Structural characterization of carbohydrate sequence, linkage, and branching in a quadrupole Ion trap mass spectrometer: neutral oligosaccharides and N-linked glycans

Several oligosaccharide and N-linked glycan samples have been utilized to evaluate structural detail obtained with an ion trap mass spectrometer (ITMS). Using multistage MS/MS (MSn) in a commercial instrument, linear sequence, linkage, and branching have been studied, as well as the positional isomers within branched glycans. Samples were prepared as methylated derivatives, which amplifies the structurally informative cross-ring and double glycosidic cleavage products associated with interresidue linkage and oligomer branching detail. Spectral analysis of a linear glucose homopolymer demonstrated ITMS to be comparable with data obtained from a triple-quadrupole instrument, and in a study of complex glycans, the feature of MSn enabled the assignment of linkages that cannot be assessed by conventional triple-quadrupole tandem instrumentation. The longer time scale of CID experiments enhances the abundance of multiple bond ruptures important for a complete understanding of structure. This technology appears to provide a significant step toward the goal of characterizing all aspects of carbohydrate structure using a single instrument.     

The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin

Large quantities of recombinant acid alpha-glucosidase are needed for in vivo experimentation of enzyme replacement therapy in Pompe disease. We describe a new purification method for the purification of this recombinant enzyme from tissue culture medium consisting of concanavalin A affinity chromatography, hydrophobic interaction chromatography, affinity chromatography on Superdex, and anion exchange chromatography. The new method is amenable to scale up, and has increased speed, and improved reproducibility with similar high yield and purification efficiency when compared to previous methods.     

Specific features of the structural state in radiation-resistant structural materials

The factors of accelerating the recombination of vacancies and interstitial atoms in the structure of structural materials during irradiation are considered. The efficiencies of the effects of the distortion factors induced by substitutional and interstitial atoms and short-range ordering in a crystal lattice on the recombination of point defects are estimated and compared.     

Consistency in structural energetics of protein folding and peptide recognition

We report a new free energy decomposition that includes structure-derived atomic contact energies for the desolvation component, and show that it applies equally well to the analysis of single-domain protein folding and to the binding of flexible peptides to proteins. Specifically, we selected the 17 single-domain proteins for which the three-dimensional structures and thermodynamic unfolding free energies are available. By calculating all terms except the backbone conformational entropy change and comparing the result to the experimentally measured free energy, we estimated that the mean entropy gain by the backbone chain upon unfolding (delta Sbb) is 5.3 cal/K per mole of residue, and that the average backbone entropy for glycine is 6.7 cal/K. Both numbers are in close agreement with recent estimates made by entirely different methods, suggesting a promising degree of consistency between data obtained from disparate sources. In addition, a quantitative analysis of the folding free energy indicates that the unfavorable backbone entropy for each of the proteins is balanced predominantly by favorable backbone interactions. Finally, because the binding of flexible peptides to receptors is physically similar to folding, the free energy function should, in principle, be equally applicable to flexible docking. By combining atomic contact energies, electrostatics, and sequence-dependent backbone entropy, we calculated a priori the free energy changes associated with the binding of four different peptides to HLA-A2, 1 MHC molecule and found agreement with experiment to within 10% without parameter adjustment.     

The six N-linked carbohydrates of the lutropin/choriogonadotropin receptor are not absolutely required for correct folding, cell surface expression, hormone binding, or signal transduction

Using two separate methods, we have determined that all six potential sites for N-linked glycosylation on the rat lutropin/choriogonadotropin receptor (rLHR) contain carbohydrates. The functional roles of the carbohydrates were analyzed initially through the use of two nonglycosylated receptor mutants rLHR(N(77,152,173,269,277,291)Q) and rLHR(N(77,152,269,277,291)Q;T(175)A). Although Western blot analyses demonstrated both mutant receptors to be stably expressed, little or no hCG binding activity could be detected in detergent solubilized extracts of 293 cells expressing either nonglycosylated LHR mutant. Although this loss of hCG binding was concluded to be due to misfolding, it was unknown whether this misfolding was due to the absence of carbohydrates or to the multiple amino acid substitutions that had been introduced into the polypeptide. To differentiate between these possibilities, hCG binding assays were performed with nonglycosylated receptors obtained after tunicamycin treatment of cells expressing the wild-type rLHR. Even though these wild-type receptors were confirmed to be devoid of all N-linked carbohydrates by Western blots, they were found to bind hCG with a normal high affinity. In addition, tunicamycin-derived, nonglycosylated LHRs were present at the cell surface and exhibited a phenotype consistent with mature receptors due to their capability to mediate hCG-stimulated cAMP production as well as bind oLH with high affinity. These results indicate that the loss of high affinity hormone binding by rLHR(N(77,152,173,269,277,291)Q) and rLHR(N(77,152,269,277,291)Q;T(175)A) is simply due to the collective amino acid substitutions rather than to the absence of carbohydrates. Therefore, N-linked carbohydrates are not absolutely required for the proper folding of the rLHR into a mature receptor capable of binding hormone and signaling. These results are in marked contrast to the follitropin receptor (FSHR), a very similar receptor which has been shown to strictly require N-linked carbohydrates for folding of the nascent protein.     

