5 research outputs found
Lectin-Array Blotting: Profiling Protein Glycosylation in Complex Mixtures
By combining electrophoretic protein separation with
lectin-array-based glycan profiling into a single experiment, we have
developed a high-throughput method for the rapid analysis of protein
glycosylation in biofluids. Fluorescently tagged proteins are separated
by SDS-PAGE and transferred by diffusion to a microscope slide covered
with multiple copies of 20 different lectins, where they are trapped
by specific carbohydrate protein interactions while retaining their
relative locations on the gel. A fluorescence scan of the slide then
provides an affinity profile with each of the 20 lectins containing
a wealth of structural information regarding the present glycans.
The affinity of the employed lectins toward <i>N</i>-glycans
was verified on a glycan array of 76 structures. While current lectin-based
methods for glycan analysis provide only a picture of the bulk glycosylation
in complex protein mixtures or are focused on a few specific known
biomarkers, our array-based glycoproteomics method can be used as
a biomarker discovery tool for the qualitative exploration of protein
glycosylation in an unbiased fashion
Negatively Charged Glyconanoparticles Modulate and Stabilize the Secondary Structures of a gp120 V3 Loop Peptide: Toward Fully Synthetic HIV Vaccine Candidates
The
third variable region (V3 peptide) of the HIV-1 gp120 is a
major immunogenic domain of HIV-1. Controlling the formation of the
immunologically active conformation is a crucial step to the rational
design of fully synthetic candidate vaccines. Herein, we present the
modulation and stabilization of either the α-helix or β-strand
conformation of the V3 peptide by conjugation to negatively charged
gold glyconanoparticles (GNPs). The formation of the secondary structure
can be triggered by the variation of the buffer concentration and/or
pH as indicated by circular dichoism. The peptide on the GNPs shows
increased stability toward peptidase degradation as compared to the
free peptide. Moreover, only the V3β-GNPs bind to the anti-V3
human broadly neutralizing mAb 447-52D as demonstrated by surface
plasmon resonance (SPR). The strong binding of V3β-GNPs to the
447-52D mAb was the starting point to address its study as immunogen.
V3β-GNPs elicit antibodies in rabbits that recognize a recombinant
gp120 and the serum displayed low but consistent neutralizing activity.
These results open up the way for the design of new fully synthetic
HIV vaccine candidates
Chemo-Enzymatic Synthesis of <sup>13</sup>C Labeled Complex N‑Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry
Methods for the absolute quantification
of glycans are needed in
glycoproteomics, during development and production of biopharmaceuticals
and for the clinical analysis of glycan disease markers. Here we present
a strategy for the chemo-enzymatic synthesis of <sup>13</sup>C labeled
N-glycan libraries and provide an example for their use as internal
standards in the profiling and absolute quantification of mAb glycans
by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)
mass spectrometry. A synthetic biantennary glycan precursor was <sup>13</sup>C-labeled on all four amino sugar residues and enzymatically
derivatized to produce a library of 15 glycan isotopologues with a
mass increment of 8 Da over the natural products. Asymmetrically elongated
glycans were accessible by performing enzymatic reactions on partially
protected UV-absorbing intermediates, subsequent fractionation by
preparative HPLC, and final hydrogenation. Using a preformulated mixture
of eight internal standards, we quantified the glycans in a monoclonal
therapeutic antibody with excellent precision and speed
Chemo-Enzymatic Synthesis of <sup>13</sup>C Labeled Complex N‑Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry
Methods for the absolute quantification
of glycans are needed in
glycoproteomics, during development and production of biopharmaceuticals
and for the clinical analysis of glycan disease markers. Here we present
a strategy for the chemo-enzymatic synthesis of <sup>13</sup>C labeled
N-glycan libraries and provide an example for their use as internal
standards in the profiling and absolute quantification of mAb glycans
by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)
mass spectrometry. A synthetic biantennary glycan precursor was <sup>13</sup>C-labeled on all four amino sugar residues and enzymatically
derivatized to produce a library of 15 glycan isotopologues with a
mass increment of 8 Da over the natural products. Asymmetrically elongated
glycans were accessible by performing enzymatic reactions on partially
protected UV-absorbing intermediates, subsequent fractionation by
preparative HPLC, and final hydrogenation. Using a preformulated mixture
of eight internal standards, we quantified the glycans in a monoclonal
therapeutic antibody with excellent precision and speed
Engineering Erg10 Thiolase from <i>Saccharomyces cerevisiae</i> as a Synthetic Toolkit for the Production of Branched-Chain Alcohols
Thiolases
catalyze the condensation of acyl-CoA thioesters through
the Claisen condensation reaction. The best described enzymes usually
yield linear condensation products. Using a combined computational/experimental
approach, and guided by structural information, we have studied the
potential of thiolases to synthesize branched compounds. We have identified
a bulky residue located at the active site that blocks proper accommodation
of substrates longer than acetyl-CoA. Amino acid replacements at such
a position exert effects on the activity and product selectivity of
the enzymes that are highly dependent on a protein scaffold. Among
the set of five thiolases studied, Erg10 thiolase from <i>Saccharomyces
cerevisiae</i> showed no acetyl-CoA/butyryl-CoA branched condensation
activity, but variants at position F293 resulted the most active and
selective biocatalysts for this reaction. This is the first time that
a thiolase has been engineered to synthesize branched compounds. These
novel enzymes enrich the toolbox of combinatorial (bio)chemistry,
paving the way for manufacturing a variety of α-substituted
synthons. As a proof of concept, we have engineered <i>Clostridium</i>’s 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol
that is interesting as a branched model compound