α-l-fucosidase from bovine kidney that cross-reacted with the human colon enzyme. The method is able to detect as little as 0.75 ng α-l-fucosidase. To illustrate the direct application of this technique, we analysed and quantified α-l-fucosidase protein levels in 18 human colon crude samples. This technique could prove useful in clinical pathology, allowing fast and accurate measurement of α-l-fucosidase in crude extracts.     

Purification and characterization of α-l-fucosidase from the liver of a fucosidosis patient

Intact viable 13762 mammary-adenocarcinoma ascites cells hydrolyse added ATP. The localization of hydrolysis product and inactivation by the slowly penetrating chemical reagent diazotized sulphanilic acid indicate that this ATPase is at the external surface of the cell. A number of features differentiate this enzyme from mitochondrial, myosin and cation-transport ATPases. It is stimulated by either Ca2+ or Mg2+ and has little or no activity in their absence. It is insensitive to ouabain, oligomycin and azide. It is the major ATPase activity found in homogenates of gently disrupted 13762 cels. The ATPase activity is inhibited at high substrate concentrations and shows an apparent stimulation by concanavalin A in isolated membranes, but not in intact cells. The stimulation by concanavalin A results predominantly from a release from substrate inhibition.     

A novel synthesis of 2-deoxy-α-glycosides

The key step of the synthesis involves the reaction of glycals [3,4,6-tri-O-acetyl-d-glucal (1), the new glycal derivative 4-O-acetyl-1,5-anhydro-2,6-dideoxy-3-C-methyl-3-O-methyl-l-ribo-hex-l-enitol (2), and 3-acetamido-4,6,-di-O-acetyl-1,5-anhydro-2,3-dideoxy-d-arabino-hex-l-enitol (3)] with 1.5 molar equivalents of several alcohols in the presence ofN-bromosuccinimide in acetonitrile to give mainly the corresponding 2-bromo-2-deoxy-α-glycopyranosides (4-21). The glycopyranosides (4-8 and16-21) from1 and3 have the α-d-manno configuration and those (10-15) from2 have the α-l-altro configuration. The yields are high from1, virtually quantitative from2, and moderate from3. Debromination of the 2-bromo-2-deoxy compounds with tributylstannane and a radical initiator gives the corresponding 2-deoxy-α-glycopyranosides (22-38) in quantitative yields. In particular, the branched-chain glycal2 reacts with alcohols to give exclusively the corresponding α-glycopyranosides (27-32) of cladinose in strikingly high overall yields. The stereoselectivity and regiospecificity of the bromination reaction are described. 1,3-Dibromo-5,5-dimethylhydantoin andN-bromoacetamide are also found to be useful for the reaction.     

A new synthesis of 4-O-α-d-galactopyranosyl-d-galactopyranose

The bromide-catalyzed condensation of 2,3,4,6-tetra-O-benzyl-d-galactopyranosyl bromide (11) with methyl 2,3,6-tri-O-benzoyl-α-d-galactopyranoside (3) gave methyl 2,3,6-tri-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzyl-α-d-galactopyranosyl)-α-d-galactopyranoside (12) in 83% yield. The yield of this glycosidation reaction was high, despite the axial orientation of the 4-hydroxyl group of 3. Stepwise deprotection of 12 afforded methyl 4-O-α-d-galactopyranosyl-α-d-galactopyranoside (15). Acetylation of 15, followed by acetolysis, gave the known α-octaacetate 17. This scheme constituted a total synthesis of 4-O-α-d-galactopyranosyl-d-galactopyranose (2) in 25% yield from 3. The disaccharide 2 is the terminal disaccharide of the ceramide trisaccharide related to Fabry's disease.     

A preparation of N-Fmoc-N-methyl-α-amino acids and N-nosyl-N-methyl-α-amino acids

A convenient route for the synthesis of lipophilic N-Fmoc-N-methyl-α-amino acids and N-nosyl-N-methyl-α-amino acids, interesting building blocks to be used for the preparation of N-methylated peptides, is presented. Both nosyl- and Fmoc-protected monomers are accessible, so these compounds can be used in solution as well as in solid phase peptide synthesis. The methodology is based on the use of benzhydryl group to protect temporarily the carboxyl function of N-nosyl-α-amino acids and on the subsequent methylation of the N-nosyl-α-amino acid benzhydryl esters with diazomethane. The benzhydryl esters offer several beneficial features such as simple preparation, stability to methylation and selective deprotection under mild conditions. The overall procedure is highly efficient in that the adopted conditions keep the chiral integrity of amino acid precursors and the process does not require chromatographic purification of the methylated products.     

