Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry.

A conserved structural feature of human IgG molecules is the presence of an oligosaccharide moiety within the Fc region at Asn297. In addition, 15-20% of normal polyclonal IgG molecules bear N-linked oligosaccharides in the variable (V) regions of the light (L) and/or heavy (H) chains. Electrospray ionization mass spectrometry (ESI-MS) has been applied to the glycan analysis of two IgG1 myeloma proteins (Wid and Cri) after mild reduction and acidification. Heterogeneous ion peaks were observed for both the H and L chains of Wid in contrast to Cri whose L chain peak was homogeneous. Site-specific deglycosylation of the H and L chains of IgG Wid was achieved under native conditions with peptide-N-glycosidase F and endoglycosidase F2, respectively. The Fc glycoforms differed between the two proteins in that Cri-Fc bears diantennary complex-type glycans that are fully core-fucosylated and partially sialylated while Wid-Fc glycans are non-fucosylated, partially galactosylated and non-sialylated. In contrast to the Fc glycans, the L chain glycans of Wid were shown to be fucosylated, fully galactosylated and sialylated, indicating that the glycosylation machinery of the Wid-producing myeloma cells is intact. Thus, combination of the two endoglycosidases can provide a simple means of glycan analysis of both Fab and Fc by ESI-MS, which may contribute to the development of therapeutic IgG with customized glycan profiles.

Multiple proteins present in purified porcine sperm apical plasma membranes interact with the zona pellucida of the oocyte.

An important step in fertilization is the recognition and primary binding of the sperm cell to the zona pellucida (ZP). Primary ZP binding proteins are located at the apical plasma membrane of the sperm head. In order to exclusively study primary zona binding proteins, plasma membranes of sperm heads were isolated, highly purified and subsequently solubilized with a mild or a strong solubilization procedure. Native, highly purified ZP ghosts were used as the binding substrate for solubilized sperm plasma membrane proteins, and a proteomic approach was employed to identify ZP binding proteins. Two-dimensional gel electrophoresis of ZP fragments with bound sperm proteins showed very reproducibly 24 sperm protein spots to be associated to the zona ghosts after mild plasma membrane solubilization whereas only three protein spots were detected after strong plasma membrane solubilization. This indicates the involvement of multiple sperm proteins in ZP binding. The three persistently bound proteins were identified by a tandem mass spectrometry as isoforms of AQN-3 and probably represent the main sperm protein involved in ZP binding. P47, fertilin beta and peroxiredoxin 5 were also conclusively identified. None of the identified proteins has a known acrosomal origin, which further indicated that there was no sample contamination with secondary ZP binding proteins from the acrosomal matrix. In this study, we showed and identified multiple zona binding proteins involved in primary sperm-zona binding. Although we were not able to identify all of the proteins involved, this is a first step in understanding the event of primary sperm-zona interactions and the relevance of this for fertilization is discussed.

Controlled assembly of luminescent racks based on heteroleptic dinuclear lanthanide complexes.

Luminescent lanthanide racks are formed in solution through supramolecular assembly of lanthanide ions with a rigid bis-didentate sensitiser ligand and octadentate aminopolycarboxylate ligands.

Tubercle bacilli generate a novel cell wall-associated pigment after long-term anaerobic culture.

Many cases of tuberculosis result from reactivation of previously acquired latent infections. Models to study such persister forms often involve gradual depletion of oxygen during culture as poor aeration is a characteristic of non-progressive TB granulomas. Anaerobically cultured bacilli develop a thickened outer-most cell wall layer. Here, we analyzed this layer from anaerobically cultured Mycobacterium tuberculosis and Mycobacterium bovis BCG. By six weeks of anaerobiosis a pigment was detected at levels > 60-fold higher in anaerobic than aerobic bacilli. This pigment was responsible for the electron-dense appearance of the thickened cell wall layer and gave an electrospray mass spectrometry peak at 409 Da (M+Na)+ or (M+H)+. We termed this pigment APP1, anaerobically produced pigment 1, the first pigment identified in M. tuberculosis.

Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: case report.

The aim of this study was to compare the sperm protein expression profile (proteome map) from a patient who experienced failed fertilization at IVF with fertile controls. One patient and three fertile donor sperm samples were characterized using two-dimensional electrophoresis. Differences in protein expression were established using gel analysis software before attempted protein identification. Gel analysis of the fertile donor proteome maps revealed excellent reproducibility as well as very low intra-donor and inter-donor variability in the presence of protein spots. In the patient samples, we have noted 20 consistent differences in protein expression (six spots missing, three additional spots, four less abundant, seven more abundant) compared with the controls. Two proteins that were more intense in the patient have been conclusively identified as secretory actin-binding protein and outer dense fibre protein 2/2. In conclusion proteome variation between different fertile donors was very low. In contrast, the patient proteome exhibited 20 differences compared with controls, which we believe is an underestimate. These proteins merit further investigation to determine whether failed fertilization at IVF might be caused by abnormalities in their expression. This case report represents a proof of principle that proteomics may be useful to study defects in sperm function.

