Serum antibody screening by surface plasmon resonance using a natural glycan microarray

A surface plasmon resonance (SPR) based natural glycan microarray was developed for screening of interactions between glycans and carbohydrate-binding proteins (CBPs). The microarray contained 144 glycan samples and allowed the real-time and simultaneous screening for recognition by CBPs without the need of fluorescent labeling. Glycans were released from their natural source and coupled by reductive amination with the fluorescent labels 2-aminobenzamide (2AB) or anthranilic acid (AA) followed by high-performance liquid chromatography (HPLC) fractionation making use of the fluorescent tag. The released and labeled glycans, in addition to fluorescently labeled synthetic glycans and (neo)glycoproteins, were printed on an epoxide-activated chip at fmol amounts. This resulted in covalent immobilization, with the epoxide groups forming covalent bonds to the secondary amine groups present on the fluorescent glycoconjugates. The generated SPR glycan array presented a subset of the glycan repertoire of the human parasite Schistosoma mansoni. In order to demonstrate the usefulness of the array in the simultaneous detection of glycan-specific serum antibodies, the anti-glycan antibody profiles from sera of S. mansoni-infected individuals as well as from non-endemic uninfected controls were recorded. The SPR screening was sensitive for differences between infection sera and control sera, and revealed antibody titers and antibody classes (IgG or IgM). All SPR analyses were performed with a single SPR array chip, which required regeneration and blocking of the chip before the application of a serum sample. Our results indicate that SPR-based arrays constructed from glycans of natural or synthetic origin, pure or as mixture, can be used for determining serum antibody profiles as possible markers for the infection status of an individual.     

Shotgun glycomics: a microarray strategy for functional glycomics

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.     

The salt-tolerance gene rstB can be used as a selectable marker in plant genetic transformation

The salt-tolerance gene rstB under the control of the cauliflower mosaic virus 35S promoter was used as a selectable marker gene in the Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum cv. Xanthi). The selective agent for plant regeneration was tolerance to 170 mM sodium chloride. The highest selection efficiency was 83.3%. No obvious differences in selection efficiencies were observed when those obtained using the standard selectable marker gene hpt and a selection regime of 10 mg l⁻¹ hygromycin. Transgenic events were confirmed by PCR, Southern blot, RT-PCR and green fluorescent protein studies. The rstB transgenic plants showed improved salt tolerance and a normal phenotype. Based on these results, we suggest that the rstB gene may be used as a promising selectable marker and an alternative to the antibiotic- or herbicide-resistance genes in plant transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The use of a proteinaceous “cushion” with a polystyrene‐binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a “cushion” between the PS‐affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS‐affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527–534, 2016     

Is heterogeneity of catchability in capture–recapture studies a mere sampling artifact or a biologically relevant feature of the population?

The heterogeneity of catchability (HC) among the individuals encountered during a capture–recapture study has long been regarded as a liability. However, heterogeneous capture probabilities may reflect interesting but hidden features of the population, such as social status. The difficulty is to distinguish between this intrinsic heterogeneity and the extrinsic heterogeneity induced by the study itself. So far, population ecologists have not been able to distinguish between these two sources of variation in capture heterogeneity because, in the presence of heterogeneity of capture in the data, they have frequently used a too simple approach. This traditional approach, which consists of incorporating two common sources of lack of fit (transience and trap-dependence), does not directly model the HC and thus cannot investigate its biological meaning. In this context, we propose, for open populations, to directly model the HC by employing multievent models. Multievent models make it possible to break HC into two classes of catchability viewed as uncertain states. With the introduction of a coefficient of heterogeneity to model proportional probabilities of capture over time in the two classes, our approach allows the investigation of HC in a parsimonious way. In this paper, we apply both this new approach and the traditional approach to a long-term data set of male deer mice Peromyscus maniculatus. We then compare 13 candidate models separately for each approach. Our results indicate that the new approach is superior to the traditional approach. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protein engineering towards biotechnological production of bifunctional polyester beads

Microbial polyester inclusions have previously been demonstrated to be applicable as versatile beads outside the bacterial cell. Engineering of proteins selectively binding to the polyester inclusions was conceived to produce polyester beads simultaneously displaying two protein-based functions suitable for applications in, for example, fluorescence activated cell sorting (FACS). The polyester synthase and the phasin protein were fused to the green fluorescent protein (GFP) and the murine myelin oligodendrocyte glycoprotein (MOG), respectively, or GFP and MOG were fused to the N- and C-terminus, respectively, of only the phasin. In both cases, fusion proteins were found to be attached to isolated polyester inclusions while displaying both functionalities per bead. Functionalities at the bead surface were assessed by ELISA, FACS and fluorescence microscopy. The respective double fusion protein was identified by peptide fingerprinting using MALDI-TOF/MS. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New active site oriented glyoxyl-agarose derivatives of <Emphasis Type="Italic">Escherichia coli</Emphasis>penicillin G acylase

Background  

Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic β-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem.     

