Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation.

Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.

Synthesis of methyl 2-O-allyl-(and 3-O-allyl)-5-O-benzyl-beta-D- ribofuranoside.

D-Ribose was converted into methyl 5-O-benzyl-beta-D-ribofuranoside and this, on tin-mediated allylation, gave a mixture of the 2-O-allyl and 3-O-allyl derivatives which were separated by chromatography. The more polar isomer was characterised as the 3-O-allyl derivative after conversion via 3-O-allyl-5-O-benzyl-1,2-O-isopropylidene-alpha-D-ribofuranose (which was also synthesised from 3-O-allyl-1,2:5,6-di-O-isopropylidene-alpha-D-allofuranose) into the known 5-O-benzyl-1,2-O-isopropylidene-alpha-D-ribofuranose. Methyl 3-O-allyl-5-O-benzyl-beta-D-ribofuranoside was converted into methyl 2-O-allyl-5-O-benzyl-beta-D-ribofuranoside via methyl 2-O-allyl-5-O-benzyl-3-O-(prop-1-enyl)-beta-D-ribofuranoside.

Activation of PRK1 by phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. A comparison with protein kinase C isotypes.

As potential targets for polyphosphoinositides, activation of protein kinase C (PKC) isotypes (beta 1, epsilon, zeta, nu) and a member of the PKC-related kinase (PRK) family, PRK1, has been compared in vitro. PRK1 is shown to be activated by both phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) as well as phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) either as pure sonicated lipids or in detergent mixed micelles. When presented as sonicated lipids, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were equipotent in activating PRK1, and, furthermore, sonicated phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) were equally effective. In detergent mixed micelles, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 also showed a similar potency, but PtdIns and PtdSer were 10-fold less effective in this assay. Similarly, PKC-beta 1, -epsilon, and -nu were all activated by PtdIns-4,5-P2 and PtdIns-3,4,5-P3 in detergent mixed micelles. The activation constants for PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were essentially the same for all the kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were found to be inhibited at 10 mM Mg2+ and mimicked by high concentrations of inositol hexaphosphate and inositol hexasulfate. The similar responses of these two classes of lipid-activated protein kinase to these phosphoinositides are discussed in light of their potential roles as second messengers.

2-Hydroxyethyl-alpha-D-glucopyranoside-2,3',4'-trisphosphate, a novel, metabolically resistant, adenophostin A and myo-inositol-1,4,5-trisphosphate analogue, potently interacts with the myo-inositol-1,4,5-trisphosphate receptor.

The novel, synthetic, adenophostin A analogue 2-hydroxyethyl-alpha-D-glucopyranoside-2,3',4'-trisphosphate [Glu(2,3',4')P3] was synthesized to probe the structure-activity relationship at the D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R]. This study was stimulated by the recent observation that the fungal isolates adenophostins A and B were very potent, metabolically resistant, Ins(1,4,5)P3R agonists [J. Biol. Chem. 269:369-372 (1994)]. Gluc(2,3',4')P3 can be visualized as a truncated version of adenophostin A, in which the 2'- and 3'-carbons of the ribose ring, with their terminal phosphate groups, are retained and the remainder of the adenosine residue is excised. Gluc(2,3',4')P3 specifically displaced [3H]Ins(1,4,5)P3 from pig cerebellar Ins(1,4,5)P3 binding sites, with an affinity (IC50 = 130 nM) only 5-fold weaker than that of Ins(1,4,5)P3 (IC50 = 27 nM). Gluc(2,3',4')P3 was also a full agonist for Ca2+ release, being only 10-12-fold less potent than Ins(1,4,5)P3 in saponin-permeabilized SH-SY5Y neuroblastoma cells [EC50 = 647 nM; Ins(1,4,5)P3 EC50 = 52 nM] and Madin-Darby canine kidney cells [EC50 = 2484 nM; Ins(1,4,5)P3 EC50 = 247 nM]. Gluc(2,3',4')P3 did not significantly interact with recombinant Ins(1,4,5)P3 3-kinase and 5-phosphatase enzymes and was also poorly metabolized by saponin-permeabilized SH-SY5Y cells. However, Gluc(2,3',4')P3 was a considerably weaker ligand (approximately 500-fold) and agonist (approximately 1000-fold) than adenophostin A, suggesting that the partial excision of the adenosine residue compromised structural motifs that have favorable interactions with the Ins(1,4,5)P3R. Indeed, molecular dynamics simulations revealed that the potencies of the three compounds show a correlation with the relative distance of the two vicinal ring phosphates from the remaining phosphate. Gluc(2,3',4')P3, with its alpha-glucoside ring, is the first synthetic Ins(1,4,5)P3 analogue that is not structurally based on a phosphorylated inositol isomer and that exhibits potent activity at the Ins(1,4,5)P3R. This, combined with the metabolic resistance of Gluc(2,3',4')P3, thus affords a novel approach for the investigation of the cellular role of Ins(1,4,5)P3 and its receptor.

