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Biomedical subjects

Julie A Leary

Publications and source records attributed to Julie A Leary.

At least 19 recordsLinked to original sources
PubMed2006

Detection and purification of tyrosine-sulfated proteins using a novel anti-sulfotyrosine monoclonal antibody.

Protein tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independently of sequence context. We show that it can detect tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2(-/-) mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2(-/-) males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms.

Animals↗
PubMed2006

Substrate recognition, protein dynamics, and iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5'-phosphosulfate reductase.

APS reductase catalyzes the first committed step of reductive sulfate assimilation in pathogenic bacteria, including Mycobacterium tuberculosis, and is a promising target for drug development. We report the 2.7 A resolution crystal structure of Pseudomonas aeruginosa APS reductase in the thiosulfonate intermediate form of the catalytic cycle and with substrate bound. The structure, high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, and quantitative kinetic analysis, establish that the two chemically discrete steps of the overall reaction take place at distinct sites on the enzyme, mediated via conformational flexibility of the C-terminal 18 residues. The results address the mechanism by which sulfonucleotide reductases protect the covalent but labile enzyme-intermediate before release of sulfite by the protein cofactor thioredoxin. P. aeruginosa APS reductase contains an [4Fe-4S] cluster that is essential for catalysis. The structure reveals an unusual mode of cluster coordination by tandem cysteine residues and suggests how this arrangement might facilitate conformational change and cluster interaction with the substrate. Assimilatory 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductases are evolutionarily related, homologous enzymes that catalyze the same overall reaction, but do so in the absence of an [Fe-S] cluster. The APS reductase structure reveals adaptive use of a phosphate-binding loop for recognition of the APS O3' hydroxyl group, or the PAPS 3'-phosphate group.

Amino Acid Sequence↗
PubMed2006

Fatty acid analysis tool (FAAT): An FT-ICR MS lipid analysis algorithm.

Electrospray ionization mass spectrometry is becoming an established tool for the investigation of lipids. As the methods for lipid analysis become more mature and their throughput increases, computer algorithms for the interpretation of such data will become a necessity. Toward this end, an algorithm dedicated to the analysis of Fourier transform mass spectral data from lipid extracts has been developed. The algorithm, Fatty Acid Analysis Tool, termed FAAT, has been successfully used to investigate complex lipid extracts containing thousands of components, from various species of mycobacteria including M. tuberculosis and M. abscessus. FAAT is rapid, generally taking tens of seconds to interpret multiple spectra, and accessible to most users as it is implemented in Microsoft Excel Visual Basic Software. In the reduction of data, FAAT begins by scaling spectra (i.e., to account for dilution factors), identifying monoisotopic ions, and assigning isotope packets. Unique features of FAAT include the following: (1) overlapping saturated and unsaturated lipid species can be distinguished, (2) known ions are assigned from a user-defined library including species that possess methylene heterogeneity, (3) and isotopic shifts from stable isotope labeling experiments are identified and assigned (up to a user-defined maximum). In addition, abundance differences between samples grown under normal and stressed conditions can be determined. In the analysis of mycobacterial lipid extracts, FAAT has successfully identified isotopic shifts from incorporation of 15N in M. abscessus. Additionally, FAAT has been used to successfully determine differences in lipid abundances between M. tuberculosis wild-type and mutant strains.

Algorithms↗
PubMed2006

N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry.

Intercellular adhesion molecule-1 (ICAM-1) is a heavily N-glycosylated transmembrane protein comprising five extracellular Ig-like domains. The soluble isoform of ICAM-1 (sICAM-1), consisting of its extracellular part, is elevated in the cerebrospinal fluid of patients with severe brain trauma. In mouse astrocytes, recombinant mouse sICAM-1 induces the production of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 induction is glycosylation dependent, as it is strongly enhanced when sICAM-1 carries sialylated, complex-type N-glycans as synthesized by wild-type Chinese hamster ovary (CHO) cells. The present study was aimed at elucidating the N-glycosylation of mouse sICAM-1 expressed in wild-type CHO cells with regard to sialylation, N-glycan profile, and N-glycosylation sites. Ion-exchange chromatography and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the released N-glycans showed that sICAM-1 mostly carried di- and trisialylated complex-type N-glycans with or without one fucose. In some sialylated N-glycans, one N-acetylneuraminic acid was replaced by N-glycolylneuraminic acid, and approximately 4% carried a higher number of sialic acid residues than of antennae. The N-glycosylation sites of mouse sICAM-1 were analyzed by MALDI-Fourier transform ion cyclotron resonance (FTICR)-MS and nanoLC-ESI-FTICR-MS of tryptic digests of mouse sICAM-1 expressed in the Lec1 mutant of CHO cells. All nine consensus sequences for N-glycosylation were found to be glycosylated. These results show that the N-glycans that enhance the MIP-2-inducing activity of mouse sICAM-1 are mostly di- and trisialylated complex-type N-glycans including a small fraction carrying more sialic acid residues than antennae and that the nine N-glycosylation sites of mouse sICAM-1 are all glycosylated.