Glycoblotting enables seamless and straightforward workflow for MALDI-TOF/MS-based sulphoglycomics of N- and O-glycans

Sulfated N- and O-glycans exist in trace levels which are challenging to detect, especially when abundant neutral and sialylated glycans are present. Current matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based sulfoglycomics approaches effectively utilize permethylation to discriminate sulfated glycans from sialyl-glycans. And a charge-based separation to isolate the sulfated glycans from the rest of the permethylated neutral and sialyl-glycans. However, these approaches suffer from concomitant sample losses during cleanup steps. Herein, we describe Glycoblotting as a straightforward complementary method with seamless glycan purification, enrichment, methylation, and labeling on a single platform to address sulfated glycan enrichment, sialic acid methylation, and sample loss. Glycoblottings’ on-bead chemoselective ligation of reducing sugars with hydrazide showed excellent recovery of sulfated glycans, allowing the detection of more sulfated glycan species. On-bead methyl esterification of sialic acid using 3-methyl-1-p-tolyltriazene (MTT) effectively discriminates sulfated glycans from sialyl-glycans. Furthermore, we have shown that using MTT as a methylating agent allowed us to simultaneously detect and differentiate sulfate from phosphate groups in isobaric N-glycan species. We believe that Glycoblotting will contribute significantly to the MALDI-TOF MS-based Sulphoglycomics workflow.     

AtGRP5, a vacuole-located glycine-rich protein involved in cell elongation

Although several glycine-rich protein (GRP) genes were isolated and characterized, very little is known about their function. The primary structure of AtGRP5 from Arabidopsis thaliana has a signal peptide followed by a region with high glycine content. In this work, green fluorescent protein fusions were obtained in order to characterize the sub-cellular localization of the AtGRP5 protein. The results indicated that this protein is the first described vacuolar GRP. Sense, antisense and RNAi transgenic A. thaliana plants were generated and analyzed phenotypically. Plants overexpressing AtGRP5 showed longer roots and an enhanced elongation of the inflorescence axis, while antisense and RNAi plants demonstrated the opposite phenotype. The analysis of a knockout T-DNA line corroborates the phenotypes obtained with the antisense and RNAi plants. Altogether, these results suggest that this vacuolar GRP could be involved in organ growth by promoting cell elongation processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Amanda Mangeon and Claudia Magioli contributed equally to this work.     

Insight into the salt tolerance factors of a wild halophytic rice, <Emphasis Type="Italic">Porteresia coarctata</Emphasis>: a physiological and proteomic approach

Salinity poses a serious threat to yield performance of cultivated rice in South Asian countries. To understand the mechanism of salt-tolerance of the wild halophytic rice, Porteresia coarctata in contrast to the salt-sensitive domesticated rice Oryza sativa, we have compared P. coarctata with the domesticated O. sativa rice varieties under salinity stress with respect to several physiological parameters and changes in leaf protein expression. P. coarctata showed a better growth performance and biomass under salinity stress. Relative water content was conserved in Porteresia during stress and sodium ion accumulation in leaves was comparatively lesser. Scanning electron microscopy revealed presence of two types of salt hairs on two leaf surfaces, each showing a different behaviour under stress. High salt stress for prolonged period also revealed accumulation of extruded NaCl crystals on leaf surface. Changes induced in leaf proteins were studied by two-dimensional gel electrophoresis and subsequent quantitative image analysis. Out of more than 700 protein spots reproducibly detected and analyzed, 60% spots showed significant changes under salinity. Many proteins showed steady patterns of up- or downregulation in response to salinity stress. Twenty protein spots were analyzed by MALDI-TOF, leading to identification of 16 proteins involved in osmolyte synthesis, photosystem functioning, RubisCO activation, cell wall synthesis and chaperone functions. We hypothesize that some of these proteins confer a physiological advantage on Porteresia under salinity, and suggest a pattern of salt tolerance strategies operative in salt-marsh grasses. In addition, such proteins may turn out to be potential targets for recombinant cloning and introgression in salt-sensitive plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fabrication of a surface imprinted hydrogel shell over silica microspheres using bovine serum albumin as a model protein template

