The AMP deaminase of the mollusk Helix pomatia is an unexpected member of the adenosine deaminase-related growth factor (ADGF) family.

We report here the first occurrence of an adenosine deaminase-related growth factor (ADGF) that deaminates adenosine 5' monophosphate (AMP) in preference to adenosine. The ADGFs are a group of secreted deaminases found throughout the animal kingdom that affect the extracellular concentration of adenosine by converting it to inosine. The AMP deaminase studied here was first isolated and biochemically characterized from the roman snail Helix pomatia in 1983. Determination of the amino acid sequence of the AMP deaminase enabled sequence comparisons to protein databases and revealed it as a member of the ADGF family. Cloning and expression of its cDNA in Pichia pastoris allowed the comparison of the biochemical characteristics of the native and recombinant forms of the enzyme and confirmed they correspond to the previously reported activity. Uncharacteristically, the H. pomatia AMP deaminase was determined to be dissimilar to the AMP deaminase family by sequence comparison while demonstrating similarity to the ADGFs despite having AMP as its preferred substrate rather than adenosine.

Defining the filarial N-glycoproteome by glycosite mapping in the human parasitic nematode Brugia malayi.

N-linked glycosylation is a critical post translational modification of eukaryotic proteins. N-linked glycans are present on surface and secreted filarial proteins that play a role in host parasite interactions. Examples of glycosylated Brugia malayi proteins have been previously identified but there has not been a systematic study of the N-linked glycoproteome of this or any other filarial parasite. In this study, we applied an enhanced N-glyco FASP protocol using an engineered carbohydrate-binding protein, Fbs1, to enrich N-glycosylated peptides for analysis by LC-MS/MS. We then mapped the N-glycosites on proteins from three host stages of the parasite: adult female, adult male and microfilariae. Fbs1 enrichment of N-glycosylated peptides enhanced the identification of N-glycosites. Our data identified 582 N-linked glycoproteins with 1273 N-glycosites. Gene ontology and cell localization prediction of the identified N-glycoproteins indicated that they were mostly membrane and extracellular proteins. Comparing results from adult female worms, adult male worms, and microfilariae, we find variability in N-glycosylation at the protein level as well as at the individual N-glycosite level. These variations are highlighted in cuticle N-glycoproteins and adult worm restricted N-glycoproteins as examples of proteins at the host parasite interface that are well positioned as potential therapeutic targets or biomarkers.

Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation in mammalian nuclear genome.

In mammalian cells, SET8 mediated Histone H4 Lys 20 monomethylation (H4K20me1) has been implicated in regulating mitotic condensation, DNA replication, DNA damage response, and gene expression. Here we show SET8, the only known enzyme for H4K20me1 is post-translationally poly ADP-ribosylated by PARP1 on lysine residues. PARP1 interacts with SET8 in a cell cycle-dependent manner. Poly ADP-ribosylation on SET8 renders it catalytically compromised, and degradation via ubiquitylation pathway. Knockdown of PARP1 led to an increase of SET8 protein levels, leading to aberrant H4K20me1 and H4K20me3 domains in the genome. H4K20me1 is associated with higher gene transcription levels while the increase of H4K20me3 levels was predominant in DNA repeat elements. Hence, SET8 mediated chromatin remodeling in mammalian cells are modulated by poly ADP-ribosylation by PARP1.

Polyglutamylation: biology and analysis.

