Kajiura, H. | bioRxiv Science

bioRxiv2023

Discovery of the apiosyltransferase, celery UGT94AX1 that catalyzes the biosynthesis of a flavone glycoside, apiin

Apiose is a unique branched-chain pentose found in plant glycosides and a key component of the cell wall-polysaccharide pectin and other specialized metabolites. More than 1,200 plant-specialized metabolites contain apiose residues, represented by apiin, a distinctive flavone glycoside found in celery and parsley in the family Apiaceae. The physiological functions of apiin remain obscure, partly due to our lack of knowledge on apiosyltransferase during apiin biosynthesis. Here, we identified celery UGT94AX1 (AgApiT) as a novel apiosyltransferase, responsible for catalyzing the last sugar-modification step in apiin biosynthesis. AgApiT showed strict substrate specificity for the sugar donor, UDP-apiose, and moderate specificity for acceptor substrates, thereby producing various apiose-containing flavone glycosides in celery. Homology modeling of AgApiT with UDP-apiose, followed by site-directed mutagenesis experiments, identified unique Ile139, Phe140, and Leu356 residues in AgApiT, which are seemingly crucial for the recognition of UDP-apiose in the sugar donor pocket. Sequence comparison and molecular phylogenetic analysis of celery glycosyltransferases paralogous to AgApiT suggested that AgApiT is the sole apiosyltransferase-encoding gene in the celery genome. This is the first report on the identification of a plant apiosyltransferase gene that will enhance our understanding of the physio-ecological functions of apiose and apiose-containing compounds.

biochemistry↗

bioRxiv2022

Impact of glycoengineering and immunogenicity on the anti-cancer activity of a plant-made lectin-Fc fusion protein

Plants are an efficient production platform for manufacturing glycoengineered monoclonal antibodies and antibody-like molecules. Avaren-Fc (AvFc) is a lectin-Fc fusion protein or lectibody produced in Nicotiana benthamiana, which selectively recognizes cancer-associated high-mannose glycans. In this study, we report the generation of a glycovariant of AvFc that is devoid of plant glycans, including the core 1,3-fucose and {beta}1,2-xylose residues. The successful removal of these glycans was confirmed by glycan analysis using HPLC. This variant, AvFc{Delta}XF, has significantly higher affinity for Fc gamma receptors and induces higher levels of luciferase expression in an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter assay against B16F10 murine melanoma cells without inducing apoptosis or inhibiting proliferation. In the B16F10 flank tumor mouse model, we found that systemic administration of AvFc{Delta}XF, but not an aglycosylated AvFc variant lacking affinity for Fc receptors, significantly delayed the growth of tumors, suggesting that Fc-mediated effector functions were integral. AvFc{Delta}XF treatment also significantly reduced lung metastasis of B16F10 upon intravenous challenge whereas a sugar-binding-deficient mutant failed to show efficacy. Lastly, we determined the impact of anti-drug antibodies (ADAs) on drug activity in vivo by pretreating animals with AvFc{Delta}XF before implanting tumors. Despite a significant ADA response induced by the pretreatment, we found that the activity of AvFc{Delta}XF was unaffected by the presence of these antibodies. These results demonstrate that glycoengineering is a powerful strategy to enhance AvFcs antitumor activity.

cancer biology↗