An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes

N-Glycosylation is one of the most common and important post-translational modification methods, and it plays a vital role in controlling many biological processes. Increasing discovery of abnormal alterations in N-linked glycans associated with many diseases leads to greater demands for rapid and efficient N-glycosylation profiling in large-scale clinical samples. In the workflow of global N-glycosylation analysis, enzymatic digestion is the main rate-limiting step, and it includes both protease digestion and peptide-N4–(N-acetyl-beta-glucosaminyl) asparagine amidase (PNGase) F deglycosylation. Prolonged incubation time is generally required because of the limited digestion efficiency of the conventional in-solution digestion method. Here, we propose novel thermoresponsive magnetic fluid (TMF)-immobilized enzymes (trypsin or PNGase F) for ultrafast and highly efficient proteome digestion and deglycosylation. Unlike other magnetic material-immobilized enzymes, TMF-immobilized enzymes display a unique temperature-triggered magnetic response behavior. At room temperature, a TMF-immobilized enzyme completely dissolves in an aqueous solution and forms a homogeneous system with a protein/peptide sample for efficient digestion but cannot be separated by magnetic force because of its excellent water dispersity. Above its lower critical solution temperature (LCST), thermoflocculation of a TMF-immobilized enzyme allows it to be easily recovered by increasing the temperature and magnetic force. Taking advantage of the unique homogeneous reaction of a TMF-immobilized enzyme, both protein digestion and glycopeptide deglycosylation can be finished within 3 min, and the whole sample processing time can be reduced by more than 20 times. The application of a TMF-immobilized enzyme in large-scale profiling of protein N-glycosylation in urine samples led to the successful identification of 2,197 N-glycopeptides and further demonstrated the potential of this strategy for fast and high-throughput analysis of N-glycoproteome in clinical samples.

Genetic Dissection for Maize Forage Digestibility Traits in a Multi-Parent Advanced Generation Intercross (MAGIC) Population

Forage feedstock is the greatest source of energy for livestock. Unfortunately, less than 50% of their fiber content is actually digested and assimilated by the ruminant animals. This recalcitrance is mainly due to the high concentration of plant cell wall material and to the limited digestion of the fiber by the microorganisms. A Genome-Wide Association Study (GWAS) was carried out in order to identify Single Nucleotide Polymorphisms (SNPs) associated with forage digestibility traits in a maize Multi-Parent Advanced Generation Intercross (MAGIC) population. We identified seven SNPs, corresponding to five Quantitative Trait Loci (QTL), associated to digestibility of the organic matter, 11 SNPs, clustered in eight QTLs, associated to Neutral Detergent Fiber (NDF) content and eight SNPs corresponding with four QTL associated with Acid Detergent Fiber (ADF). Candidate genes under the QTL for digestibility of the organic matter could be the ones involved in pectin degradation or phenylpropanoid pathway. Transcription factor genes were also proposed for the fiber QTL identified, in addition to genes induced by oxidative stress, or a gene involved in lignin modifications. Nevertheless, for the improvement of the traits under study, and based on the moderate heritability value and low percentage of the phenotypic variability explained by each QTL, a genomic selection strategy using markers evenly distributed across the whole genome is proposed.

Circulating Chromogranin A Is Cleaved Into Vasoregulatory Fragments in Patients With Pancreatic Ductal Adenocarcinoma

Chromogranin A (CgA), a secretory protein released in the blood by the neuroendocrine system, consists of a mixture of full-length molecules and fragments endowed of vasoregulatory activity. The extent and the role of CgA fragmentation were investigated in patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC, n=172). Multivariate analysis showed that full-length CgA was associated with better progression free and overall survival, whereas CgA C-terminal fragmentation was associated with worse prognosis. In vitro studies showed that PDAC cells can promote the cleavage of CgA C-terminal region by activating plasminogen to plasmin. Limited digestion of full-length CgA with plasmin abolished its anti-angiogenic activity and generated pro-angiogenic molecules. The fragmentation of CgA C-terminal region was increased also in murine models of PDAC. In these models, the inhibition of CgA fragmentation with aprotinin, an inhibitor of plasmin and other serine proteases, or the blockade of pro-angiogenic fragments with specific antibodies inhibited the growth of PDAC implanted subcutaneously in mice. Finally, administration of full-length CgA to mice bearing orthotopic PDAC reduced tumor perfusion, as measured by contrast-enhanced ultrasound. These findings suggest that PDAC can promote the cleavage of circulating CgA C-terminal region to generate fragments that regulate the tumor vascular biology and that may represent new potential therapeutic targets.

Universal NicE-seq for high-resolution accessible chromatin profiling for formaldehyde-fixed and FFPE tissues

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for the FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high-resolution genomic feature studies.

Universal NicE-seq for high resolution accessible chromatin profiling for formaldehyde fixed and FFPE tissues

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negate the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high resolution genomic feature studies.

