Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.02.12.580006

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

Jayachandran, D.·Parvate, A.·Brookreson, J. T.·Evans, J. E.·Chundawat, S. P. S.
View original source ↗Export metadata ↓

Abstract

Polysaccharides are a major class of natural polymers found abundantly across all major life forms and play a critical role as structural, metabolic, or functional components in biomolecular processes. Some polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs). Despite the fact that the GT-2 family has the maximum number of deposited sequences, the biochemistry of GT-2 family enzymes is still poorly understood due to difficulties associated with GT membrane protein expression, purification, and reconstitution in lipid carriers. Here, we chose Populus tremula x tremuloides cellulose synthase 8 (PttCesA8) and Streptococcus equisimilis hyaluronan synthase (SeHas) as putative family-2-GTs to be expressed in a wheat-germ-based cell-free expression (CFE) system as proteoliposomes. The cell-free products were obtained as reconstituted liposomes directly from CFE reactions at high yields and short processing times compared to other approaches. GT enzymes expression was confirmed using SDS-PAGE and immunoblotting, and the integration of GTs in lipid layers was observed using cryogenic electron microscopy. Both GTs tested were catalytically active when incubated with their respective substrates and cofactors. The Michalis-Menten kinetic constants, Km for PttCesA8, was 295.8 {micro}M, and SeHas was 321.51 {micro}M (toward UDP N-Acetyl Glucosamine) and 207.88 {micro}M (toward UDP Glucuronic Acid), respectively. UDP was found to actively inhibit both these GTs with apparent inhibition constants of 10.08 {micro}M and 24.38 {micro}M. Mutation of specific conserved residues in structure-deficit SeHas confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, wheat-germ-based CFE can be used to express functionally active and liposome-reconstituted family-2 GTs at high yields with relative ease to enable classical enzymology assays and will also enable more detailed structural studies in the near future.

Source connections

Explore related subjects

bioengineeringPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Jayachandran, D., Parvate, A., Brookreson, J. T., Evans, J. E., Chundawat, S. P. S.. 2024-02-14. Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases. https://doi.org/10.1101/2024.02.12.580006

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Comparative study of chlorophyll measurement in Physcomitrium patens moss using a conventional microscope adapted for combined 2D+1D imaging and spectral analysis

Imaging spectroscopy often requires expensive and complex equipment. Here we show a simple procedure for attaching a standard miniature fiber spectrometer to a conventional microscope, allowing easy integration of 2D imaging with 1D high-resolution spectral measurements. This combination provides much of the benefit of a full imaging spectrometer without the large equipment investment, and we provide instructions for modifying microscopes to this setup and the present measurements of living cells that demonstrate their performance. Using this setup, we compare the quantitative measurement of chlorophyll concentration in Physcomitrium patens moss using color imaging and spectral sampling.

bioengineering↗
bioRxiv2026

De novo designed single-domain antibodies protect against lethal cobra venom neurotoxicity in vivo

Generative protein design can now rapidly produce de novo binders with high affinity and functional activity against a wide range of targets, including lethal snake venom toxins. However, so far most reported successes rely on new-to-nature scaffolds with limited therapeutic precedent. Single-domain antibodies (VHHs) offer a clinically validated alternative scaffold that can bind and neutralize long-chain -neurotoxins, which are some of the most lethal components in snake venoms. Here we compare three recently established de novo design models with VHH-design capabilities (Germinal, RFantibody, and BoltzGen) for their ability to generate VHHs against the neurotoxin -cobratoxin from the monocled cobra (Naja kaouthia). Using standardized model inputs and evaluation criteria based on AlphaFold3 interface confidence (ipTM) and RMSD self-consistency, we find that Germinal was the only method to generate designs passing stringent in silico criteria for experimental testing. We therefore performed a larger Germinal design campaign employing three different VHH frameworks and experimentally validated 46 designs in vitro. Of these, 42 expressed as soluble proteins and we identified four binding hits derived from two of the three tested frameworks. Of the four binders, two lead candidates were further characterized and demonstrated high affinity (KDs of 4.1 nM and 10.8 nM), monomeric behavior and low polyreactivity, indicating favorable biophysical and developability properties, as well as functional toxin neutralization in vitro. To assess their therapeutic potential we investigated their ability to protect against -cobratoxin toxicity in vivo. Both candidates fully protected mice after -cobratoxin challenge, with 100% survival compared to a lethal control. One candidate also retained notable neutralization capacity against whole venom of Naja kaouthia with a survival of 56%, while the other protected 22% when tested in a rescue setting. Together, we demonstrate that de novo VHH design can generate high affinity single-domain antibodies with in vivo protection against lethal cobra venom neurotoxicity, and provide practical insights into method- and framework-dependent performance.

bioengineering↗
bioRxiv2026

Simple Feedback for Complex Movement: Capturing Whole-Limb Reorganization during Single-IMU Gait Retraining

Clinical gait retraining typically relies on multi-sensor arrays and high-dimensional feedback displays, imposing setup and interpretation burdens that limit routine clinical deployment. We developed a single-IMU visual biofeedback system that delivers real-time feedback of Lower Limb Trajectory Error (LLTE), a composite kinematic error metric integrating knee position and shank angle across the stance phase. Twenty able-bodied adults walked on a treadmill under two visual biofeedback targets (flexed-knee, extended-knee) while receiving either corrected (n=10) or uncorrected (n=8) feedback, where the correction accounted for limb orientation at initial contact. LLTE and stance-phase knee kinematics adapted consistently under the flexed-knee target for both feedback groups, with feedback formulation moderating the temporal trajectory of change. Adaptation toward the extended-knee target was limited, likely because participants were already operating near terminal knee extension and because the scalar error metric provided limited directional information for correction. Ankle range of motion (ROM) changed significantly across the stance phase under both target conditions, while hip ROM did not. Multiscale multivariate sample entropy (MSMVSE) increased monotonically with time scale across all conditions, with no statistically distinguishable difference between corrected and uncorrected feedback. These results suggest that single-IMU LLTE biofeedback can modify gait mechanics and that adaptation was expressed across multiple lower-limb segments rather than through changes at a single joint.

bioengineering↗