The preparation of intermediates for the synthesis of 1D-myo-inositol 1,4,5- and 2,4,5-trisphosphates, 1,4-bisphosphate 5-phosphorothioate, and 4,5-bisphosphate 1-phosphorothioate from 1D-3,6-di-O-benzyl-1,2-O-isopropylidene-myo-inositol.

The preparation of 1D-1,6-di-O-benzyl-2,5-di-O-p-methoxybenzyl-myo-inositol is described. This compound and 1D-3,6-di-O-benzyl-1,2-O-isopropylidene-myo-inositol were converted into 1D-1,3,6-tri-O-benzyl-myo-inositol which was phosphorylated to give an intermediate for the synthesis of 1D-myo-inositol 2,4,5-trisphosphate. 1D-3,6-Di-O-benzyl-1,2-O-isopropylidene-myo-inositol was converted into 1D-2,3,6-tri-O-benzyl-myo-inositol (an intermediate for the synthesis of 1D-myo-inositol 1,4,5-trisphosphate) and 1D-2,3,6-tri-O-benzyl-1-O-p-methoxybenzyl-myo-inositol (an intermediate for the synthesis of the 1-phosphorothioate analogue of 1D-myo-inositol 1,4,5-trisphosphate). 1D-3,6-Di-O-benzyl-1,2-O-isopropylidene-myo-inositol was also converted into 1D-2,3,6-tri-O-benzyl-5-O-p-methoxybenzyl[and -5-O(cis-prop-1-enyl)]-myo- inositol both of which are intermediates for the synthesis of the 5-phosphorothioate analogue of 1D-myo-inositol 1,4,5-trisphosphate. The synthesis of 1D-2,3,6-tri-O-benzyl-myo-inositol 1,4-bis(dibenzyl phosphate) 5-(dibenzyl phosphorothioate) from 1D-2,3,6-tri-O-benzyl-myo-inositol 1,4-bis(dibenzyl phosphate) is described.

Purification and characterization of inositol-1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.

The metabolism of inositol 1,3,4-trisphosphate is a pivotal branch point of inositol phosphate turnover; its dephosphorylation replenishes cellular inositol pools, its phosphorylation at the 6-position supports the synthesis of inositol pentakisphosphate, and its phosphorylation at the 5-position produces inositol 1,3,4,5-tetrakisphosphate (Shears, S.B. (1989) J. Biol. Chem. 264, 19879-19886). In order to increase understanding of the control of inositol-1,3,4-trisphosphate kinase activity, the enzyme was highly purified from rat liver by precipitation with polyethylene glycol, MonoQ ion-exchange chromatography, heparin-agarose affinity chromatography, and a novel affinity chromatography procedure that utilized Affi-Gel resin to which InsP6 was coupled (Marecek, J.F., and Prestwich, G.D. (1991) Tetrahedron Lett. 32, 1863-1866). The final purification was about 26,000-fold, with a 6% yield. This final preparation performed both 5- and 6-kinase activities in the ratio of approximately 1:5. The affinity of the enzyme for inositol 1,3,4-trisphosphate was 0.04 microM, the highest yet determined for an inositol phosphate kinase. Both inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate were competitive inhibitors of the kinase (Ki values of 2-4 microM). The enzyme was determined to have a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity was unaffected by Ca2+/calmodulin, protein kinase A, or protein kinase C.