Surface imprinting is an effective approach to improve the template transfer efficiency in applications of molecularly imprinted polymers as biosensors and separation materials. In this paper, we tried to fabricate a surface imprinted hydrogel over silica microspheres for selective recognition of bovine serum albumin by covalent immobilization of a water-soluble UV sensitive initiator onto the surface of silica beads. The polymerization was initiated by UV radiation with N-[3-(dimethylamino)propyl]methacrylamide and N-isopropylacrylamide as the functional monomer and assistant monomer, respectively, and a thin coat of stimuli-responsive hydrogel yielded over the silica gels. The surface imprinted hydrogels exhibited specific affinity toward the template protein with an association constant (K_a) of 2.2 × 10⁵ L mol⁻¹ and a maximum binding capacity (Q_max) of 27.3 mg g⁻¹ in Tris–HCl buffer (pH 7.0). The rebinding and desorption kinetics of the surface imprinted hydrogels were determined and proven to be extremely fast (about 1 min compared to 3 h for the previously prepared bulk imprinted hydrogel). Besides, the hydrogel-silica core-shell particles inherit both the stimuli-responsive property of the hydrogel and the good mechanical strength of the silica beads based on the on-line evaluation with high-performance liquid chromatography. The above comprehensive merits of the obtained surface imprinted hydrogel suggest the presented approach an attractive and broadly applicable way of developing biosensors and high-performance protein separation materials.     

Generation of a natural glycan microarray using 9-fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag

Glycan microarray technology has become a successful tool for studying protein–carbohydrate interactions, but a limitation has been the laborious synthesis of glycan structures by enzymatic and chemical methods. Here we describe a new method to generate quantifiable glycan libraries from natural sources by combining widely used protease digestion of glycoproteins and Fmoc chemistry. Glycoproteins including chicken ovalbumin, bovine fetuin, and horseradish peroxidase (HRP) were digested by Pronase, protected by FmocCl, and efficiently separated by 2D-HPLC. We show that glycans from HRP glycopeptides separated by HPLC and fluorescence monitoring retained their natural reducing end structures, mostly core α1,3-fucose and core α1,2-xylose. After simple Fmoc deprotection, the glycans were printed on NHS-activated glass slides. The glycans were interrogated using plant lectins and antibodies in sera from mice infected with Schistosoma mansoni, which revealed the presence of both IgM and IgG antibody responses to HRP glycopeptides. This simple approach to glycopeptide purification and conjugation allows for the development of natural glycopeptide microarrays without the need to remove and derivatize glycans and potentially compromise their reducing end determinants.     

Covalent immobilization of recombinant Citrobacter koseri transaminase onto epoxy resins for consecutive asymmetric synthesis of L-phosphinothricin

Transaminase responsible for alienating prochiral ketone compound is applicable to asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). In this work, the covalent immobilization of recombinant transaminase from Citrobacter koseri (CkTA) was investigated on different epoxy resins. Using optimum ES-105 support, a higher immobilized activity was obtained via optimizing immobilization process in terms of enzyme loading, coupling time and initial PLP concentration. Crucially, due to blocking unreacted epoxy groups on support surface with amino acids, the reaction temperature of blocked immobilized biocatalyst was enhanced from 37 to 57 °C. Its thermostability at 57 °C was also found to be superior to that of free CkTA. The K_m value was shifted from 36.75 mM of free CkTA to 39.87 mM of blocked immobilized biocatalyst, demonstrating that the affinity of enzyme to the substrate has not been apparently altered. Accordingly, the biocatalyst performed the consecutive synthesis of L-PPT for 11 cycles (yields>91%) with retaining more than 91.13% of the initial activity. The seemingly the highest reusability demonstrates this biocatalyst has prospective for reducing the costs of consecutive synthesis of L-PPT with high conversion.

