5 research outputs found
Synthesis of Biologically Active <i>N</i>- and <i>O</i>‑Linked Glycans with Multisialylated Poly‑<i>N</i>‑acetyllactosamine Extensions Using <i>P. damsela</i> α2‑6 Sialyltransferase
Sialosides
on <i>N</i>- and <i>O</i>-linked glycoproteins
play a fundamental role in many biological processes, and synthetic
glycan probes have proven to be valuable tools for elucidating these
functions. Though sialic acids are typically found α2-3- or
α2-6-linked to a terminal nonreducing end galactose, poly-LacNAc
extended core-3 <i>O</i>-linked glycans isolated from rat
salivary glands and human colonic mucins have been reported to contain
multiple internal Neu5Acα2-6Gal epitopes. Here, we have developed
an efficient approach for the synthesis of a library of <i>N</i>- and <i>O</i>-linked glycans with multisialylated poly-LacNAc
extensions, including naturally occurring multisialylated core-3 <i>O</i>-linked glycans. We have found that a recombinant α2-6
sialyltransferase from <i>Photobacterium damsela</i> (Pd2,6ST)
exhibits unique regioselectivity and is able to sialylate internal
galactose residues in poly-LacNAc extended glycans which was confirmed
by MS/MS analysis. Using a glycan microarray displaying this library,
we found that Neu5Acα2-6Gal specific influenza virus hemagglutinins,
siglecs, and plant lectins are largely unaffected by adjacent internal
sialylation, and in several cases the internal sialic acids are recognized
as ligands. Polyclonal IgY antibodies specific for internal sialoside
epitopes were elicited in inoculated chickens
Heparin Binding to an Engineered Virus-like Nanoparticle Antagonist
The
anticoagulant activity of heparin administered during medical
interventions must be reversed to restore normal clotting, typically
by titrating with protamine. Given the acute toxicity associated with
protamine, we endeavored to generate safer heparin antagonists by
engineering bacteriophage Qβ virus-like particles (VLPs) to
display motifs that bind heparin. A particle bearing a single amino
acid change from wild-type (T18R) was identified as a promising candidate
for heparin antagonism. Surface potential maps generated through molecular
modeling reveal that the T18R mutation adds synergistically to adjacent
positive charges on the particle surface, resulting in a large solvent-accessible
cationic region that is replicated 180 times over the capsid. Chromatography
using a heparin-sepharose column confirmed a strong interaction between
heparin and the T18R particle. Binding studies using fluorescein-labeled
heparin (HepFL) resulted in a concentration-dependent change in fluorescence
intensity, which could be perturbed by the addition of unlabeled heparin.
Analysis of the fluorescence data yielded a dissociation constant
of approximately 1 nM and a 1:1 binding stoichiometry for HepFL:VLP.
Dynamic light scattering (DLS) experiments suggested that T18R forms
discrete complexes with heparin when the VLP:heparin molar ratios
are equivalent, and in vitro clotting assays confirmed the 1:1 binding
stoichiometry as full antagonism of heparin is achieved. Biolayer
interferometry and backscattering interferometry corroborated the
strong interaction of T18R with heparin, yielding <i>K</i><sub>d</sub> ∼ 1–10 nM. These biophysical measurements
further validated T18R, and VLPs in general, for potential clinical
use as effective, nontoxic heparin antagonists