Biology subjects

Nibbering, P.

Publications and source records attributed to Nibbering, P..

4 recordsLinked to original sources

bioRxiv2025

Precision Glycoform Engineering: Combining plant and in vitro systems for tailored biopharmaceutical production

Protein biopharmaceuticals play a key role in providing effective, targeted, and personalized therapies for diverse diseases, while also preventing and mitigating a broad range of infections. N-glycosylation is a key post-translational modification influencing the biological activity of many protein-based therapeutics, yet structure-function relationships of N-glycans remain poorly understood due to challenges in producing homogeneous glycoforms. Current go-to production hosts, mammalian and yeast cells, often yield heterogeneous glycan profiles and require extensive genetic manipulation. Alternative production hosts such as the plant Nicotiana benthamiana, provide more homogeneous glycosylation and flexibility through transient expression, but are limited in the generation of certain glycoforms. In vitro glycoengineering can overcome these limitations but is time consuming and requires expensive resources. In this study, we show that by combining in planta and in vitro glycoengineering strategies, we can quickly produce a wide range of homogeneous glycoforms of pharmaceutical proteins with high mannose, paucimannose, hybrid and complex N-glycan structures. Using N. benthamiana as a transient expression host, we produced two pharmaceutical glycoproteins -- the monoclonal antibody rituximab and the helminth vaccine candidate OoASP-1 -- and modified them in vitro using Escherichia coli produced glycoenzymes. The combination of these two glycoengineering systems minimizes the amount of time and resources required, while maintaining high glycan homogeneity. This scalable, flexible, and cost-effective platform opens the door to glycan structure-function relationship studies and can support rational design of next-generation biopharmaceuticals.

bioengineering↗

bioRxiv2023

WOX11-mediated cell size control in Arabidopsis attenuates fecundity of endoparasitic cyst nematodes

O_LICyst nematodes establish permanent feeding structures called syncytia inside host root vasculature, disrupting the flow of water and minerals. In response, plants form WOX11-mediated adventitious lateral roots at nematode infection sites. WOX11-adventitious lateral rooting modulates tolerance to nematode infections, however, whether this also benefits nematode parasitism remains unknown. C_LIO_LIHere, we report on bioassays using a 35S::WOX11-SRDX transcriptional repressor mutant to investigate whether WOX11-adventitious lateral rooting promotes syncytium development and thereby female fecundity. Moreover, we chemically inhibited cellulose biosynthesis to verify if WOX11 directly modulates cell wall plasticity in syncytia. Finally, we performed histochemical analyses to test if WOX11 mediates syncytial cell wall plasticity via reactive oxygen species (ROS). C_LIO_LIRepression of WOX11-mediated transcription specifically enhanced the radial expansion of syncytial elements, increasing both syncytium size and female offspring. The enhanced syncytial hypertrophy observed in the 35S::WOX11-SRDX mutant could be phenocopied by chemical inhibition of cellulose biosynthesis and was associated with elevated levels of ROS at nematode infection sites. C_LIO_LIWe therefore conclude that WOX11 restricts radial expansion of nematode feeding structures and female fecundity, likely by modulating ROS-mediated cell wall plasticity mechanisms. Remarkably, this novel role of WOX11 in plant cell size control is independent of WOX11-adventitious rooting underlying disease tolerance. C_LI

plant biology↗

bioRxiv2021

Golgi-localized GALT7 and GALT8 participate in cellulose biosynthesis

Arabinogalactan protein (AGP) glycan biosynthesis in the Golgi apparatus contributes to plant cell wall assembly, but the mechanisms underlying this process are largely unknown. Here, we show that two putative galactosyltransferases -named GALT7 and GALT8 -from the glycosyltransferase family 31 (GT31) of Arabidopsis thaliana participate in cellulose biosynthesis. galt7galt8 mutants show primary cell wall defects manifesting as impaired growth and cell expansion in seedlings and etiolated hypocotyls, along with secondary cell wall defects, apparent as collapsed xylem vessels and reduced xylem wall thickness in the inflorescence stem. These phenotypes were associated with a [~]30% reduction in cellulose content, a [~]50% reduction in secondary cell wall CELLULOSE SYNTHASE (CESA) protein levels and reduced cellulose biosynthesis rate. CESA transcript levels were not significantly altered in galt7galt8 mutants, suggesting that the reduction in CESA levels was caused by a post-transcriptional mechanism. We provide evidence that both GALT7 and GALT8 localise to the Golgi apparatus, while quantitative proteomics experiments revealed reduced levels of the entire FLA subgroup B in the galt7galt8 mutants. This leads us to hypothesize that a defect in FLA subgroup B glycan biosynthesis reduces cellulose biosynthesis rate in galt7galt8 mutants.

plant biology↗

bioRxiv2020

Golgi-localized exo-β1,3-galactosidases involved in AGP modification and root cell expansion in Arabidopsis

Plant arabinogalactan proteins (AGPs) are a diverse group of cell surface- and wall-associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships between their glycosylation, processing, and functionality are poorly understood. Here we report the identification and functional characterization of two Golgi-localized exo-{beta}-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana. GH43 loss of function mutants exhibit root cell expansion defects in sugar-containing growth media. This root phenotype is associated with an increase in the extent of AGP cell wall association, as demonstrated by Yariv phenylglycoside dye quantification and comprehensive microarray polymer profiling of sequentially extracted cell walls. Recombinant GH43 characterization showed that the exo-{beta}-1,3-galactosidase activity of GH43s is hindered by {beta}-1,6 branches on {beta}-1,3-galactans. In line with this steric hindrance, the recombinant GH43s did not release galactose from cell wall extracted glycoproteins or AGP rich gum arabic. These results show that Arabidopsis GH43s are involved in AGP glycan biosynthesis in the Golgi, and suggest their exo-{beta}-1,3-galactosidase activity influences AGP and cell wall matrix interactions, thereby adjusting cell wall extensibility.

plant biology↗