Animals↗
PubMed2006

Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding.

Despite the wide range of sequence diversity among chemokines, their tertiary structures are remarkably similar. Furthermore, many chemokines form dimers or higher order oligomers, but all characterized oligomeric structures are based primarily on two dimerization motifs represented by CC-chemokine or CXC-chemokine dimer interfaces. These observations raise the possibility that some chemokines could form unique hetero-oligomers using the same oligomerization motifs. Such interactions could modulate the overall signaling response of the receptors, thereby providing a general mechanism for regulating chemokine function. For some chemokines, homo-oligomerization has also been shown to be coupled to glycosaminoglycan (GAG)-binding. However, the effect of GAG binding on chemokine hetero-oligomerization has not yet been demonstrated. In this report, we characterized the heterodimerization of the CCR2 ligands MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), and eotaxin (CCL11), as well as the effects of GAG binding, using electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Strong heterodimerization was observed between CCL2 and CCL8 at the expense of homodimer formation. Using NMR, we showed that the heterodimer is predominant in solution and forms a specific CC chemokine-like dimer. By contrast, only moderate heterodimer formation was observed between CCL2.CCL13, CCL2.CCL11 and CCL8.CCL13, and no heterodimerization was observed when any other CCR2 ligand was added to CCL7. To investigate the effect of a highly sulfated GAG on the formation of heterodimers, each chemokine pair was mixed with the heparin pentasaccharide, Arixtra, and assayed by ESI-FTICR mass spectrometry. Although no CCL8.CCL11 heterodimer was observed in the absence of GAG, abundant ions corresponding to the ternary complex, CCL8.CCL11.Arixtra, were observed upon addition of Arixtra. Heterodimerization between CCL2 and CCL11 was also enhanced in the presence of Arixtra. In summary, these results indicate that some CCR2 ligands can form stable heterodimers in preference to homodimers and that these interactions, like those of homo-oligomers, can be influenced by some GAGs.

Chemokine CCL8↗
PubMed2006

Effects of sulfate position on heparin octasaccharide binding to CCL2 examined by tandem mass spectrometry.

Chemokine-glycosaminoglycan (GAG) interactions have been shown to be essential for in vivo chemokine signaling, which functions in such diverse processes as inflammation, development, and cancer metastasis. Despite the importance of these interactions, the saccharide sequence dependency of chemokine-GAG interactions is poorly understood. In a recent study, FT-ICR mass spectrometry was used to show that the chemokine CCL2 (monocyte chemoattractant protein 1) binds only to the 11- and 12-sulfated components of a heparin octasaccharide library. Although the exact structure of the fully sulfated, 12-sulfated octasaccharide is known, the 11-sulfated species could have a number of sulfated disaccharide sequences. In the current study, the composition of the 11-sulfated heparin octasaccharides, as well as the composition of CCL2 affinity purified 11-sulfated heparin octasaccharides, were examined by tandem MS. Of the three possible singly desulfated disaccharides, one species, III-S, is enriched by CCL2 affinity purification, indicating that the 11-sulfated heparin octasaccharides containing this disaccharide are preferentially bound to CCL2. These data suggest that 2-O and N sulfation of heparin may be of greater importance to CCL2-heparin binding than 6-O sulfation.

Binding Sites↗
PubMed2006

A sulfated metabolite produced by stf3 negatively regulates the virulence of Mycobacterium tuberculosis.

Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells. Mycobacterium tuberculosis, the causative agent of tuberculosis, produces sulfated molecules that have eluded functional characterization for decades. We demonstrate here that a previously uncharacterized sulfated molecule, termed S881, is localized to the outer envelope of M. tuberculosis and negatively regulates the virulence of the organism in two mouse infection models. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate and a previously uncharacterized sulfotransferase, stf3. These findings extend the known functions of sulfated molecules as general modulators of cell-cell interactions to include those between a bacterium and a human host.