Contrasting roles for symmetrically disposed beta-turns in the folding of a small protein

To investigate the role of turns in protein folding, we have characterized the effects of combinatorial and site-directed mutations in the two beta-turns of peptostreptococcal protein L on folding thermodynamics and kinetics. Sequences of folded variants recovered from combinatorial libraries using a phase display selection method were considerably more variable in the second turn than in the first turn. These combinatorial mutants as well as strategically placed point mutants in the two turns had a similar range of thermodynamic stabilities, but strikingly different folding kinetics. A glycine to alanine substitution in the second beta-turn increased the rate of unfolding more than tenfold but had little effect on the rate of folding, while mutation of a symmetrically disposed glycine residue in the first turn had little effect on unfolding but slowed the rate of folding nearly tenfold. These results demonstrate that the role of beta-turns in protein folding is strongly context-dependent, and suggests that the first turn is formed and the second turn disrupted in the folding transition state.     

The multiple roles of Cyk1p in the assembly and function of the actomyosin ring in budding yeast

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino terminal portion containing the calponin homology domain binds to actin filaments and is required for the assembly of actin filaments to the ring. This result supports the hypothesis that Cyk1p/Iqg1p plays a direct role in F-actin recruitment. Deletion of the domain harboring the eight IQ motifs abolishes the localization of Cyk1p/Iqg1p to the bud neck, suggesting that Cyk1p/Iqg1p may be localized through interactions with a calmodulin-like protein. Interestingly, deletion of the COOH-terminal GTPase-activating protein-related domain does not affect Cyk1p/Iqg1p localization or actin recruitment to the ring but prevents actomyosin ring contraction. In vitro binding experiments show that Cyk1p/Iqg1p binds to calmodulin, Cmd1p, in a calcium-dependent manner, and to Tem1p, a small GTP-binding protein previously found to be required for the completion of anaphase. These results demonstrate the critical function of Cyk1p/Iqg1p in regulating various steps of actomyosin ring assembly and cytokinesis.     

The biosynthesis of oligosaccharides in intact Golgi preparations from rat liver. Analysis of N-linked and O-linked glycans labeled by UDP-[6-3H]N-acetylgalactosamine

Endogenous acceptors in a Golgi apparatus-enriched subcellular fraction from rat liver were labeled with UDP-[3H]GalNAc. The great majority of these acceptors were protected from protease degradation in the absence of detergent. These molecules are therefore present in intact vesicles of the correct topological orientation, which are likely to be similar to the Golgi compartments of the intact cell. Several distinct glycoproteins are labeled, but most are different from those labeled with UDP-[3H]GlcNAc. The enzyme peptide-N4(N-acetyl-beta-glucosiminyl)asparagine amidase releases label from a few specific proteins, indicating that [3H]GalNAc is transferred to N-linked oligosaccharides. Both neutral and anionic N-linked oligosaccharides are found, the great majority of which do not bind to ConA-Sepharose. Most of the [3H]GalNAc found in neutral oligosaccharides is terminal and beta-linked. The negative charge on the anionic molecules is due to sialic acid, and phosphate. A major portion of the [3H] GalNAc in this fraction is acid labile, and is released with kinetics consistent with it being in a phosphodiester linkage. These results show the existence of a whole new class of GalNAc-containing N-linked oligosaccharides, and demonstrates that this in vitro approach can detect previously undescribed structures. O-linked oligosaccharide biosynthesis was also studied in the same labeled rat liver Golgi apparatus preparations. beta-Elimination releases approximately 95% of the peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)-resistant label which, in the absence of other added nucleotides, is almost exclusively [3H] GalNAcitol. If other unlabeled sugar nucleotides and adenosine 3'-phosphate,5'-phosphosulfate are added during the chase period two anionic O-linked oligosaccharides are synthesized, indicating that the UDP-GalNAc:peptide-N-acetylgalactosaminyltransferase is at least in part functionally co-localized with enzymes that extend and modify O-linked oligosaccharides.     