Polyglutamylation is a posttranslational modification (PTM) that adds several glutamates on glutamate residues in the form of conjugated peptide chains by a family of enzymes known as polyglutamylases. Polyglutamylation is well documented in microtubules. Polyglutamylated microtubules consist of different α- and ß-tubulin subunits with varied number of added glutamate residues. Kinetic control and catalytic rates of tubulin modification by polyglutamylases influence the polyglutamylation pattern of functional microtubules. The recent studies uncovered catalytic mechanisms of the glutamylation enzymes family, particularly tubulin tyrosine ligase-like (TTLL). Variable length polyglutamylation of primary sequence glutamyl residues have been mapped with a multitude of protein chemistry and proteomics approaches. Although polyglutamylation was initially considered a tubulin-specific modification, the recent studies have uncovered a calmodulin-dependent glutamylase, SidJ. Nano-electrospray ionization (ESI) proteomic approaches have identified quantifiable polyglutamylated sites in specific substrates. Indeed, conjugated glutamylated peptides were used in nano-liquid chromatography gradient delivery due to their relative hydrophobicity for their tandem mass spectrometry (MS/MS) characterization. The recent polyglutamylation characterization has revealed three major sites: E445 in α-tubulin, E435 in ß-tubulin, and E860 in SdeA. In this review, we have summarized the progress made using proteomic approaches for large-scale detection of polyglutamylated peptides, including biology and analysis.

A Broad-Specificity O-Glycoprotease That Enables Improved Analysis of Glycoproteins and Glycopeptides Containing Intact Complex O-Glycans.

Characterization of mucin-type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constraints, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain types of glycoproteins, or protein sequence bias. These factors limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. Peptide sequence selectivity and its impact on IMPa activity were interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomic workflow for glycoprofiling of the purified glycoproteins granulocyte colony-stimulating factor and receptor-type tyrosine-protein phosphatase C without the need for glycopeptide enrichment. In these examples, IMPa enabled both the identification of O-glycosites and the range of complex O-glycan structures at each site.

Enhanced expression and purification of nucleotide-specific ribonucleases MC1 and Cusativin.

Combinations of ribonucleases (RNases) are commonly used to digest RNA into oligoribonucleotide fragments prior to liquid chromatography-mass spectrometry (LC-MS) analysis. The distribution of the RNase target sequences or nucleobase sites within an RNA molecule is critical for achieving a high mapping coverage. Cusativin and MC1 are nucleotide-specific endoribonucleases encoded in the cucumber and bitter melon genomes, respectively. Their high specificity for cytidine (Cusativin) and uridine (MC1) make them ideal molecular biology tools for RNA modification mapping. However, heterogenous recombinant expression of either enzyme has been challenging because of their high toxicity to expression hosts and the requirement of posttranslational modifications. Here, we present two highly efficient and time-saving protocols that overcome these hurdles and enhance the expression and purification of these RNases. We first purified MC1 and Cusativin from bacteria by expressing and shuttling both enzymes to the periplasm as MBP-fusion proteins in T7 Express lysY/IqE. coli strain at low temperature. The RNases were enriched using amylose affinity chromatography, followed by a subsequent purification via a C-terminal 6xHIS tag. This fast, two-step purification allows for the purification of highly active recombinant RNases significantly surpassing yields reported in previous studies. In addition, we expressed and purified a Cusativin-CBD fusion enzyme in P. pastoris using chitin magnetic beads. Both Cusativin variants exhibited a similar sequence preference, suggesting that neither posttranslational modifications nor the epitope-tags have a substantial effect on the sequence specificity of the enzyme.

Genome analysis of Salmonella enterica serovar Typhimurium bacteriophage L, indicator for StySA (StyLT2III) restriction-modification system action.

Bacteriophage L, a P22-like phage of Salmonella enterica sv Typhimurium LT2, was important for definition of mosaic organization of the lambdoid phage family and for characterization of restriction-modification systems of Salmonella. We report the complete genome sequences of bacteriophage L cI-40 13-am43 and L cII-101; the deduced sequence of wildtype L is 40,633 bp long with a 47.5% GC content. We compare this sequence with those of P22 and ST64T, and predict 72 Coding Sequences, 2 tRNA genes and 14 intergenic rho-independent transcription terminators. The overall genome organization of L agrees with earlier genetic and physical evidence; for example, no secondary immunity region (immI: ant, arc) or known genes for superinfection exclusion (sieA and sieB) are present. Proteomic analysis confirmed identification of virion proteins, along with low levels of assembly intermediates and host cell envelope proteins. The genome of L is 99.9% identical at the nucleotide level to that reported for phage ST64T, despite isolation on different continents ∼35 years apart. DNA modification by the epigenetic regulator Dam is generally incomplete. Dam modification is also selectively missing in one location, corresponding to the P22 phase-variation-sensitive promoter region of the serotype-converting gtrABC operon. The number of sites for SenLTIII (StySA) action may account for stronger restriction of L (13 sites) than of P22 (3 sites).