Animals↗
PubMed2006

Potential inhibitors of chemokine function: analysis of noncovalent complexes of CC chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry.

Chemokines play a critical role in inducing chemotaxis, extravasation, and activation of leukocytes both in routine immunosurveillance and autoimmune diseases. Traditionally, to disrupt chemokine function, strategies have focused on blockage of its interaction with the receptor. Recently, it has been demonstrated that binding to glycosaminoglycans (GAGs) is also required for the in vivo activity of many chemokines. Thus, interference with the GAG-binding of chemokines may offer an alternative, valid, anti-inflammatory strategy. However, the potential of using small polyanions to inhibit the interactions between chemokines and cell surface GAGs has not been fully explored. In this study, a mass spectrometry based filtration trapping assay was utilized to study the interactions between two CCR 2 ligands (MCP-1/CCL2 and MCP-3/CCL7) and a series of low molecular weight, polyanionic molecules. Findings were confirmed by using a hydrophobic trapping assay. The results indicated that Arixtra (fondaparinux sodium), sucrose octasulfate, and suramin were specific binders of the chemokines, while cyclodextrin sulfate, although the most highly sulfated molecule among the ones investigated, showed no binding. The binding stoichiometry of the small molecule ligand was determined from the measured molecular weight of the noncovalent complex. Furthermore, the dissociation constant between MCP-3 and Arixtra was determined by using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, which compared favorably with the result of the isothermal titration calorimetry (ITC) assay. The relative binding affinity of these ligands to MCP-3 was also determined using a competitive filtration trapping assay.

Calorimetry↗
PubMed2005

Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: implications for substrate binding and catalysis.

The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron-sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron-sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster.

Adenosine Monophosphate↗
PubMed2005

Analysis of hyaluronan content in chondroitin sulfate preparations by using selective enzymatic digestion and electrospray ionization mass spectrometry.

Two important glycosaminoglycans (GAGs) with structural roles in the body's cartilage are hyaluronan (HA) and chondroitin sulfate (CS). A simple mass spectrometric method for determining the amount of HA that may be present in isolated CS samples is presented in this article. Samples are subjected to selective enzymatic digestion using a bacterial hyaluronidase (HA lyase, EC 4.2.2, from Streptococcus dysgalactiae) specific for HA. Undigested CS GAG is then removed by centrifugal filtration, and digested HA left in the filtrate is quantified by electrospray ionization mass spectrometry using an internal standard and selected ion monitoring. The described method was applied to the analysis of several CS samples prepared for use in nutritional supplements.

Carbohydrate Sequence↗
PubMed2005

Heparin sequencing using enzymatic digestion and ESI-MSn with HOST: a heparin/HS oligosaccharide sequencing tool.

Mass spectrometry, and specifically sequential stages of mass spectrometry (MSn), is an established tool for the analysis of carbohydrates, proteins, and more recently glycosaminoglycans. As this trend continues, the development of algorithms for the rapid and automatic interpretation of mass spectra to identify glycan structure is also expected to grow as an active field of research. The methodology described herein utilizes a combination of enzymatic digestion, ESI-MS, and MSn for the sequencing of small heparin oligosaccharides. The heparin oligosaccharide sequencing tool (HOST) is a basic software application that was developed to aid in the integration and analysis of the data generated from these experiments, facilitating the process involved in arriving at sequence information. The sequences of several heparin oligosaccharides were determined using this method to illustrate proof of principle. Tandem MS is a very rapid and efficient tool for oligosaccharide analysis when limited amounts of material are available. Having a means, such as HOST, for automating the interpretation of the MSn data generated from glycosaminoglycans, provides a practical methodology for the future analysis of heparin/HS oligosaccharides of unknown structure.

