Measurement of the anchorage force between GPI-anchored alkaline phosphatase and supported membranes by AFM force spectroscopy

Mammalian alkaline phosphatases (AP) are glycosylphosphatidylinositol (GPI) anchored proteins that are localized on the outer layer of the plasma membrane. The GPI anchors are covalently attached to the C-termini of proteins and consist of a glycan chain bonded to phosphatidylinositol with two acyl chains anchored into the membrane bilayer. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the adhesion of alkaline phosphatase in the absence and presence of anchors. The GPI anchors increase markedly the adhesion frequency (i.e., the protein affinity for the membrane). An adhesion force of 350 +/- 200 pN is measured between GPI-anchored AP (AP(GPI)) and supported phospholipid bilayers of dipalmitoylphosphatidylcholine (DPPC) presenting structural defects (holes). In the absence of defects, the adhesion force (103 +/- 17 pN) and the adhesion frequency are reduced. These results indicate that AP(GPI) poorly spontaneously insert into membranes in vivo and open new perspectives for the characterization of the interactions between GPI proteins and membranes.     

糖基磷脂酰肌醇及其锚定蛋白的合成研究进展

在真核生物中,蛋白质的C-末端以共价键形式与糖基磷脂酰肌醇（GPI）相连是一种常见的翻译后修饰,GPI修饰的蛋白质可以通过GPI锚定在细胞膜的外叶.GPI锚及其锚定蛋白的结构复杂、多样,在众多生物学过程中扮演着不可或缺的重要作用.化学合成结合酶催化反应是获得结构明确、纯度高的GPI锚及GPI锚定蛋白的重要方法,为在分子水平上深入探索此类化合物的结构和生物学功能奠定了基础.本文对此合成领域中所涉及的光学纯且差异性保护的肌-肌醇衍生物的制备、天然来源GPI的合成策略、以结构多样性为导向的GPI衍生物的合成,以及GPI锚定蛋白的合成策略进行综述.     

Inositolphosphoglycan mediators structurally related to glycosyl phosphatidylinositol anchors: synthesis, structure and biological activity

The preparation of the pseudopentasaccharide 1a, an inositol-phosphoglycan (IPG) that contains the conserved linear structure of glycosyl phosphatidylinositol anchors (GPI anchors), was carried out by using a highly convergent 2+3-block synthesis approach which involves imidate and sulfoxide glycosylation reactions. The preferred solution conformation of this structure was determined by using NMR spectroscopy and molecular dynamics simulations prior to carrying out quantitative structure--activity relationship studies in connection with the insulin signalling process. The ability of 1a to stimulate lipogenesis in rat adipocytes as well as to inhibit cAMP dependent protein kinase and to activate pyruvate dehydrogenase phosphatase was investigated. Compound 1a did not show any significant activity, which may be taken as a strong indication that the GPI anchors are not the precursors of the IPG mediators.     

Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers

Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified proteins in the outer leaflet of the plasma membrane. GPI-anchored proteins play vital roles in signal transduction, the vertebrate immune response, and the pathobiology of trypanosomal parasites. While many GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. We synthesized a series of GPI-protein analogues bearing modified anchor structures that were designed to dissect the contribution of various glycan components to the GPI-protein's membrane behavior. These anchor analogues were similar in length to native GPI anchors and included mimics of the native structure's three domains. A combination of expressed protein ligation and native chemical ligation was used to attach these analogues to the green fluorescent protein (GFP). These modified GFPs were incorporated in supported lipid bilayers, and their mobilities were analyzed using fluorescence correlation spectroscopy. The data from these experiments suggest that the GPI anchor is more than a simple membrane-anchoring device; it also may prevent transient interactions between the attached protein and the underlying lipid bilayer, thereby permitting rapid diffusion in the bilayer. The ability to generate chemically defined analogues of GPI-anchored proteins is an important step toward elucidating the molecular functions of this interesting post-translational modification.     