Hairpin-shaped heterometallic luminescent lanthanide complexes for DNA intercalative recognition.

Luminescent Ln-Pt2 metallohairpin complexes have been developed, and their intercalative recognition with DNA has been demonstrated with linear dichroism spectroscopy. The heterotrimetallic complexes were formed in a one-step reaction, by assembly of an aminopolycarboxylate ligand, a platinum terpyridine unit, and the lanthanide salt. The metallohairpin complexes bear a neutral lanthanide moiety and two positively charged platinum-containing intercalating units. The Nd(III) analogues are luminescent in the near infrared, and this near-IR luminescence is retained upon binding to DNA. The DNA recognition was demonstrated by linear dichroism spectroscopy. The linear dichroism spectra suggested that the complexes bind perpendicular to the DNA helical axis, confirming intercalative recognition accompanied by dramatic stiffening of DNA, which suggests bis-intercalation of the complex.

Germacrene A is a product of the aristolochene synthase-mediated conversion of farnesylpyrophosphate to aristolochene.

The biosynthesis of several sesquiterpenes has been proposed to proceed via germacrene A. However, to date, the production of germacrene A has not been proven directly for any of the sesquiterpene synthases for which it was postulated as an intermediate. We demonstrate here for the first time that significant amounts of germacrene A (7.5% of the total amount of products) are indeed released from wild-type aristolochene synthase (AS) from Penicillium roqueforti. Germacrene A was identified through direct GC-MS comparison to an authentic sample and through production of beta-elemene in a thermal Cope rearrangement. AS also produced a small amount of valencene through deprotonation of C6 rather than C8 in the final step of the reaction. On the basis of the X-ray structure of AS, Tyr 92 was postulated to be the active-site acid responsible for protonation of germacrene A (Caruthers, J. M.; Kang, I.; Rynkiewicz, M. J.; Cane, D. E.; Christianson, D. W. J. Biol. Chem. 2000, 275, 25533-25539). The CD spectra of a mutant protein, ASY92F, in which Tyr 92 was replaced by Phe, and of AS were very similar. ASY92F was approximately 0.1% as active as nonmutated recombinant AS. The steady-state kinetic parameters were measured as 0.138 min(-1) and 0.189 mM for k(cat) and K(M), respectively. Similar to a mutant protein of 5-epi-aristolochene (Rising, K. A.; Starks, C. M.; Noel, J. P.; Chappell, J. J. Am. Chem. Soc. 2000, 122, 1861-1866), the mutant released significant amounts of germacrene A (approximately 29%). ASY92F also produced various amounts of a further five hydrocarbons of molecular weight 204, valencene, beta-(E)-farnesene, alpha- and beta-selinene, and selina-4,11-diene.

Ferrocene-containing carbohydrate dendrimers.

Aliphatic amines, incorporating one or three (branched) acylated beta-D-glucopyranosyl residues, were coupled with the acid chloride of ferrocenecarboxylic acid and with the diacid chloride of 1,1'-ferrocenedicarboxylic acid to afford four dendrimer-type, carbohydrate-coated ferrocene derivatives in good yields (54-92%). Deprotection of the peracylated beta-D-glucopyranosyl residues was achieved quantitatively by using Zemplén conditions, affording four water-soluble ferrocene derivatives. When only one of the two cyclopentadienyl rings of the ferrocene unit is substituted, strong complexes are formed with beta-cyclodextrin in H2O, as demonstrated by liquid secondary ion mass spectrometry (LSIMS), 1H NMR spectroscopy, electrochemical measurements, and circular dichroism spectroscopy. Molecular dynamics calculations showed that the unsubstituted cyclopentadienyl ring is inserted through the cavity of the toroidal host in these complexes. The electrochemical behavior of the protected and deprotected ferrocene-containing dendrimers was investigated in acetonitrile and water, respectively. The diffusion coefficient decreases with increasing molecular weight of the compound. The potential for oxidation of the ferrocene core, the rate constant of heterogeneous electron transfer, and the rate constant for the energy-transfer reaction with the luminescent excited state of the [Ru(bpy)3]2+ complex (bpy = 2,2'-bipyridine) are strongly affected by the number (one or two) of substituents and by the number (one or three) of carbohydrate branches present in the substituents. These effects are assigned to shielding of the ferrocene core by the dendritic branches. Electrochemical evidence for the existence of different conformers for one of the dendrimers in aqueous solution was obtained.

A Molecular Chameleon: Chromophoric Sensing by a Self-Complexing Molecular Assembly.

A color change from purple to green takes place on addition of tetrathiafulvalene (TTF) to the macrobicyclic receptor 14+ , which is composed of a cyclobis(paraquat-p-phenylene) tetracation that shares one of its paraphenylene rings with a 1,5-naphthoparaphenylene-[36]crown-10 macrocycle. The TTF molecule forces the macrobicycle to turn inside out (see schematic drawing below) and displaces the self-complexed 1,5-dioxynaphthalene ring system from the center of the tetracationic cyclophane.