The preparation of intermediates for the synthesis of 1D-myo-inositol 1,4,5-trisphosphate, a second messenger for signal transduction in cells.

Racemic 1,2,4-tri-O-benzyl-5,6-O-isopropylidene-myo-inositol was prepared by a new route involving crotyl (but-2-enyl) ethers and converted into the (-)-omega-camphanates to give the pure crystalline 1L-diastereoisomer and the chirally impure, syrupy 1D-diastereoisomer. The latter was converted via the 1-O-allyl or 1-O-p-methoxybenzyl ethers into chirally pure 1D-2,3,6-tri-O-benzyl-myo-inositol [required as an intermediate for the synthesis of 1D-myo-inositol 1,4,5-trisphosphate (1,4,5-IP3)], which was also prepared by de-p-methoxybenzylation of 1D-2,3,6-tri-O-benzyl-1,5-di-O-p-methoxybenzyl-myo-inositol. Racemic 2,4-di-O-benzyl-5,6-O-isopropylidene-1-O-p-methoxybenzyl-myo-inositol was prepared in a similar way to the analogous tribenzyl ether (using crotyl ethers) and the omega-camphanate esters behaved similarly, allowing efficient resolution by crystallisation of the (-)- and (+)-omega-camphanates. Racemic 1,2,4-tri-O-allyl-3-O-(but-2-enyl)-myo-inositol was resolved via the (-)-omega-camphanates and was also converted into 1,2,4-tri-O-(cis-prop-1-enyl)-myo-inositol, an alternative intermediate for the synthesis of 1,4,5-IP3.

The preparation and phosphorylation of 2,5- and 1D-2,6-di-O-benzyl-myo-inositol.

1,3,4,6-Tetra-O-allyl-myo-inositol was converted into the 2,5-di-O-benzyl- and 2,5-di-O-p-methoxybenzyl ethers, and the products were deallylated to give the 2,5-di-O-benzyl (and p-methoxybenzyl) ethers of myo-inositol, which were converted into the mono-O-isopropylidene derivatives. Both the 2,5-di-O-benzyl ether and its mono-O-isopropylidene derivative were converted into the crystalline octa(2-cyanoethyl) ester of 2,5-di-O-benzyl-myo-inositol 1,3,4,6-tetrakisphosphate. (+-)-1,3,4,5-Tetra-O-allyl-myo-inositol was converted into (+-)-2,4-di-O-benzyl-myo-inositol which gave a separable mixture of the 1,6- and 5,6-O-isopropylidene derivatives. The 1,6-O-isopropylidene derivative was resolved via (-)- and (+)-omega-camphanates and was also converted into (+-)-2,6-di-O-benzyl-1,5-di-O-p-methoxybenzyl-myo-inositol, which was resolved via the (-)-omega-camphanates. The 5,6-O-isopropylidene derivative and 1,3-di-O-allyl-myo-inositol were converted into (+-)-1,3-di-O-allyl-2,6-di-O-benzyl-myo-inositol, which was resolved as the (-)-omega-camphanates. 1D-1,3,4,5-Tetra-O-allyl-myo-inositol and the above described, relevant diaste reoisomers were converted into 1D-2,6-di-O-benzyl-myo-inositol which gave the syrupy octabenzyl ester of 1D-2,6-di-O-benzyl-myo-inositol 1,3,4,5-tetrakisphosphate.

Interactions between inositol tris- and tetrakis-phosphates. Effects on intracellular Ca2+ mobilization in SH-SY5Y cells.