Phospholipase cleavage of D- and L-chiro-glycosylphosphoinositides asymmetrically incorporated into liposomal membranes

The nature of chiro-inositol-containing inositolphosphoglycans (IPGs), reported to be putative insulin mediators, was studied by examination of the substrate specificities of the phosphatidylinositol-specific phospholipase C (PI-PLC) and the glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) by using a series of synthetic D- and L-chiro-glycosylphosphoinositides. 3-O-alpha-D-Glucosaminyl- (3) and -galactosaminyl-2-phosphatidyl-L-chiro-inositol (4), which show the maximum stereochemical similarity to the 6-O-alpha-D-glucosaminylphosphatidylinositol pseudodisaccharide motifs of GPI anchors, were synthesized and asymmetrically incorporated into phospholipid bilayers in the form of large unilamellar vesicles (LUVs). Similarly, 2-O-alpha-D-glucosaminyl- (5) and -galactosaminyl-1-phosphatidyl-D-chiro-inositol (6), which differ from the corresponding pseudodisaccharide motif of the GPI anchors only in the axial orientation of the phosphatidyl moiety, were also synthesized and asymmetrically inserted into LUVs. The cleavage of these synthetic molecules in the liposomal constructs by PI-PLC from Bacillus cereus and by GPI-PLD from bovine serum was studied with the use of 6-O-alpha-D-glucosaminylphosphatidylinositol (7) and the conserved GPI anchor structure (8) as positive controls. Although PI-PLC cleaved 3 and 4 with about the same efficiency as 7 and 8, this enzyme did not accept 5 or 6. GPI-PLD accepted both the L-chiro- (3 and 4) and the D-chiro- (5 and 6) glycosylinositolphosphoinositides. Therefore, IPGs containing L-chiro-inositol only are expected to be released from chiro-inositol-containing GPIs if the cleavage is effected by a PI-PLC, whereas GPI-PLD cleavage could result in both L-chiro- and D-chiro-inositol-containing IPGs.     

A variable concept for the preparation of branched glycosyl phosphatidyl inositol anchors

A variable concept for the synthesis of branched glycosyl phosphatidyl inositol (GPI) anchors was established. Its efficiency could be shown by the successful synthesis of the GPI anchor of rat brain Thy-1 and of the scrapie prion protein both in the water soluble 1c and lipidated form 1a. Retrosynthesis led to building blocks 2-6 of which 5 could be further disconnected to building blocks 7-9. Trichloroacetimidate 5 was built up in a straightforward manner starting from glycosyl acceptor 9 using known glycosyl donors 7 and 8. The carbohydrate backbone was then assembled by glycosylation of pseudodisaccharide acceptor 6 with donor 5. To ensure high stereoselectivity and good yields in the glycosylation reactions, anchimeric assistance was employed. Successive deprotection and introduction of the various phosphate residues gave the fully protected GPI anchors. Catalytic hydrogenation and acid-catalyzed cleavage of the Boc protecting groups afforded the target molecules, which could be fully structurally assigned.     

1,2-Diacetals in synthesis: total synthesis of a glycosylphosphatidylinositol anchor of Trypanosoma brucei

A full account on a total synthesis of GPI anchor 1 employing butanediacetal (BDA) groups and a chiral bis(dihydropyran) is presented. The reactivity of selenium and thio glycosides was tuned by the use of BDA groups. This allowed the assembly of an appropriately protected GPI anchor precursor 2 in just six steps from the six building blocks 5-10 including only one protecting group manipulation. myo-Inositol was desymmetrised with the bis(dihydropyran) derivative 15 and appropriately protected to give inositol acceptor 21 in nine steps and 17% overall yield. The use of common starting materials and BDA-protections give efficient access to building blocks 5, 6, 7 and 8. A new and improved synthesis of the glucosamine donor 28 is included. In summary, a highly convergent and efficient synthesis of GPI anchor 1, which is clearly adaptable to other GPI anchors, has been reported.     

Recent Advances in the Chemical Synthesis of Glycosylphosphatidylinositols (GPIs): Expanding Synthetic Versatility for Investigating GPI Biology

Carbohydrate chemists have been remarkably successful at developing methods for the chemical synthesis of glycosylphosphatidylinositols (GPIs), a highly complex and structurally diverse family of glycolipids that anchor proteins to eukaryotic cell surfaces. With the goal of generating new tools for GPI biological research, several groups in this field have recently shifted their attention from narrowly focused target-oriented total synthesis to the development of more versatile synthetic strategies that allow access to a broad variety of GPIs, GPI analogs, and GPI-anchored proteins. These recent efforts are the topic of this review article.     