Supramolecular Daisy Chains.

Our childhoods may be recalled when a self-complementary cation, endowed with both a dibenzo[24]crown-8 macroring and a secondary dialkylammonium sidearm, self-assembles to form a two-component supramolecular architecture that is reminiscent of a daisy chain (depicted schematically on the right). This daisy-chain-like superarchitecture is stabilized by a combination of [N+ -H⋅⋅⋅O] hydrogen bonds and aryl-aryl stacking interactions.

A Chemically and Electrochemically Switchable [2]Catenane Incorporating a Tetrathiafulvalene Unit.

A mechanical switch in a [2]catenane, made up of a cyclobis(paraquat-p-phenylene) tetracation interlocked with a macrocyclic polyether containing a redox-active tetrathiafulvalene (TTF) unit and a 1,5-dioxynaphthalene ring system, can be thrown either chemically or electrochemically. The neutral TTF unit resides "inside" the tetracationic cyclophane in the reduced state and "alongside" it in the oxidized species (TTF+ / TTF2+ ). Switching between the reduced (I4+ ) and oxidized state (I5+ (I6+ )) is accompanied by a dramatic color change.

Translational Isomerism in Some Two- and Three-Station [2]Rotaxanes.

The template-directed syntheses of three [2]rotaxanes are described. They all have dumbbell components, with both hydroquinone and resorcinol rings inserted into polyether chains terminated by tetraarylmethane stoppers, that become encircled during the key self-assembly processes by the tetracationic cyclophane, cyclobis(paraquat-p-phenylene), with its two pi-electron deficient bipyridinium units. It has been demonstrated by low-temperature (1)H NMR spectroscopy that the pi-electron deficient tetracationic cyclophane has a remarkably high preference to reside around the hydroquinone ring in these molecular shuttles. This observation illustrates how a very small constitutional difference-hydroquinone versus resorcinol recognition sites-can lead to the overwhelming preference for one translational isomer over another in this particular range of [2]rotaxanes.

Recognition of Bipyridinium-Based Derivatives by Hydroquinone- and/or Dioxynaphthalene-Based Macrocyclic Polyethers: From Inclusion Complexes to the Self-Assembly of [2]Catenanes.

A range of pi-electron-rich macrocyclic polyethers incorporating dioxybenzene (hydroquinone) and/or dioxynaphthalene units have been synthesized in good yields by simple two-step procedures. These macrocycles are able to bind bipyridinium-based guests as a result of a series of cooperative noncovalent bonding interactions. These molecular recognition events can be extended to the self-assembly of [2]catenanes incorporating the bipyridinium-based cyclophane, cyclobis(paraquat-p-phenylene), and the macrocyclic polyethers incorporating dioxybenzene and -naphthalene units. The efficiencies of these self-assembly processes were found to depend upon the stereoelectronic features of the pi-electron-rich macrocycles-namely, the nature and the substitution pattern of the aromatic units. X-ray crystallographic analysis of some of these [2]catenanes proved unequivocally the relative geometries of the interlocked components. In addition, in the case of those asymmetric [2]catenanes incorporating two different aromatic units within their macrocyclic polyether components, only one of the expected two translational isomers was observed in the solid state. In particular, in all the structures examined, the 1,4-dioxybenzene and 1,5-dioxynaphthalene units are located within the cavity of the tetracationic cyclophane component in preference to other regioisomeric dioxynaphthalene units that reside alongside. Variable-temperature (1)H NMR spectroscopic investigation of the geometries adopted by these [2]catenanes in solution revealed the same selectivity that was observed for one translational isomer over another in the solid state.

Synthetic Cyclic Oligosaccharides-Syntheses and Structural Properties of a Cyclo[(1 → 4)-α-L-rhamnopyranosyl-(1 → 4)-α-D-mannopyranosyl]trioside and -tetraoside.

An efficient polycondensation-cyclization approach to the synthesis of cyclodextrin analogues is demonstrated by the preparation of cyclohexaoside 1 and cyclooctaoside 2. The key intermediate, disaccharide 3, bearing the cyanoethylidene group as a glycosyl donor function and the trityloxy group as a glycosyl acceptor function was prepared in 15 steps starting from L-rhamnose and D-mannose. The crucial cyclooligomerization of the disaccharide monomer 3 was carried out in the presence of TrClO4 as a promoter with the use of ultra-dry conditions at normal concentrations. This reaction led to formation of the cyclic oligosaccharides 28 and 29 (in 34 and 31% yield, respectively), which were deprotected to afford 1 and 2, respectively. The X-ray crystal structural analysis of the cyclooctaoside 2 reveals a cylindrical shape for the cyclic oligosaccharide with C4 symmetry. Individual molecules of 2 are arranged perfectly in stacks that form nanotubes in the solid state.