The potential Ca2(+)-releasing activity of the inositol tetrakisphosphates Ins(1,3,4,6)P4 and DL-Ins(1,4,5,6)P4 and the inositol pentakisphosphate Ins(1,3,4,5,6)P5 and their effect on Ins(1,4,5)P3- and DL-Ins (1,3,4,5)P4-mediated Ca2+ release were examined in permeabilized SH-SY5Y human neuroblastoma cells. Neither DL-Ins(1,4,5,6)P4 nor Ins(1,3,4,5,6)P5 exhibit Ca2(+)-releasing activity at concentrations up to 10 microM, but Ins(1,3,4,6)P4 releases Ca2+ dose-dependently, with an EC50 value (conen, giving half-maximal effect) of 5.92 +/- 0.47 microM. Maximal response by this tetrakisphosphate (49 +/- 2.5%) is significantly less than that seen with Ins(1,4,5)P3 (60 +/- 3%) and is achieved at a concentration of 30 microM. In the presence of this concentration of Ins(1,3,4,6)P4 the EC50 value for Ins(1,4,5)P3-mediated Ca2+ release increases from 0.12 +/- 0.02 microM to 2.11 +/- 0.51 microM, providing evidence that this naturally occurring inositol tetrakisphosphate may recognize and exhibit its Ca2(+)-releasing activity via the Ins(1,4,5)P3 receptor. DL-Ins(1,3,4,5)P4, however, at its maximally effective concentration (10 microM) does not significantly affect Ins(1,4,5)P3-mediated Ca2+ release, and therefore appears to mediate its Ca2(+)-mobilizing action through a receptor distinct from that for Ins(1,4,5)P3.

Inositol 1,3,4,6-tetrakisphosphate mobilizes calcium in Xenopus oocytes with high potency.

Injection of Ins(1,3,4,6)P4 into Xenopus oocytes evoked Ca2(+)-dependent membrane currents with a potency 5-10 times less than Ins(1,4,5)P3, whereas Ins(1,3,4)P3 and Ins(1,3,4,5,6)P5 were almost ineffective. Responses to Ins(1,3,4,6)P4 arose through liberation of intracellular Ca2+ and through entry of extracellular Ca2+. These results, together with the observation that Ins(1,3,4,6)P4 facilitated responses to Ins(1,4,5)P3, suggests that both of these compounds may act on the same intracellular receptors.

The synthesis and resolution of (+/-)-1,5,6-tri-O-benzyl-myo-inositol.

Racemic 1,5,6-tri-O-benzyl-myo-inositol was prepared by five routes and converted into 1,5,6-tri-O-benzyl-2,3-O-isopropylidene-myo-inositol, the camphanates of which were readily separated by chromatography. The absolute configurations of the chiral derivatives were established by their conversion into the known chiral 1,4,5,6-tetra-O-benzyl-myo-inositols. 1D-1,5,6-Tri-O-benzyl-2,3-O-isopropylidene-myo-inositol was converted into 1D-1,3,5,6-tetra-O-benzyl-myo-inositol and thence into 1D-2,4-di-O-methyl-myo-inositol. 1D-1,5,6-Tri-O-benzyl-myo-inositol was converted into 1D-1,2,5,6-tetra-O-benzyl-myo-inositol, the diacetate of which is a chiral analogue of "thermosalient crystals". The potential of the above compounds for the synthesis of natural products is surveyed.

Stereospecific mobilization of intracellular Ca2+ by inositol 1,4,5-triphosphate. Comparison with inositol 1,4,5-trisphosphorothioate and inositol 1,3,4-trisphosphate.

The stereo specificity of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize Ca2+ from an intracellular store has been examined in permeabilized rat pituitary-tumour GH3 and Swiss 3T3 cells. A comparison of D-Ins(1,4,5)P3 with the synthetic enantiomer L-Ins(1,4,5)P3 and the racemate DL-Ins(1,4,5)P3 clearly demonstrates the marked stereospecificity of the response. Whereas D-Ins(1,4,5)P3 released 30-50% of non-mitochondrially-bound Ca2+ with a EC50 (concentration producing 50% of maximal response) of 200 nM, the L isomer was both substantially less potent and efficacious. A high concentration of the L isomer (10 microM) did not significantly shift the dose-response curve for the D isomer in Swiss 3T3 cells, suggesting that the less active isomer is probably a very weak agonist. Other studies revealed, in contrast with previous work, that the other naturally occurring isomer, D-Ins(1,3,4)P3, was essentially inactive in releasing Ca+, whereas a novel 5-phosphatase-resistant analogue, DL-myo-inositol 1,4,5-trisphosphorothioate, was a relatively potent full agonist in GH3 cells. These data reveal, for the first time, the stereoselectivity of the intracellular receptor associated with Ca2+ release. They also provide evidence for the activity of the novel phosphorothioate analogue of Ins(1,4,5)P3, but suggest that D-Ins(1,3,4)P3 is not involved in cellular Ca2+ mobilization.