Inositolphosphoglycan Mediators: An Effective Synthesis of the Conserved Linear GPI Anchor Structure*

An effective new preparative synthesis of the conserved linear pseudopentasaccharide structure of the GPI anchors and of the full GPI structure has been carried out that has permitted obtaining both molecules in sufficient quantities as to perform further structural and biologic studies. The synthesis involves a 3+2 block synthesis strategy in which a conveniently protected Man α(1→4) GlcN₃ α(1→6) myo‐Ins building block, previously used in the synthesis of inositolphosphoglycan (IPG) mediators, is glycosylated with a protected Man α(1→2) Man trichloroacetimidate.     

Convergent synthesis of a fully lipidated glycosylphosphatidylinositol anchor of Plasmodium falciparum

A highly convergent strategy for the synthesis of fully lipidated GPI anchors of malarial origin is reported. This strategy utilized three orthogonal protecting groups, which can be chemoselectively deprotected and functionalized in the late stage of the synthesis. Rapid access to the target GPIs in a highly efficient manner in sufficient quantities for the biological studies has been achieved.     

Studies on analgesic oligopeptides. VI. Further studies of synthesis and biological properties of tripeptide alkylamides, Tyr-D-Arg-Phe-X

Nine analogs based on a structure of Tyr-D-Arg-Phe-X (X = alkylamides or alkylhydrazide containing electron-withdrawing atoms or groups) were newly synthesized and their biological properties were examined by the opioid receptor binding properties of mu-, delta- and kappa-receptors, guinea-pig ileum (GPI) assay and analgesic activity in the tail pinch test after subcutaneous administration in mice. Analogs with X = NHCF2CF3, Sar-ol, or NH(CH2)2CN showed potent activities in the GPI and analgesic assays and high affinity for mu-receptor. An analog with X = taurinamide was found to possess 4-fold higher mu-receptor selectivity than that of [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAGO). The receptor binding properties of previously reported analogs [Chem. Pharm. Bull., 33, 1528 (1985); ibid., 33, 4865 (1985); ibid., 36, 4834 (1988)] were also examined for overall discussion of the structure-activity relationships of this series of tripeptide amides.     

Synthesis of lipidated green fluorescent protein and its incorporation in supported lipid bilayers

Herein we report a semisynthetic method of producing membrane-anchored proteins. Ligation of synthetic lipids with designed anchor structures to proteins was performed using native chemical ligation (NCL) of a C-terminal peptide thioester and an N-terminal cysteine lipid. This strategy mimics the natural glycosylphosphatidylinositol (GPI) linkage found in many natural membrane-associated proteins; however, the synthetic method utilizes simple lipid anchors without glycans. Synthetically lipidated recombinant green fluorescent protein (GFP) was shown to be stably anchored to the membrane, and its lateral fluidity was quantitatively characterized by direct fluorescence imaging in supported membranes. Circumventing the steps of purification from native cell membranes, this methodology facilitates the reconstitution of membrane-associated proteins.     

Synthesis of a malaria candidate glycosylphosphatidylinositol (GPI) structure: a strategy for fully inositol acylated and phosphorylated GPIs

A congener of the glycosylphosphatidylinositol (GPI) membrane anchor present on the cell surface of the malaria pathogen Plasmodium falciparum has been synthesized. This GPI is an example of a small number of such membrane anchors that carry a fatty acyl group at O-2 of the inositol. Although the acyl group plays crucial roles in GPI biosynthesis, it rarely persits in mature molecules. Other notable examples are the mammalian GPIs CD52 and AchE. The presence of bulky functionalities at three contiguous positions of the inositol moiety creates a very crowded environment that poses difficulties for carrying out selective chemical manipulations. Thus installations of the axial long-chain acyl group and neighboring phosphoglyceryl complex were fraught with obstacles. The key solution to these obstacles in the successful synthesis of the malarial candidate and prototype structures involved stereoelectronically controlled opening of a cyclic ortho ester. The reaction proceeds in very good yields, the desired axial diastereomer being formed predominantly, even more so in the case of long-chain acyl derivatives. The myoinositol precursor was prepared from methyl alpha-d-glucopyranoside by the biomimetic procedure of Bender and Budhu. For the glycan array, advantage was taken of the fact that (a). n-pentenyl ortho ester donors are rapidly and chemospecifically activated upon treatment with ytterbium triflate and N-iodosuccinimide and (b). coupling to an acceptor affords alpha-coupled product exclusively. A strategy for obtaining the GPI's alpha-glucosaminide component from the corresponding alpha-mannoside employed Deshong's novel azide displacement procedure. Thus all units of the glycan array were obtained from a beta-d-manno-n-pentenyl ortho ester, this being readily prepared from d-mannose in three easy, high-yielding steps. The "crowded environment" at positions 1 and 2, noted above, could conceivably be relieved by migration of the acyl group to the neighboring cis-O-3-hydroxyl in the natural product. However, study of our synthetic intermediates and prototypes indicate that the O-2 acyl group is quite stable, and that such migration does not occur readily.     

