Mark Nitz

Study of Cell Antigens and Intracellular DNA by Identification of Element-Containing Labels and Metallointercalators Using Inductively Coupled Plasma Mass Spectrometry

The enumeration of absolute cell numbers and cell proliferation in clinical samples is important for diagnostic and research purposes. Detection of cellular DNA with fluorescent dyes is the most commonly used approach for cell enumeration in cytometry. Inductively coupled plasma mass spectrometry (ICPMS) has been recently introduced to the field of protein and cell surface antigen identification via ICPMS-linked immunoassays using element-labeled affinity reagents such as gold and lanthanide-conjugated antibodies. In the present work, we describe novel methods for using metallointercalators that irreversibly bind DNA and low concentrations of rare earth metals added to cell growth media for rapid and sensitive measurement of cell numbers by mass spectrometry. We show that Ir- and Rh-containing metallointercalators are useful reagents for labeling cells and normalizing signals when studying antigen expression on different types and numbers of cells. Results are presented for solution analysis performed by conventional ICPMS and compared to measurements obtained on the novel flow cytometer mass spectrometer (FC-MS) instrument, designed to analyze multiple antigens and DNA simultaneously in single cells.

Flow cytometer with mass spectrometer detection for massively multiplexed single-cell biomarker assay

This paper describes the development and application of new metal-tagging reagents and a novel mass spectrometer (MS) detector for a flow cytometer that enables highly multiplexed measurement of many biomarkers in individual cells. A new class of tagging reagents, based on an acrylic polymer backbone that incorporates a reproducible number of lanthanide elements, has been developed. When linked to antibodies that specifically recognize target proteins of interest, determination of the tag elements is diagnostic for the presence and quantification of the antigen. The use of enriched stable isotope tags provides the opportunity for multiparametric assay. The new instrument uses inductively coupled plasma (ICP) to vaporize, atomize, and ionize individual cells that have been probed using the metal-labeled antibodies. The elemental composition, specifically of the metal tags, is recorded simultaneously using a time-of-flight (TOF)-MS that has been specifically designed for high-speed analysis during the short transient corresponding to the individual cell event. The detector provides for well-resolved atomic fingerprints of many elemental and isotopic tags, with little overlap of neighboring signals (high abundance sensitivity) and wide dynamic range both for a single antigen and between antigens.

Synthesis of a functional metal-chelating polymer and steps toward quantitative mass cytometry bioassays.

We describe the synthesis and characterization of metal-chelating polymers with a degree of polymerization of 67 and 79, high diethylenetriaminepentaacetic acid (DTPA) functionality, M(w)/M(n) ≤ 1.17, and a maleimide as an orthogonal functional group for conjugation to antibodies. The polymeric disulfide form of the DP(n) = 79 DTPA polymer was analyzed by thermogravimetric analysis to determine moisture and sodium-ion content and by isothermal titration calorimetry (ITC) to determine the Gd(3+) binding capacity. These results showed each chain binds 68 ± 7 Gd(3+) per chain. Secondary goat antimouse IgG was covalently labeled with the maleimide form of the DTPA polymer (DP(n) = 79) carrying (159)Tb. Conventional ICPMS analysis of this conjugate showed each antibody carried an average of 161 ± 4 (159)Tb atoms. This result was combined with the ITC result to show there are an average of 2.4 ± 0.3 polymer chains attached to each antibody. Eleven monoclonal primary antibodies were labeled with different lanthanide isotopes using the same labeling methodology. Single cell analysis of whole umbilical cord blood stained with a mixture of 11 metal-tagged antibodies was performed by mass cytometry.

Pattern-based recognition of heparin contaminants by an array of self-assembling fluorescent receptors.

Tracking down potential killers: Strong host-guest interactions enable the facile combination of polycationic cyclodextrin binding motifs (blue) with fluorescent reporters (orange) tethered to a hydrophobic guest molecule (dark green). An array of supramolecular fluorescent receptors prepared by this modular approach was used for the pattern-based recognition of negatively charged contaminants in the anticoagulant drug heparin.

Designing fluorescent sensors of heparin.