Comparison of the properties of concanavalin A and anti-α-d-glucose antibodies

Concanavalin A and anti-α-d-glucose antibodies form precipitin complexes with antigens havingα-d-glucose as terminal units. The sedimentation rates, molecular weights, gel electrophoretic mobilities, isoelectric points, and immunoglobulin type of Con A andα-Ab have been determined. The interactions of the compounds with antigens in the presence of potential inhibitors have been compared. The data show that the interaction of Con A with glucose units occurs with hydrogen bonding at hydroxyl groups at C1, 3,4, and 6 and van der Waals bonding at the pyranose ring oxygen. In theα-Ab complex with glucose units, in addition to the above bond types, a hydrogen bond at the hydroxyl at C2 occurs and this bond is essential for interaction.     

Three dimensional structure of a bacterial α-l-fucosidase with a 5-membered iminocyclitol inhibitor

Fucosidases, enzymes that cleave fucose from the non-reducing end of a glycan, represent promising medicinal targets reflecting their roles in cancer metastasis, inflammation, host-parasite interactions and the lysosomal storage disorder fucosidosis. The X-ray crystal structures of Bacteroides thetaiotaomicron GH29 α-l-fucosidase (BtFuc2970) in a new crystal form (at a resolution of 1.59 Å) and liganded with a 5-membered iminocyclitol inhibitor (1.73 Å) are reported herein. The 5-membered iminocyclitol binds in a ³E conformation, mimicking the proposed ³H₄ half chair transition-state of the enzyme catalysed reaction, and its K_i for BtFuc2970 was determined as 2 μM. Structural analysis of fucosidase inhibition through 5-membered iminocyclitols will aid in the rational design of more potent fucosidase inhibitors for treatment of a range of medical conditions.     

α-Trifluoromethylhistamine: A mechanism-based inhibitor of mammalian histidine decarboxylase

α-Trifluoromethylhistamine (1), proposed as a suicide inhibitor of histidine decarboxylase, has been prepared from β-trifluoromethyl-β-alanine. Histidine decarboxylase from hamster placenta is inhibited in a time-dependent manner by 1; however, the adduct formed between inhibitor and enzyme is labile. 1 inhibits stomach histidine decarboxylase activity in vivo in rats, but has no antisecretory effect in the pyloric-ligated stomach of the mouse.     

A chemoenzymatic synthesis of 5a-carba-α-D-mannose-6-phosphate

An efficient chemical synthesis of 5a-carba-α-D-mannose and its enzymatic elaboration to 5a-carba-α-D-mannose-6-phosphate, using yeast hexokinase, is described.     

Biosynthesis,processing, and extracellular release of α-l-fucosidase in lymphoid cell lines of different genetic origins

In humans, the quantity of α-l-fucosidase in serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. Lymphoid cell lines derived from individuals with either low, intermediate, or high α-l-fucosidase in serum were established. Steady-state levels of intracellular and extracellular α-l-fucosidase as well as rates of synthesis and secretion of enzyme overlapped among the cell lines. Thus,_vivo} serum phenotypes were not expressed in this system. No appreciable differences in the qualitative processing of newly made α-l-fucosidase were observed among these lymphoid cell lines. Cells pulse-labeled with³⁵S-methionine from 0.25 to 2 hr had an intracellular form of enzyme with aM _r=58,000. Cells pulsed for 1.5 hr and chased for 21 hr with unlabeled methionine had an intracellular form ofM _r=60,000 and an extracellular form ofM _r=62,000. All three enzyme forms were glycoproteins with a common polypeptide chain ofM _r=52,000 but with different carbohydrate moieties. No evidence for a high molecular mass precursor form of α-l-fucosidase was found. Fucosidosis is a rare, inherited disease in which α-l-fucosidase activity in tissues and body fluids is low or absent. The mutations for fucosidosis and the serum polymorphism map separately. Lymphoid cells from two siblings with fucosidosis had 8-fold to 341-fold less intracellular α-l-fucosidase protein with 11-fold to 56-fold lower specific activities than control cells. Residual mutant enzyme was a glycoprotein with a polypeptide chain virtually the same size (M _r=52,000) as control enzyme. However, residual mutant enzyme was hypoglycosylated and hypersecreted as compared to control enzyme. This research was supported by National Institutes of Health Grant DK 32161.     