Characterization of Recombinant β2GPI Using Hydrogen-Deuterium Exchange and X-Ray Crystallography

Introduction: Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). However, there remains significant inconsistency in measurements of anti-β2GPI antibodies, possibly due to different sources of β2GPI. To address this issue and others, we have developed a system in which recombinant β2GPI (rβ2GPI) is expressed in mammalian (HEK293) cells using a lentiviral construct with a CMV promoter, in which the signal peptide of β2GPI has been mutated. In previous work, we demonstrated that patient-derived anti-β2GPI antibodies recognize rβ2GPI similarly to purified plasma β2GPI. Plasma Β2GPI has been crystallized and found to be in a "fishhook" or "J" conformation, however the protein was isolated under harsh conditions that included perchloric acid precipitation. Moreover, electron microscopy and other approaches have suggested that β2GPI may adopt several conformations in addition to the fishhook, including circular and other intermediate forms. Methods: cDNA encoding full length APOH (the gene encoding β2GPI), was cloned into pLenti CMV Puro DEST. Lentivirus was produced using the Lentiviral Gateway Expression kit and stable cell lines developed by transducing HEK293 cells with lentivirus encoding APOH. Cell lines were grown in suspension, and supernatants containing secreted full length β2GPI were purified using a hitrap-heparin column. Anti-β2GPI antibody binding to rβ2GPI was determined using a standard β2GPI ELISA. For hydrogen-deuterium exchange (HDX) mass spectrometry, proteins were in 10 mM phosphate buffer, 99.9% D2O in an autosampler at 20 °C. After 10-10,000 seconds of incubation, the reaction was quenched. Sequences of peptic fragments were identified and mass assignment for each peptide without deuterium exchange was checked manually. The average of relative fractional deuterium uptake for each amide proton residue was calculated using HDsite equations. Crystallization of plasma derived and recombinant β2GPI-WT was performed using a hanging-drop vapor diffusion method at 4°C in 150 mM NaCl and 50 mM Tris-HCl pH 7.3. Results: After transduction with lentiviral β2GPI constructs, HEK-293 cells efficiently secreted rβ2GPI-WT, site-directed mutants and several domains constructs. Hydrogen-deuterium exchange (HDX) analysis of plasma derived and rβ2GPI-WT revealed that both have very similar surface deuterium exchange rates and heat map profiles (Figure 1A, B). In both plasma derived and rβ2GPI, limited digestion of domains D-I, II and III by pepsin was observed, consistent with other sushi domain containing proteins. However, solid coverage of domain IV and V was obtained and the heat maps generated for both plasma derived and rβ2GPI-WT were very similar, suggesting similar structure and conformation. Moreover, although crystallization of plasma derived and rβ2GPI-WT revealed differences in crystal size and appearance, X-ray structural analysis to a resolution of 3.1 Å (Figure 1C, D) demonstrated that both plasma derived and rβ2GPI had the same structure and conformation under the conditions employed. Conclusion: These studies demonstrate that recombinant β2GPI produced in mammalian cells following lentiviral transduction has a similar structure and conformation as plasma-derived β2GPI. This methodology may be used for additional mutagenesis studies to better define the underlying structural features regulating the conformation of β2GPI as well as its interactions with other proteins, diagnostic utility and functions. Disclosures McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Glycoside Hydrolase Genes Are Required for Virulence ofAgrobacterium tumefaciensonBryophyllum daigremontianaand Tomato

ABSTRACTAgrobacterium tumefaciensis a rhizosphere bacterium that can infect wound sites on plants. The bacterium transfers a segment of DNA (T-DNA) from the Ti plasmid to the plant host cell via a type IV secretion system where the DNA becomes integrated into the host cell chromosomes. The expression of T-DNA in the plant results in tumor formation. Although the binding of the bacteria to plant surfaces has been studied previously, there is little work on possible interactions of the bacteria with the plant cell wall. Seven of the 48 genes encoding putative glycoside hydrolases (Atu2295,Atu2371,Atu3104,Atu3129,Atu4560,Atu4561, andAtu4665) in the genome ofA. tumefaciensC58 were found to play a role in virulence on tomato andBryophyllum daigremontiana. Two of these genes (pglAandpglB;Atu3129andAtu4560) encode enzymes capable of digesting polygalacturonic acid and, thus, may play a role in the digestion of pectin. One gene (arfA;Atu3104) encodes an arabinosylfuranosidase, which could remove arabinose from the ends of polysaccharide chains. Two genes (bglAandbglB;Atu2295andAtu4561) encode proteins with β-glycosidase activity and could digest a variety of plant cell wall oligosaccharides and polysaccharides. One gene (xynA;Atu2371) encodes a putative xylanase, which may play a role in the digestion of xylan. Another gene (melA;Atu4665) encodes a protein with α-galactosidase activity and may be involved in the breakdown of arabinogalactans. Limited digestion of the plant cell wall byA. tumefaciensmay be involved in tumor formation on tomato andB. daigremontiana.IMPORTANCEA. tumefaciensis used in the construction of genetically engineered plants, as it is able to transfer DNA to plant hosts. Knowledge of the mechanisms of DNA transfer and the genes required will aid in the understanding of this process. Manipulation of glycoside hydrolases may increase transformation and widen the host range of the bacterium.A. tumefaciensalso causes disease (crown gall tumors) on a variety of plants, including stone fruit trees, grapes, and grafted ornamentals such as roses. It is possible that compounds that inhibit glycoside hydrolases could be used to control crown gall disease caused byA. tumefaciens.

Limited proteolysis in porous membrane reactors containing immobilized trypsin

Trypsin-containing membranes effect limited digestion to identify facile digestion sites in protein structures.