The critical N-linked glycan of murine leukemia virus envelope protein promotes both folding of the C-terminal domains of the precursor polyprotein and stability of the postcleavage envelope complex

The infectivity of Friend ecotropic murine leukemia virus was previously shown to be highly sensitive to modification in its envelope protein (Env) at only one of the eight signals for N-linked glycan attachment, the fourth from the N terminus (gs4). In the present study, a set of six single-amino-acid substitutions in or near gs4 was used to determine the function of this region of Env and the role played by the glycan itself. One mutant that lacked the gs4 glycan was fully infectious, while one that retained this glycan was completely noninfectious, indicating that the gs4 glycan per se is not required for Env function. Infectivity correlated with the level of mature Env complex incorporated into virus particles, which was determined by the severity of defects in transport of the envelope precursor protein (gPrEnv) from the endoplasmic reticulum into the Golgi apparatus, in cleavage of gPrEnv into the two envelope subunits (the surface protein [SU] and the transmembrane protein [TM]), and in the association of SU with cellular membranes. All of the mutants induced the wild-type level of superinfection interference, indicating that the gs4 region mutations did not interfere with proper folding of the N-terminal domain of SU. These results suggest that the gs4 region mediates folding of the C-terminal domains of gPrEnv and stability of the interaction between SU and TM. Although the gs4 glycan was not essential for infectivity, processing of all mutant Envs lacking this glycan was significantly impaired, suggesting that efficient folding of gPrEnv requires a glycan at this position. The conservation of a glycosylation site homologous to gs4 across a broad range of retroviruses suggests that this sequence may play a similar role in many retroviral Envs.     

The development of the reticular lamina in the hamster: an examination of transitory features and their functional roles

This study examines the development of the reticular lamina in the Syrian golden hamster postriatally from birth to adulthood at 2 day intervals using the scanning electron microscope. During this period, numerous transitory features emerged whose roles were concerned primarily with the development of the tectorial membrane (TM). The principal findings were as follows. (1) The surface of the developing organ of Corti produced all the fibrous material composing the minor tectorial membrane (mTM) including radial and longitudinal fiber bundles which formed the skeleton of the TM, and spongy, amorphous material which formed its intervening ground substance. (2) Throughout most of the cochlear spiral, radial fiber bundles were seen extending from the microvilli of supporting cells and projecting toward the major tectorial membrane (MTM). In most of the basal turn, but not in the apical turn, these radial bundles were interwoven with longitudinal fiber bundles which emerged from the surface of Hensen's cells. These findings indicate that the architecture of the TM is more complex in the basal turn than in the apex. (3) Increases in the dimensions of the reticular lamina resulted from the emergence of pillar cell headplates and growth in the diameter of hair cells and supporting cells. The emergence of pillar cell headplates was the principal factor contributing to increases in the radial dimension of the reticular lamina. This emergence was most dramatic between 10 and 12 days after birth (DAB) after the mTM completed its growth. Since the mTM appears to be bound medially to the MTM and laterally to the marginal pillars by 10 DAB, it seems likely that the growth of the reticular lamina after 10 DAB causes some stretching of the mTM both radially and longitudinally. (4) Completion of outer hair cell stereocilia growth at 8 DAB was followed by loss of supporting cell attachments of the TM (trabeculae) by 10 DAB, and coincided with the formation of marginal pillars from the third row of supporting cells. It is suggested that the formation of marginal pillars may be required for coupling of the TM to the tips of outer hair cell stereocilia and for induction of radial tension of the mTM. (5) Removal of the marginal pillar attachments occurred following completion of hair cell growth. (6) All structures on the reticular lamina appeared to have adult-like characteristics by 20 DAB.     

Molecular and structural aspects of fimbriae biosynthesis and assembly in Escherichia coli

Fimbriae are long filamentous polymeric protein structures located at the surface of bacterial cells. They enable the bacteria to bind to specific receptor structures and thereby to colonise specific surfaces. Fimbriae consist of so-called major and minor subunits, which form, in a specific order, the fimbrial structure. In this review emphasis is put on the genetic organisation, regulation and especially on the biosynthesis of fimbriae of enterotoxigenic Escherichia coli strains, and more in particular on K88 and related fimbriae, with ample reference to well-studied P and type 1 fimbriae. The biosynthesis of these fimbriae requires two specific and unique proteins, a periplasmic chaperone and an outer membrane located molecular usher ('doorkeeper'). Molecular and structural aspects of the secretion of fimbrial subunits across the cytoplasmic membrane, the interaction of these subunits with periplasmic molecular chaperone, their translocation to the inner site of the outer membrane and their interaction with the usher protein, as well as the (ordered) translocation of the subunits across the outer membrane and their assembly into a growing fimbrial structure will be described. A model for K88 fimbriae is presented.     

