Biology subjects

Archer-Hartmann, S.

Publications and source records attributed to Archer-Hartmann, S..

2 recordsLinked to original sources

bioRxiv2025

Mass Spectrometric Determination of Site-Specific O-Acetylation in Rhamnogalacturonan-I Oligomers

O-Acetylation, a common modification in rhamnogalacturonan I (RG-I), is critical for various biological processes, including plant growth, stress responses, and pathogen defense. Precise determination of the degree and specific positions of acetylation is therefore essential. To date, nuclear magnetic resonance (NMR) and tandem mass spectrometry have been employed to identify acetyl positions in pectin oligosaccharides. Although NMR is effective, it requires pure, high-concentration samples. Tandem mass spectrometry (MS), which uses lower sample amounts, faces challenges due to acetyl migration between monosaccharide positions. The multiple steps in pectin sample analysis can further promote O-acetyl migration, especially near free hydroxyl groups. Moreover, during tandem MS, acetyl groups may detach, complicating accurate tracking. This study presents an approach to lock O-acetyl groups by introducing trideuteroacetyl and propionyl substituents onto free hydroxyls of RG-I or partially acetylated RG-I. By combining matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS and electrospray ionization (ESI) MS with MS/MS or tandem mass spectrometry (MSn), we devised a way to determine the monosaccharide sequence in the oligomer and precise positions of acetyl groups in partially acetylated RG-I. This method enables the study of the regiospecificity of recombinant pectin O-acetyltransferases and can be applied to other oligosaccharides to determine acyl positions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/694011v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1384347org.highwire.dtl.DTLVardef@c8f21forg.highwire.dtl.DTLVardef@8fed52org.highwire.dtl.DTLVardef@125c4a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Glycoengineering of the hepatitis C virus E2 glycoprotein leads to improved biochemical properties and enhanced immunogenicity

An effective vaccine against hepatitis C virus (HCV) must elicit the production of broadly neutralizing antibodies (bnAbs) reproducibly against the E1E2 glycoprotein complex. Little is known about how glycan content affects this process. Ideally, glycans would maximize epitope exposure without compromising antigen stability or exposing new epitopes. However, typical recombinant vaccines contain considerable heterogeneity in glycan content, which can affect the antibody response and neutralization potency. Here we employed glycoengineered Chinese hamster ovary (geCHO) cell lines that impart nearly homogeneous glycosylation as a means to test how specific glycan features influence antigenicity and immunogenicity for the secreted HCV E2 ectodomain (sE2). Specific geCHO antigens exhibited a modest but reproducible increase in affinity for some mAbs relative to CHO- and HEK293-produced sE2. Surprisingly, one geCHO sE2 antigen failed to bind the CD81 receptor, indicating the potential for significant glycan effects on biochemical properties. We immunized mice with the four antigens and found the total antibody response to be the same for all groups. However, sera from one geCHO group exhibited a 7-fold improvement in neutralization against the homologous HCV pseudovirus and had the most mice whose sera exhibited neutralization activity against genotypes 1b, 2a, 2b, and 3. Further analysis identified beneficial and deleterious glycan features, and the glycan that correlated the most with decreased potency was relatively small. However, size was not the sole determinant of glycan-driven effects on the antibody response. In summary, glycan content impacts biochemical properties of antigens to varying degrees and such effects can influence immune response quality and uniformity.

immunology↗