A bi-specific lectin from the mushroom Boletopsis grisea and its application in glycoanalytical workflows.

The BLL lectin from the edible Japanese "Kurokawa" mushroom (Boletopsis leucomelaena) was previously reported to bind to N-glycans harboring terminal N-acetylglucosamine (GlcNAc) and to induce apoptosis in a leukemia cell line. However, its gene has not been reported. In this study, we used a transcriptomics-based workflow to identify a full-length transcript of a BLL functional ortholog (termed BGL) from Boletopsis grisea, a close North American relative of B. leucomelaena. The deduced amino acid sequence of BGL was an obvious member of fungal fruit body lectin family (Pfam PF07367), a highly conserved group of mushroom lectins with a preference for binding O-glycans harboring the Thomsen-Friedenreich antigen (TF-antigen; Galß1,3GalNAc-α-) and having two ligand binding sites. Functional characterization of recombinant BGL using glycan microarray analysis and surface plasmon resonance confirmed its ability to bind both the TF-antigen and ß-GlcNAc-terminated N-glycans. Structure-guided mutagenesis of BGL's two ligand binding clefts showed that one site is responsible for binding TF-antigen structures associated with O-glycans, whereas the second site specifically recognizes N-glycans with terminal ß-GlcNAc. Additionally, the two sites show no evidence of allosteric communication. Finally, mutant BGL proteins having single functional bindings site were used to enrich GlcNAc-capped N-glycans or mucin type O-glycopeptides from complex samples in glycomics and glycoproteomics analytical workflows.

Mapping of polyglutamylation in tubulins using nanoLC-ESI-MS/MS.

Tubulin polyglutamylation is a polymeric modification that extends from the carboxyl-terminus of tubulins. Molecular description of amino acids and their branching polyglutamyls is a hallmark of tubulin in microtubules. There are different chemical approaches for detecting these polymeric structures, mostly reported prior to development of nESI peptide analysis. Here we demonstrate a novel and simple approach to detect shared regions of amino acid ions from tubulin polyglutamylated peptides in nanoLC-MS/MS. This involves two parallel in gel digestions with trypsin and subtilisin followed by mapping of di- and triglutamyl modifications of α- and ß-tubulins using a routine proteomics assay. We present three levels of information: i) identification of proteomics MS/MS data, ii) description of internal fragment ion series common across digests, and iii) extracted ion chromatograms mapped relative to retention time standards for confirmation of relative hydrophobicity values. Our nanoLC assay positive ion ESI detects up to 3 conjugated glutamates in tubulins. We implemented an analytical column only bottom up approach that characterizes molecular features of polyglutamylated tubulins.

Plasmid replication-associated single-strand-specific methyltransferases.

Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E. coli O104:H4. Pacific Biosciences SMRT sequencing was used to investigate DNA methyltransferases M.BceJIII and M.EcoGIX, using artificial constructs. Mating properties of engineered pESBL derivatives were also investigated. Both MTases yield promiscuous m6A modification of single strands, in the context SAY (where S = C or G and Y = C or T). Strikingly, this methylation is asymmetric in vivo, detected almost exclusively on one DNA strand, and is incomplete: typically, around 40% of susceptible motifs are modified. Genetic and biochemical studies suggest that enzyme action depends on replication mode: DNA Polymerase I (PolI)-dependent ColE1 and p15A origins support asymmetric modification, while the PolI-independent pSC101 origin does not. An MTase-PolI complex may enable discrimination of PolI-dependent and independent plasmid origins. M.EcoGIX helps to establish pESBL in new hosts by blocking the action of restriction enzymes, in an orientation-dependent fashion. Expression and action appear to occur on the entering single strand in the recipient, early in conjugal transfer, until lagging-strand replication creates the double-stranded form.