Remodeling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase

Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.     

Modification of β2 I by glutardialdehydeI by glutardialdehyde

Autoantibodies from patients with antiphospholipid syndrome (APS) recognize an epitope on β₂glycoprotein I (β₂GPI) only when native β₂GPI is adsorbed on surfaces composed of anionic phospholipids or oxidized polystyrene, β₂GPI was modified with the crosslinking agent, glutardialdehyde (GDA), which induced exposure of the anti-β₂GPI epitope at GDA: β₂GPI mol ratios in the range of 500–2000. A second crosslinking agent, dimethyl-suberimidate (DMS), did not expose the epitope, which may be a consequence of its having less tendency than GDA to form intermolecular links. SDS-PAGE experiments demonstrate that GDA does promote extensive intermolecular crosslinking of β₂GPI, and DMS does not. Formaldehyde also reacts with the lysine residues of β₂GPI, but does not expose the epitope. The circular dichroism spectra of native and modified β₂GPI confirm that GDA induces changes in conformation that are qualitatively different from those caused by formaldehyde. These data provide evidence that binding of lysine residues is not a sufficient condition for exposure of the autoepitope, and also support the likelihood that β₂GPI antibodies bind only to aggregates of the protein. Thus, by synthesizing an active holoantigen of β₂GPI, conditions were defined that are necessary for binding of human autoantibodies. The authors also suggest that treatment of phospholipid-binding proteins with chemical agents might provide a strategy to modify their structure and permit exposure of epitopes, resulting in synthetic antigens for therapeutic and diagnostic use.     

Computational prediction of the effects of non-synonymous single nucleotide polymorphisms on the GPI-anchor transamidase subunit GPI8p of Plasmodium falciparum

Drug resistance is increasingly evolving in malaria parasites; hence, it is important to discover and establish alternative drug targets. In this context, GPI-anchor transamidase (GPI-T) is a potential drug target primarily of its crucial role in the development and survival of the parasite in the GPI anchor biosynthesis pathway. The present investigation was undertaken to explore the plausible effects of nsSNP on the structure and functions of GPI-T subunit GPI8p of Plasmodium falciparum. The GPI8p (PF3D7_1128700) was analyzed using various sequence-based and structure-based computational tools such as SIFT, PROVEAN, PredictSNP, SNAP2, I-Mutant, MuPro, ConSurf, NetSurfP, MUSTER, COACH server and STRING server. Of the 34 nsSNPs submitted for functional analysis, 18 nsSNPs (R124 L, N143 K, Y145 F, V157I, T195S, K379E, I392 K, I437 T, Y438H, N439D, Y441H, N442D, N448D, N451D, D457A, D457Y, I458 L and N460 K) were predicted to have deleterious effects on the protein GPI8p. Additionally, I-Mutant 2.0 and MuPro both showed a decrease in stability after mutation as a result of these nsSNPs, suggesting the destabilization of protein. ConSurf findings suggest that most of the regions were highly conserved. In addition, COACH server was used to predict the ligand binding sites. It was found that no mutation was present at the predicted ligand binding site. The results of the STRING database showed that the protein GPI8p interacts with those proteins which either involve the biosynthetic process of attaching GPI anchor to protein or GPI anchor. The present study suggested that the GPI8p could be a novel target for anti-malarial drugs, which provides significant details for further experimentation.     