Arrest of subunit folding and assembly of nicotinic acetylcholine receptors in cultured muscle cells by dithiothreitol

In this study we have used cultured muscle cells to investigate the role of disulfide bond formation in the sequence of molecular events leading to nicotinic acetylcholine receptor (AChR) assembly and surface expression. We have observed that disulfide bond formation in newly synthesized AChR alpha-subunits occurs 5-20 min after translation and that this modification can be blocked by dithiothreitol (DTT), a membrane-permeant thiol-reducing agent. DTT treatment was found to arrest AChR alpha-subunit conformational maturation, assembly, and appearance on the cell surface, showing that these events are dependent on prior formation of disulfide bonds. Subunits prevented from maturation by the reducing agent do not irreversibly misfold or aggregate, since upon removal of DTT, AChR alpha-subunits undergo formation of disulfide bonds and resume folding, oligomerization, and surface expression. We have previously found that nascent alpha-subunits form transient complexes with the molecular chaperone calnexin immediately after subunit synthesis (Gelman, M.S., Chang, W., Thomas, D. Y., Bergeron, J. J. M., and Prives, J. M. (1995) J. Biol. Chem. 270, 15085-15092) and have now observed that both the formation and the subsequent dissociation of these complexes are unaffected by DTT treatment. Thus, alpha-subunits appear to dissociate from calnexin independently of their undergoing disulfide bond formation and achieving conformational maturation. This finding together with the absence of irreversible misfolding of DTT-arrested alpha-subunits suggests that calnexin may act to prevent misfolding by aiding in the initial folding events and is not an essential participant in the late stages of alpha-subunit maturation.     

An IgG monoclonal antibody against Dictyostelium discoideum glycoproteins specifically recognizes Fucalpha1,6GlcNAcbeta in the core of N-linked glycans. Localized expression of core-fucosylated glycoconjugates in human tissues

Core fucosylation of N-linked oligosaccharides (GlcNAcbeta1, 4(Fucalpha1,6)GlcNAcbeta1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideum glycoproteins (Crandall, I. E. and Newell, P. C. (1989) Development 107, 87-94), specifically recognize fucose residues in alpha1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing alpha-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core alpha1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice.     

The modern face of prejudice and structural features that moderate the effect of cooperation on affect

Facial muscle activity and self-reports were examined for racial bias in 3 studies. In the first 2 experiments, While participants imagined cooperating with a Black or White partner. Experiment 1 manipulated reward structure in the context of cooperating with a deficient partner. Experiment 2 manipulated partner deficiency and willingness to expend compensatory effort. On both facial EMG and self-report measures, joint rewards produced more negative affect than independent rewards. However, all partners were liked more when they were willing to try to compensate for their deficits. In addition, more liking was reported for Black partners, but EMG activity indicated bias against Blacks. Experiment 3 investigated individual differences in prejudice. Again, a greater preference for Blacks than Whites occurred on self-report measures, but in their facial muscle activity, high-prejudiced participants exhibited bias against Blacks.     

Personality, self-estimated intelligence, and uses of music: A Spanish replication and extension using structural equation modeling.

This study replicates and extends a recent study on personality, intelligence and uses of music [Chamorro-Premuzic, T., & Furnham, A. (2007). Personality and music: Can traits explain how people use music in everyday life? British Journal of Psychology, 98, 175–185] using Spanish participants and structural equation modeling. Data from 245 university students showed that, in line with our hypotheses, individuals higher in Neuroticism were more likely to use music for emotional regulation (influencing their mood states), those higher in Extraversion were more likely to use music as background to other activities, and those higher in Openness were more likely to experience music in a cognitive or intellectual way. As predicted, self-estimates of intelligence were also linked to cognitive use of music, though not when individual differences were considered. On other hand, contrasting with initial predictions, Extraversion was positively rather than negatively linked to emotional use of music. Small incremental effects of gender (over personality) were also found on the emotional use of music. Results are discussed in regards to previous findings on personality traits as determinants of uses of music. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

New minimal substrate structural requirements in the enzymatic peroxidation of alkenes with soybean lipoxygenase

A longitudinal study was conducted to investigate the association between human immunodeficiency virus (HIV) infection, history of major depressive disorder (MDD), and persistent or recurrent MDD among intravenous drug users. Psychiatric disorders were assessed in a sample of HIV-positive (HIV+) and HIV-negative (HIV-) intravenous drug users every 6 months for 3 years. Results indicated that HIV status and baseline MDD independently predicted persistent or recurrent episodes of MDD after gender, drug use, ethnicity, income, and the presence other psychiatric disorders were controlled statistically. Among HIV+ intravenous drug users with baseline MDD, 90% experienced at least one subsequent episode of MDD and 47% experienced at least three subsequent episodes of MDD. However, less than 40% of intravenous drug users with current MDD received treatment for emotional problems. These findings indicate that intravenous drug users with HIV infection and a history of MDD are at considerable risk for future episodes of MDD or recurrent MDD, and that increased provision of treatment for intravenous drug users with MDD may be necessary.     

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.     

Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist

kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.