In-Vitro Sorbent-Mediated Removal of Edoxaban from Human Plasma and Albumin Solution.

BACKGROUND AND OBJECTIVE: Based on previous experience of sorbent-mediated ticagrelor, dabigatran, and radiocontrast agent removal, we set out in this study to test the effect of two sorbents on the removal of edoxaban, a factor Xa antagonist direct oral anticoagulant. METHODS: We circulated 100 mL of edoxaban solution during six first-pass cycles through 40-mL sorbent columns (containing either CytoSorb in three passes or Porapak Q 50-80 mesh in the remaining three passes) during experiments using human plasma and 4% bovine serum albumin solution as drug vehicles. Drug concentration was measured by liquid chromatography-tandem mass spectrometry. RESULTS: Edoxaban concentration in two experiments performed with human plasma dropped from 276.8 to 2.7 ng/mL and undetectable concentrations, respectively, with CytoSorb or Porapak Q 50-80 mesh (p = 0.0031). The average edoxaban concentration decreased from 407 ng/mL ± 216 ng/mL to 3.3 ng/mL ± 7 ng/mL (p = 0.017), for a removal rate of 99% across all six samples of human plasma (two samples) and bovine serum albumin solution (four samples). In four out of the six adsorbed samples, the drug concentrations were undetectable. CONCLUSION: Sorbent-mediated technology may represent a viable pathway for edoxaban removal from human plasma or albumin solution.

The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin.

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein-protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311 Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.

Computational and experimental analysis of the glycophosphatidylinositol-anchored proteome of the human parasitic nematode Brugia malayi.

Further characterization of essential systems in the parasitic filarial nematode Brugia malayi is needed to better understand its biology, its interaction with its hosts, and to identify critical components that can be exploited to develop novel treatments. The production of glycophosphatidylinositol-anchored proteins (GPI-APs) is essential for eukaryotic cellular and physiological function. In addition, GPI-APs perform many important roles for cells. In this study, we characterized the B. malayi GPI-anchored proteome using both computational and experimental approaches. We used bioinformatic strategies to show the presence or absence of B. malayi GPI-AP biosynthetic pathway genes and to compile a putative B. malayi GPI-AP proteome using available prediction programs. We verified these in silico analyses using proteomics to identify GPI-AP candidates prepared from the surface of intact worms and from membrane enriched extracts. Our study represents the first description of the GPI-anchored proteome in B. malayi and lays the groundwork for further exploration of this essential protein modification as a target for novel anthelmintic therapeutic strategies.

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance.

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication. SAHH enhances DNMT1 activity in vitro, and its overexpression in mammalian cells led to hypermethylation of the genome, whereas its inhibition by adenosine periodate or siRNA-mediated knockdown resulted in hypomethylation of the genome. Hypermethylation was consistent in both gene bodies and repetitive DNA elements leading to aberrant gene regulation. Cells overexpressing SAHH specifically up-regulated metabolic pathway genes and down-regulated PPAR and MAPK signaling pathways genes. Therefore, we suggest that alteration of SAHH level affects global DNA methylation levels and gene expression.

An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides.

A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.

Ticagrelor Removal From Human Blood.

The authors devised an efficient method for ticagrelor removal from blood using sorbent hemadsorption. Ticagrelor removal was measured in 2 sets of in vitro experiments. The first set was a first-pass experiment using bovine serum albumin (BSA) solution pre-incubated with ticagrelor, whereas the second set, performed in a recirculating manner, used human blood mixed with ticagrelor. Removal of ticagrelor from BSA solution reached values >99%. The peak removal rate was 99% and 94% from whole blood and 99.99% and 90% from plasma during 10 h and 3 to 4 h of recirculating experiments, respectively. In conclusion, hemadsorption robustly removes ticagrelor from BSA solution and human blood samples.

