4 research outputs found
Detection of Cellular Sialic Acid Content Using Nitrobenzoxadiazole Carbonyl-Reactive Chromophores
The selective ligation of hydrazine and amino-oxy compounds
with
carbonyls has gained popularity as a detection strategy with the recognition
of aniline catalysis as a way to accelerate the labeling reaction
in water. Aldehydes are a convenient functional group choice since
there are few native aldehydes found at the cell surface. Aldehydes
can be selectively introduced into sialic acid containing glycoproteins
by treatment with dilute sodium periodate. Thus, the combination of
periodate oxidation with aniline-catalyzed ligation (PAL) has become
a viable method for detection of glycoconjugates on live cells. Herein
we examine two fluorescent nitrobenzoxadiazole dyes for labeling of
glycoproteins and cell surface glycoconjugates. We introduce a novel
4-aminooxy-7-nitro-benz-[2,1,3-<i>d</i>]-oxadiazole (NBDAO)
(<b>5</b>) fluorophore, and offer a comparison to commercial
dyes including the known 4-hydrazino-7-nitro-benz-[2,1,3-<i>d</i>]-oxadiazole (NBDH) (<b>2</b>) and Bodipy FL hydrazide. We
confirm specificity for sialic acid moieties and that both dyes are
suitable for in vitro and in vivo labeling studies using PAL and fluorescence
spectroscopy. The dyes examined here are attractive labeling agents
for microscopy, as they can be excited by a 488 nm laser line and
can be made in a few synthetic steps. These carbonyl-reactive chromophores
provide a one step alternative to avidinābiotin labeling strategies
and simplify the detection of sialic acid in cells and glycoproteins
Synergic āClickā Boronate/Thiosemicarbazone System for Fast and Irreversible Bioorthogonal Conjugation in Live Cells
Fast,
high-yielding, and selective bioorthogonal āclickā reactions
employing nontoxic reagents are in high demand for their great utility
in the conjugation of biomolecules in live cells. Although a number
of click reactions were developed for this purpose, many are associated
with drawbacks and limitations that justify the development of alternative
systems for both single- or dual-labeling applications. Recent reports have highlighted the potential
of boronic ester formation as a bioorthogonal click reaction between
abiotic boronic acids and diols. Boronic ester formation is a fast
dehydrative process; however it is intrinsically reversible in aqueous
medium. We designed and optimized a synergic system based on two bifunctional
reagents, a thiosemicarbazide-functionalized nopoldiol and an <i>ortho</i>-acetyl arylboronic acid. Both reagents were shown
to be chemically stable and nontoxic to HEK293T cells at concentrations
as high as 50 Ī¼M. The resulting boronate/thiosemicarbazone adduct
is a medium-sized ring that forms rapidly and irreversibly without
any catalyst at low Ī¼M concentrations, in neutral buffer, with
a rate constant of 9 M<sup>ā1</sup> s<sup>ā1</sup> as
measured by NMR spectroscopy. Control experiments in the presence
of competing boronic acids showed no crossover side-products and confirmed
the stability and lack of reversibility of the boronate/thiosemicarbazone
conjugates. Formation of the conjugates is not affected by the presence
of biological diols such as fructose, glucose, and catechol, and the
thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles
of the sort found on protein-bound glyoxylyl units. The suitability
of this system in the cell-surface labeling of live cells was demonstrated
using a SNAP-tag approach to install the boronic acid reagent onto
the extracellular domain of the Beta-2 adrenergic receptor in HEK293T
cells, followed by incubation with the optimal thiosemicarbazide-functionalized
nopoldiol reagent labeled with fluorescein dye. Successful visualization
by fluorescence microscopy was possible with a reagent concentration
as low as 10 Ī¼M, thus confirming the potential of this system
in biological applications
ProteināGlycosphingolipid Interactions Revealed Using Catch-and-Release Mass Spectrometry
Glycosphingolipids (GSL) on the surface of cells are
important
receptors in antigen/microbial recognition and cell adhesion. However,
their functional characterization is often challenging. We have developed
a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS)
assay for the identification of specific interactions between water-soluble
proteins or protein complexes with GSL incorporated into nanodiscs.
The specificity and sensitivity of the assay is demonstrated for interactions
involving cholera toxin and Shiga toxin, with their natural GSL receptors,
the ganglioside GM1, and the globotriaosylceramide Gb3, respectively.
The detection of binding between cholera toxin and GM1 within a mixture
of lipids extracted from cell membranes highlights the potential of
this assay for the discovery of biologically relevant proteināGSL
interactions
Conjugation of A and B Blood Group Structures to Silica Microparticles for the Detection of Antigen-Specific B Cells
Silica
microparticles were functionalized with A and B blood group
carbohydrate antigens (A type I, A type II, B type I, and B type II)
to enable the detection and monitoring of ABO antigen-specific B cells.
Microparticles were prepared via the StoĢber synthesis, labeled
with an Alexafluor fluorescent dye, and characterized via TEM and
fluorescence microscopy. The silica microparticles were functionalized
with (3-aminopropyl)Ātrimethoxysilane (APTMS), followed by the use
of an established fluorenylmethyloxycarbonyl (Fmoc)-protected PEG-based
linker. The terminal Fmoc moiety of the PEG-based linker was then
deprotected, yielding free amino groups, to which the A and B antigens
were coupled. The carbohydrate antigens were synthesized with a <i>p</i>-nitrophenol ester to enable conjugation to the functionalized
silica microparticles via an amide bond. The number of free amine
groups available for coupling for a given mass of PEG-functionalized
silica microparticles was quantified via reaction with Fmoc-glycine.
The antigen-functionalized microparticles were then evaluated for
their specificity in binding to A and B antigen-reactive B-cells via
flow cytometry, and for blocking of naturally occurring antibodies
in human serum. Selective binding of the functionalized microparticles
to blood group-reactive B cells was observed by flow cytometry and
fluorescence microscopy. The modular approach outlined here is applicable
to the preparation of silica microparticles containing any carbohydrate
antigen and alternative fluorophores or labels. This approach therefore
comprises a novel, general platform for screening B cell populations
for binding to carbohydrate antigens, including, in this case, the
human A and B blood group antigens