Assembly of a series of malarial glycosylphosphatidylinositol anchor oligosaccharides

We report an efficient and convergent synthesis of a series of oligosaccharides comprised of the malaria GPI glycan (2a), a promising anti-malaria vaccine candidate currently in preclinical trials and several related oligosaccharide sequences (3-8) that are possible biosynthetic precursors of the malarial GPI. A flexible synthetic strategy is disclosed that relies on a late-stage coupling between oligomannosides of varying length and pseudo-disaccharide glycosyl acceptor 11 to readily access various malarial GPI structures. Phosphorylation was accomplished by mild and efficient H-phosphonate chemistry before the final deprotection was carried out by using sodium in ammonia. The direct connection of a thiol group via a phosphate diester linkage to the inositol moiety provides a handle for easy conjugation of the GPI glycan to carrier proteins, immobilization on carbohydrate microarrays and photo-affinity labels identification. These synthetic oligosaccharides will serve as molecular probes.     

Designing of precise vaccine construct against visceral leishmaniasis through predicted epitope ensemble: A contemporary approach

Visceral leishmaniasis (VL) caused by Leishmania donovani is a fatal parasitic disease affecting primarily the poor population in endemic countries. Increasing number of deaths as well as resistant to existing drugs necessitates the development of an effective vaccine for successful treatment of VL. The present study employed a combinatorial approach for designing monomer vaccine construct against L. donovani by applying forecasted B- and T- cell epitopes from 4 genome derived antigenic proteins having secretory signal peptides and glycophosphatidylinositol (GPI) anchors with ≤ 1 transmembrane helix. The forecasted population coverage of chosen T cell epitope ensemble (combined HLA class I and II) cover 99.14 % of world-wide human population. The predicted 3D structure of vaccine constructs (VC1/VC2) were modeled using homology modeling approach and docked to innate immune receptors TLR-2 and TLR-4 with respective docking energies −1231.4/−910.3 and −1119.4/−1476 kcal/mol. Overall, the aforementioned designed vaccine constructs were found appropriate for including in self-assembly protein nanoparticles (SAPN) for further study in developing cutting-edge precision vaccine against VL in short duration with cost-effective manner.     

Total synthesis of a glycosylphosphatidylinositol anchor of the human lymphocyte CD52 antigen

The first total synthesis of a glycosylphosphatidylinositol (GPI) anchor bearing a polyunsaturated arachidonoyl fatty acid is reported. This lipid is found in mammalian GPIs that do not undergo lipid remodeling, a process that has important implications in the localization and function of GPI-anchored proteins. Incorporation of the oxidation- and reduction-sensitive arachidonoyl lipid in the target GPI was accomplished by using the para-methoxybenzyl (PMB) group for permanent hydroxyl group protection, which featured a selective, rapid, and efficient global deprotection protocol. The flexibility of this synthetic strategy was further highlighted by the inclusion of two additional GPI core structural modifications present in the GPI anchor of the human lymphocyte CD52 antigen.     

Biological properties of opioid peptides replacing Tyr at position 1 by 2,6-dimethyl-Tyr.

To understand the effect of the replacement of Tyr residue at position 1 in opioid peptides by 2,6-dimethyl-Tyr (Dmt) on the biological property, chiral (D or L) Dmt1 analogs of Leu-enkephalin (Enk) and Tyr-D-Arg-Phe-beta Ala-NH2 (YRFB) were synthesized and their enzymatic stabilities, in vitro bioactivities and receptor binding affinities compared with those of parent peptides. [L-Dmt1]Enk (1) exhibited 4-fold higher stability against aminopeptidase-M and possessed dramatically increased activities in guinea pig ilium (GPI) (187-fold) and mouse vas deferens (MVD) (131-fold) assays, and in rat brain receptor binding assays (356-fold at mu receptor and 46-fold at delta receptor) as compared to Enk. [L-Dmt1]YRFB (3) also exhibited increased activities in GPI (46-fold) and MVD (177-fold) assays, and in the binding assays (69-fold at mu receptro and 341-fold at delta receptor) as compared to the parent peptide. [D-Dmt1]Enk (2) and [D-Dmt1]YRFB (4) exhibited activities with diminished or lesser potency than the parent peptide in all assays. These results indicate that there is a tendency for mu affinity to be enhanced more than delta affinity with introduction of L-Dmt into delta ligand peptide (Enk), and for delta affinity to be enhanced more than mu affinity in case of mu ligand peptide (YRFB), resulting in reduced receptor selectivities at the receptors.