Glycosaminoglycans (GAGs) are a critically important class of linear anionic polysaccharides that are implicated in intercellular communication pathways as diverse as chemokine signaling and pathogen recognition. While recent advances in analytical techniques have allowed the determination of GAG sulfation patterns and the interrogation of GAG–protein interactions, research into the numerous biological roles of GAGs would be greatly facilitated by access to fluorescent sensors capable of real-time, accurate, and specific measurements of GAG concentrations in cellular systems. Our proposed design for such a sensor is based on the observation that the predominant driving force in protein–GAG interactions is the polyelectrolyte effect. During a GAG-binding event under physiological conditions, the anions (predominately chloride) associated with the cationic GAG binding site are displaced by the incoming polyanionic GAG (Scheme 1). By installing a halide-sensitive fluorophore within a GAG binding site, we aim to develop a generalizable approach to the synthesis of GAG sensors that rely upon the displacement of chloride ions to signal a GAG-association event. The 6-methoxyquinolinium fluorophore has been used as an efficient sensor of chloride ions in cellular assays, as it undergoes dynamic quenching with halide ions and is insensitive to many other common biological anions. Thus, correct positioning of the 6methoxyquinolinium fluorophore within a GAG binding motif will provide a sensor in which the quenching mechanism is disrupted upon GAG association (Scheme 1). Heparin is a highly sulfated GAG that has been used extensively in anticoagulant therapy for over 40 years due to its interaction with the proteins involved in the blood-clotting cascade. During heparin anticoagulant therapy it is crucial that heparin levels are closely monitored to avoid complications. Many assays for heparin monitoring have been established. The most commonly used clinical assays include activated clotting time (ACT) and antifactor Xa assays, both of which are indirect methods. It is widely known that these assays can correlate poorly to the actual blood heparin levels because of their lack of specificity and potential interference from other factors. 9] Direct assays based on potentiometry and ELISA with large protein fusions have recently been established and might prove to be useful in the clinic. Our sensor design provides a novel fluorescent approach to directly detecting heparin. Heparan is biosynthesized along the same pathway as heparin, but, unlike heparin, it remains attached to a core protein. Heparan has a domain structure that is made up of highly sulfated regions, which closely resemble the repeating unit in heparin, separated by regions with low degrees of sulfation. A fluorescent sensor for heparan would enable experiments to follow the localization and concentration changes of the sulfated domains of heparan with the aid of fluorescence microscopy in real-time settings. Access to a specific fluorescent heparan sensor could also potentially lead to assays that directly interrogate the roles of heparan in processes such as immune regulation or neuron growth. Recently, the first example of a fluorescent heparin sensor, which relies on a tripodal boronic acid motif that decreases in fluorescence upon heparin binding, was reported. Here we communicate our efforts towards the development of new fluorescent GAG sensors that are based on a previously unexplored chloride-dependent sensing mechanism. This approach will lead to sensors that increase their fluorescence signal upon heparin or heparan binding and that are amenable to application under physiological ionic strength. As a proof of principle, a 6-methoxyquinolinium-containing amino acid was synthesized to allow for facile incorporation of this fluorophore within a heparin binding peptide motif. The selectively protected quinolinium-based fluorescent amino acid (FmocQuin, 1) was synthesized from the commercially available protected aspartic acid (FmocAspOtBu) derivative in five steps (Scheme 2). The halide-sensitive fluorescent amino acid (1) was introduced into the heparin-specific peptide sequence AG73 from the laminin a1 chain (Scheme 2). Previously, the AG73 peptide had been shown to bind to heparin with micromolar affinity. Amino acid 1 was inserted at position 6 of the peptide sequence to produce AGf73, since alanine-scanning mutagenesis had demonstrated that mutation at Val6 minimally perturbs heparin binding in the AG73 sequence. The peptide was syn[a] J. C. Sauceda, R. M. Duke, Dr. M. Nitz Department of Chemistry, University of Toronto 80 St. George Street, Toronto, Ontario M5S 3H6 (Canada) Fax: (+1)416-946-0640 E-mail : mnitz@chem.utoronto.ca Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author: experimental procedures for the peptide synthesis and fluorescence measurements as well as the H NMR spectra of compounds 1 and 4 are available. Scheme 1. Changes in the counter ions around the quinolinium sensor lead to a dramatic increase in fluorescence intensity upon heparin association.