Profiling of core fucosylated N-glycans using a novel bacterial lectin that specifically recognizes α1,6 fucosylated chitobiose.

A novel fucose-binding lectin (SL2-1) from the bacterium Streptomyces rapamycinicus was identified by analysis of metagenomic DNA sequences. SL2-1 belongs to a new group of bacterial fucose-specific lectins that have no similarity to known bacterial fucose-binding proteins, but are related to certain eukaryotic fucose-binding lectins. The 17 kDa protein was expressed recombinantly in E. coli and purified by affinity chromatography. Glycan microarray analysis with fluorescently labeled recombinant SL2-1 demonstrated its ability to bind to core α1-6 fucosylated N-glycans, but not to core α1-3 fucosylated N-glycans, or other α1-2, α1-3 and α1-4 fucosylated oligosaccharides. The minimal high affinity binding epitope of SL2-1 was α1-6 fucosylated di-n-acetylchitobiose. The recombinant lectin was efficient in detection of N-glycan core fucosylation using lectin blotting and lectin ELISA assays. Finally, a workflow using SL2-1 for selective and quantitative profiling of core fucosylated N-glycans using UPLC-HILIC-FLR analysis was established. The approach was validated for selective capture and analysis of core fucosylated N-glycans present in complex glycan mixtures derived from mammalian serum IgG.

Differential connectivity of splicing activators and repressors to the human spliceosome.

BACKGROUND: During spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions. RESULTS: Here, we investigate the protein connectivity of SR and hnRNP proteins to the core spliceosome using probabilistic network reconstruction based on the integration of interactome and gene expression data. We validate our model by immunoprecipitation and mass spectrometry of the prototypical splicing factors SRSF1 and hnRNPA1. Network analysis reveals that a factor's properties as an activator or repressor can be predicted from its overall connectivity to the rest of the spliceosome. In addition, we discover and experimentally validate PPIs between the oncoprotein SRSF1 and members of the anti-tumor drug target SF3 complex. Our findings suggest that activators promote the formation of PPIs between spliceosomal sub-complexes, whereas repressors mostly operate through protein-RNA interactions. CONCLUSIONS: This study demonstrates that combining in-silico modeling with biochemistry can significantly advance the understanding of structure and function relationships in the human spliceosome.

A tool to evaluate correspondence between extraction ion chromatographic peaks and peptide-spectrum matches in shotgun proteomics experiments.

Chromatographed peptide signals form the basis of further data processing that eventually results in functional information derived from data-dependent bottom-up proteomics assays. We seek to rank LC/MS parent ions by the quality of their extracted ion chromatograms. Ranked extracted ion chromatograms act as an intuitive physical/chemical preselection filter to improve the quality of MS/MS fragment scans submitted for database search. We identify more than 4900 proteins when considering detector shifts of less than 7 ppm. High quality parent ions for which the database search yields no hits become candidates for subsequent unrestricted analysis for PTMs. Following this rational approach, we prioritize identification of more than 5000 spectrum matches from modified peptides and confirmed the presence of acetylaldehyde-modified His/Lys. We present a logical workflow that scores data-dependent selected ion chromatograms and leverage information about semianalytical LC/LC dimension prior to MS. Our method can be successfully used to identify unexpected modifications in peptides with excellent chromatography characteristics, independent of fragmentation pattern and activation methods. We illustrate analysis of ion chromatograms detected in two different modes by RF linear ion trap and electrostatic field orbitrap.

CK2 phosphorylation inactivates DNA binding by the papillomavirus E1 and E2 proteins.

Papillomaviruses have complex life cycles that are understood only superficially. Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood. Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity. Phosphorylation at multiple sites in the N-terminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins. The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity. Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication. In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication. We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1.