Use of synthetic glycoconjugates containing the Mycobacterium leprae specific and immunodominant epitope of phenolic glycolipid I in the serology of leprosy.

The high specificity of phenolic glycolipid I (PG-I) in the identifying individuals with leprosy appears to be attributable to the species-specific trisaccharide region of the molecule. Synthetic glycoconjugates were produced by coupling the corresponding terminal mono- or disaccharide to bovine serum albumin by reductive amination. Conjugates which contained only the terminal sugar maintained in its pyranose form, the terminal disaccharide with only the terminal sugar in its pyranose form and the terminal disaccharide with both the 3,6,di-o-Me-glucose and 2,3,di-o-Me-rhamnose sugars in their pyranose forms, were all highly active in the enzyme-linked immunosorbent assay (ELISA) and showed good concordance with native PG-1 in analysis of sera from leprosy patients. The antibody levels to the glycoconjugates in tuberculosis patients and patients with other mycobacterial infections were not significantly different from the levels in normal healthy control subjects. A few of the leprosy sera showed much stronger binding to conjugates which contained the disaccharide with both sugars in the pyranose form than to conjugates with only the terminal sugar in its pyranose form. Therefore a synthetic conjugate which contains the intact disaccharide region of PG-I may provide the most sensitive antigen for the large scale serodiagnosis of leprosy.

The allyl group for protection in carbohydrate chemistry. 17. Synthesis of propyl O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3- di-O-methyl-alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-alpha- L-rhamnopyranoside: the oligosaccharide portion of the major serologically active glycolipid from Mycobacterium leprae.

Allyl 4-O-benzyl-alpha-L-rhamnopyranoside was converted into allyl 4-O-benzyl-3-O-methyl-alpha-L-rhamnopyranoside and this was condensed with 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl chloride to give a disaccharide derivative which was converted into allyl 4-O-benzyl-2-O-(2,3-O-isopropylidene-alpha-L-rhamnopyranosyl)-3-O-methyl -alpha- L-rhamnopyranoside. This disaccharide derivative was condensed with 2,4-di-O-acetyl-3,6-di-O-methyl-alpha-D-glucopyranosyl chloride to give a trisaccharide derivative which was converted into the title compound. This compound represents the oligosaccharide portion of the major serologically active glycolipid from Mycobacterium leprae which is required to prepare a synthetic diagnostic agent for leprosy infection at an early stage and to investigate the specificities of monoclonal antibodies directed towards the glycolipid.

Analysis of the major antigenic determinants of the characteristic phenolic glycolipid from Mycobacterium leprae.

Antibodies to the major phenolic glycolipid purified from Mycobacterium leprae have been demonstrated previously in sera of leprosy but not tuberculosis patients using an ELISA. The major antigenic determinants on this molecule were investigated using antisera raised in rabbits to the purified glycolipid and with a pool of sera from human lepromatous leprosy patients. A small, but significant cross-reaction was observed with the glycolipids from M. bovis and M. kansasii, which contain the phenolphthiocerol dimycocerosate part of the molecule but have different sugars, and also with a semi-synthetic 'attenuation indicator lipid' which shares the phenolphthiocerol but has no sugars. There was however no cross-reaction with phthiocerol dimycocerosate. The disaccharide, corresponding to the two terminal sugars of the M. leprae glycolipid has been chemically synthesized and shown to inhibit the reaction between glycolipid and antibody in the ELISA. The cross-reactivity observed with the M. bovis and M. kansasii glycolipids was not inhibited by the synthetic disaccharide. These findings suggest that the cross-reactivity is associated with the phenol ring and implies the disaccharide may be a unique antigenic determinant of M. leprae.