The structure and metal dependent activity of Escherichia coli PgaB provides insight into the partial de-N-acetylation of poly-β-1,6-N-acetyl-D-glucosamine

Background: Polysaccharide intercellular adhesin-dependent biofilm formation in E. coli requires the de-N-acetylation of poly-β-1,6-N-acetyl-d-glucosamine by PgaB. Results: Nickel- and iron-bound structures of PgaB have been determined, and the metal-dependent de-N-acetylase activity of the enzyme has been characterized. Conclusion: PgaB has low catalytic efficiency and shows preference for Co2+, Ni2+, and Fe2+ ions. Significance: The structure of PgaB will guide inhibitor design to combat biofilm formation. Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni2+ and Fe3+ have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)x barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)4 oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co2+ and Ni2+ under aerobic conditions, and Co2+, Ni2+ and Fe2+ under anaerobic conditions, but decreased activity with Zn2+. The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.

Small molecule β-amyloid inhibitors that stabilize protofibrillar structures in vitro improve cognition and pathology in a mouse model of Alzheimer’s disease

β‐Amyloid (Aβ) peptides are thought to play a major role in the pathogenesis of Alzheimer’s disease. Compounds that disrupt the kinetic pathways of Aβ aggregation may be useful in elucidating the role of oligomeric, protofibrillar and fibrillar Aβ in the etiology of the disease. We have previously reported that scyllo‐inositol inhibits Aβ42 fibril formation but the mechanism(s) by which this occurs has not been investigated in detail. Using a series of scyllo‐inositol derivatives in which one or two hydroxyl groups were replaced with hydrogen, chlorine or methoxy substituents, we examined the role of hydrogen bonding and hydrophobicity in the structure–function relationship of scyllo‐inositol–Aβ binding. We report here that all scyllo‐inositol derivatives demonstrated reduced effectiveness in preventing Aβ42 fibrillization compared with scyllo‐inositol, suggesting that scyllo‐inositol interacts with Aβ42 via key hydrogen bonds that are formed by all hydroxyl groups. Increasing the hydrophobicity of scyllo‐inositol by the addition of two methoxy groups (1,4‐di‐O‐methyl‐scyllo‐inositol) produced a derivative that stabilized Aβ42 protofibrils in vitro. Prophylactic administration of 1,4‐di‐O‐methyl‐scyllo‐inositol to TgCRND8 mice attenuated spatial memory impairments and significantly decreased cerebral amyloid pathology. These results suggest that Aβ aggregation can be targeted at multiple points along the kinetic pathway for the improvement of Alzheimer’s disease‐like pathology.

Modulation of amyloid‐β aggregation and toxicity by inosose stereoisomers

Amyloid‐β (Aβ) aggregation and amyloid formation are key pathological features of Alzheimer’s disease, and are considered to be two of the major contributing factors to neurodegeneration and dementia. Identification of small molecule inhibitors that are orally available, have low toxicity and high central nervous system bioavailability is one approach to the potential development of a disease‐modifying treatment for Alzheimer’s disease. We have previously identified inositol stereoisomers as exhibiting stereospecific inhibition of Aβ aggregation and toxicity in vitro and in vivo. We report here the effects of inosose versus inositol stereoisomers on Aβ fibrillogenesis as determined using CD and fluorescence spectroscopy and negative‐stain electron microscopy. The inososes differ from inositols by the oxidation of one of the hydroxyl groups to a ketone. These molecules help in the further elucidation of the structure–activity relationships of inositol–Aβ interactions and identify both allo‐inositol and epi‐2‐inosose as in vitro inhibitors of Aβ aggregation.

Enantioselective synthesis and application of the highly fluorescent and environment-sensitive amino acid 6-(2-dimethylaminonaphthoyl) alanine (DANA)Electronic supplementary information (ESI) available: experimental details. See http://www.rsc.org/suppdata/cc/b2/b205224e/

6-(2-Dimethylaminonaphthoyl) alanine (DANA) was prepared via an enantioselective synthesis and incorporated into the S-peptide of RNase S establishing the large changes in fluorescence that can occur upon peptide-protein interaction.

Characterization of the poly-β-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms.

ABSTRACT We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.