Amino Acid Sequence↗
PubMed2005

Mechanism and kinetics of metalloenzyme phosphomannose isomerase: measurement of dissociation constants and effect of zinc binding using ESI-FTICR mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was used to study the noncovalent complexation of a metalloenzyme, phosphomannose isomerase (PMI), which catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The zinc cofactor binding effect and the noncovalent interactions of the holoenzyme with its two natural substrates and two inhibitors, erythrose 4-phosphate and mannitol 1-phosphate, were investigated. Under nondenaturing conditions, the intact zinc-containing monomeric protein ions were reproducibly observed with no dissociation. Molecular ions corresponding to apo-PMI monomer were obtained by dialyzing the holoenzyme against EDTA. The binding/release of the metal ion did not alter the charge-state distributions of the protein to any significant extent, but changed the binding affinity of the substrates by at least 5-fold. Using ESI-FTICR mass spectrometry, the binding stoichiometry and specificity of the enzyme-substrate and enzyme-inhibitor complexes were directly determined. The first time report of the apparent dissociation constant for the isomeric substrates of PMI was measured to be 88.8 microM. The relative dissociation constant of the two inhibitors derived from gas-phase noncovalent complexation was very similar to the relative inhibition constant derived from solution phase kinetics.

Enzyme Inhibitors↗
PubMed2005

Chemokine-glycosaminoglycan binding: specificity for CCR2 ligand binding to highly sulfated oligosaccharides using FTICR mass spectrometry.

Glycosaminoglycans (GAGs) have recently been demonstrated to be required for the in vivo activity of several chemokines. Minimally, the interaction is thought to provide a mechanism for retention at the site of secretion and the formation of chemokine gradients that provide directional cues for receptor bearing cells, particularly in the presence of shear forces. Thus, a key issue will be to determine the sequence and structure of the GAGs that bind to specific chemokines. Herein, we describe a mass spectrometry assay that was developed to detect protein-oligosaccharide noncovalent complexes, in this case chemokine-GAG interactions, and to select for high affinity GAGs. The process is facilitated by the ability of electrospray ionization to transfer the intact noncovalent complexes from solution into the gas phase. The elemental composition as well as the binding stoichiometry can be calculated from the mass of the complex. Ligands of the chemokine receptor, CCR2 (MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13, and Eotaxin/CCL11), and the CCR10 ligand CTACK/CCL27 were screened against a small, highly sulfated, heparin oligosaccharide library with limited structural variation. The results revealed heparin octasaccharides with 11 and 12 sulfates as binders. Oligomerization of some chemokines was observed upon GAG binding, whereas in other instances only the monomeric noncovalent complex was identified. The results indicate that, in contrast to the apparent redundancy in the chemokine system, where several chemokines bind and activate the same receptor, these chemokines could be differentiated into two groups based on the stoichiometry of their complexes with the heparin oligosaccharides.

Animals↗
PubMed2005

A conserved mechanism for sulfonucleotide reduction.

Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3-)) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor.

Adenosine Phosphosulfate↗
PubMed2005

Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry.

Mycobacterial carbohydrate sulfotransferase Stf0 catalyzes the sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to trehalose. The sulfation of trehalose is required for the biosynthesis of sulfolipid-1, the most abundant sulfated metabolite found in Mycobacterium tuberculosis. In this paper, an efficient enzyme kinetics assay for Stf0 using electrospray ionization (ESI) mass spectrometry is presented. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfuryl group transfer reaction was investigated in initial rate kinetics and product inhibition experiments. In addition, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was employed to detect the noncovalent complexes, the Stf0-PAPS and Stf0-trehalose binary complexes, and a Stf0-3'-phosphoadenosine 5'-phosphate-trehalose ternary complex. The results from our study strongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for Stf0 with formation of a ternary complex intermediate. In this mechanism, PAPS and trehalose bind and their products are released in random fashion. To our knowledge, this is the first detailed mechanistic data reported for Stf0, which further demonstrates the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.

Adenosine Diphosphate↗
PubMed2005

Mass spectrometric analysis of the human 40S ribosomal subunit: native and HCV IRES-bound complexes.

Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap-independent translation initiation. Although IRES-mediated translation initiation is not subject to many of the regulatory mechanisms that control cap-dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES-bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top-down and bottom-up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty-one out of a possible 32 ribosomal proteins were identified by combining top-down and bottom-up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES-bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES-mediated translation initiation complex.

Hepacivirus↗
PubMed2005

Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase.

An important class of carbohydrates studied within the field of glycobiology, heparin and heparan sulfate (HS) have been implicated in a diverse array of biological functions. Changes in their sulfation pattern and domain organization have been associated with different pathological situations such as viral infectivity, tumor growth, and metastasis. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. The method was applied to assay for the heparin substrate specificity of a newly discovered human extracellular endosulfatase, HSulf-2, which has been implicated in tumorigenesis. This new protocol incorporates 12 known heparin disaccharides, including three sets of isomers. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues.

Animals↗