Functional Coupling of the Gαolf Variant XLGαolf with the Human Adenosine A2A Receptor

A recently identified novel Gα_olf variant, XLGα_olf, is shown to functionally couple to the human adenosine A_2A receptor (A_2AR). In Sf9 cells expressing A_2AR, β1, and γ2, co-expression of XLGα_olf increased NECA-induced [³⁵S]GTPγS binding from approximately 130% to 300% of basal levels. Pharmacological characteristics of A_2AR ligands on these cells were evaluated by using [³H]ZM241385- and [³⁵S]GTPγS- binding assays. The rank order of the equilibrium binding constants (K_d or K_i) of adenosine receptor ligands were [³H]ZM241385 ≈ CGS15943 < MRS1220 < < CV1808 ≈ NECA < CGS21680 ≈ adenosine < IBMECA < HEMADO ≈ CPA ≈ CCPA. The rank order of EC₅₀ values for agonists were CV1808 ≈ NECA < adenosine ≈ CGS26180 < IBMECA < HEMADO ≈ CPA ≈ CCPA. This pharmacology is consistent with the literature for A_2AR and suggests that Sf9 cells co-expressing A_2AR, β1, γ2, and XLGα_olf could serve as a heterologous expression system for A_2AR drug screening.     

Synthesis of fucosylated oligosaccharides with α-l-fucosidase from Thermotoga maritima immobilized on Eupergit® CM

Extremophiles - Fucosylated oligosaccharides present in human milk perform various biological functions that benefit infants’ health. These compounds can be also obtained by enzymatic...     

Improvement of the transfucosylation activity of α-l-fucosidase from Thermotoga maritima for the synthesis of fucosylated oligosaccharides in the presence of calcium and sodium

The influence of CaCl₂ and NaCl in the hydrolytic activity and the influence of CaCl₂ in the synthesis of fucosylated oligosaccharides using α-l-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-l-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (a_w) of 0.9672 and of 138% in the presence of 1.1 M CaCl₂ (a_w 0.9581). In addition, the hydrolytic activity was higher when using CaCl₂ compared to NaCl at a_w of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl₂ in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl₂ favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl₂ did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl₂, the decrement in a_w and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides.

     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-α

Cytosolic phospholipase A₂-α (cPLA₂) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA₂ is dependent on a rise in cytosolic Ca²⁺ concentration, membrane association via the Ca²⁺-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA₂ via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP₂), including the role of a “pleckstrin homology (PH)-like” region of cPLA₂ (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate + the Ca²⁺ ionophore, A23187, or epidermal growth factor + A23187, expression of the PH domain of phospholipase C-δ1 (which sequesters membrane PIP₂) attenuated cPLA₂ activity. Stimulated cPLA₂ activity was also attenuated by the expression of cPLA₂ 135-366, or cPLA₂ 2-366, and expression of a PIP₂-specific 5′-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP₂, with high affinity, only cPLA₂ 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA₂s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA₂ activity can be modulated by sequestration or depletion of cellular PIP₂, although the interaction of cPLA₂ with membrane PIP₂ appears to be indirect, or of weak affinity.     

A synthesis of 7α-hydroxyandrost-4-ene-3, 17-dione

The first convenient chemical synthesis of 7α-hydroxyandrost-4-ene-3,17-dione is reported. Androsta-4,6-diene-3,17-dione was converted into its 6α,7α-epoxy-derivative; reduction of the epoxide with aluminium amalgam gave 7α-hydroxyandrost-4-ene-3,17-dione. This reducing agent is more efficient than chromous acetate for the purpose.     

Isolation of sea urchin embryo histone H2Aα1 and immunological identification of other stage-specific H2A proteins

H2A_α1, the principal H2A histone synthesized prior to the blastula stage of the sea urchin, was isolated free of other putative H2A subtypes and other histones. Its amino acid composition provides confirmation that H2A_α1 is the H2A protein encoded in the histone gene cluster carried by pCO2. An antibody prepared against this protein cross-reacts strongly with CS2A (a putative H2A synthesized only during the cleavage stage) as well as with H2A_β, H2A_γ, and H2A_δ (putative H2As synthesized principally after the blastula stage) but not with non-H2A core histones or other nuclear proteins. The data support the view that CS2A, H2A_α1, H2A_β, H2A_γ, and H2A_δ are all H2A proteins.