     

Evidence of Symbiosis Between the Soil Yeast <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> and a Sclerophyllous Medicinal Shrub, <Emphasis Type="Italic">Agathosma betulina</Emphasis> (Berg.) Pillans

The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Immobilization of lipase Eversa Transform 2.0 on poly(urea–urethane) nanoparticles obtained using a biopolyol from enzymatic glycerolysis

In this work, the free lipase Eversa^® Transform 2.0 was used as a catalyst for enzymatic glycerolysis reaction in a solvent-free system. The product was evaluated by nuclear magnetic resonance (¹H NMR) and showed high conversion related to hydroxyl groups. In sequence, the product of the glycerolysis was used as stabilizer and biopolyol for the synthesis of poly(urea–urethane) nanoparticles (PUU NPs) aqueous dispersion by the miniemulsion polymerization technique, without the use of a further surfactant in the system. Reactions resulted in stable dispersions of PUU NPs with an average diameter of 190 nm. After, the formation of the PUU NPs in the presence of concentrated lipase Eversa^® Transform 2.0 was studied, aiming the lipase immobilization on the NP surface, and a stable enzymatic derivative with diameters around 231 nm was obtained. The hydrolytic enzymatic activity was determined using ρ-nitrophenyl palmitate (ρ-NPP) and the immobilization was confirmed by morphological analysis using transmission electron microscopy and fluorescence microscopy.

     

Glycosylation-related genes are variably expressed depending on the differentiation state of a bioaminergic neuronal cell line: implication for the cellular prion protein

A striking feature of the cellular prion protein (PrP^C) is the heterogeneity of its glycoforms, whose contribution to PrP^C function has yet to be defined. Using the 1C11 neuronal bioaminergic differentiation model and a glycomics approach, we show here a correlation between differential PrP^C N-glycosylations in 1C11^5-HT serotonergic and 1C11^NE noradrenergic cells compared to their 1C11 precursor cells and a variation of the glycogenome expression status in these cells. In particular, expression of genes involved in N-glycan synthesis or in the modeling of chondroitin and heparan sulfate proteoglycans appeared to be modulated. Our results highlight that, the expression of glycosylation-related genes is regulated during bioaminergic neuronal differentiation, consistent with a participation of glycoconjugates in neuronal development and plasticity. A neuronal regulation of glycosylation processes may have direct implications on some neurospecific functions of PrP^C and may participate in specific brain targeting of prion strains. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Judging a tumor cell by its cover: a matter of mitochondrial contact sites

Glioblastoma is a highly aggressive brain tumor constituted by glioma stem cell and differentiated cell populations with distinct susceptibility to cytotoxic T lymphocytes crucial for tumor immune surveillance. In this issue of The EMBO Journal, Bassoy et al ( 2017 ) show that the surface expression of certain glycans that favor recognition and killing by T cells depends on mitochondrial contacts with the endoplasmic reticulum, extending the function of this interface even to immune recognition.     

Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody

The HIV envelope has evolved a dense array of immunologically "self" carbohydrates that efficiently protect the virus from antibody recognition. Nonetheless, one broadly neutralising antibody, IgG1 2G12, has been shown to recognise a cluster of oligomannose glycans on the HIV-1 surface antigen gp120. Thus the self carbohydrates of HIV are now regarded as potential targets for viral neutralisation and vaccine design. Here, we show that chemical inhibition of mammalian glycoprotein synthesis, with the plant alkaloid kifunensine, creates multiple HIV (2G12) epitopes on the surface of previously non-antigenic self proteins and cells, including HIV gp120. This formally demonstrates the structural basis for self/non-self discrimination between viral and host glycans, by a neutralising antibody. Moreover, this study provides an alternative protein engineering approach to the design of a carbohydrate vaccine for HIV-1 by chemical synthesis.     

Biocatalysts for fuel cells: efficient hydrogenase orientation for H<Subscript>2</Subscript> oxidation at electrodes modified with carbon nanotubes

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H₂ oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H₂ oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and validation of a FACS-based lipoprotein localization screen in the Lyme disease spirochete <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

Background  

In our previous studies on lipoprotein secretion in the Lyme disease spirochete Borrelia burgdorferi, we used monomeric red fluorescent protein 1 (mRFP1) fused to specifically mutated outer surface protein A (OspA) N-terminal lipopeptides to gather first insights into lipoprotein sorting determinants. OspA:mRFP1 fusions could be detected by epifluorescence microscopy both in the periplasm and on the bacterial surface. To build on these findings and to complement the prior targeted mutagenesis approach, we set out to develop a screen to probe a random mutagenesis expression library for mutants expressing differentially localized lipoproteins.