Assessing the activity of β1,4-galactosidases from Nicotiana benthamiana leaves to enhance glyco-engineering efficiency

Plants offer great advantages for the production of glyco-engineered biopharmaceutical glycoproteins, but these glycoproteins are subjected to a wide array of plant-native glycosidases. A consequence of glycosidase activity along the secretory pathway is that the predominant glycoform is steered away from the desired glycan structure. For example, β-galactosidase (BGAL) 1 from the glycosyl hydrolase family GH35 in Nicotiana benthamiana has recently been identified as one of the enzymes responsible for preventing efficient β1,4-galactosylation of N-glycans, as well as the ability to cleave mucin-type O-glycans. Strikingly, galactosylation is still not complete in plants lacking BGAL1 activity, indicating that more members of the GH35 family are reducing galactosylation efficiency. In this study, we cloned several novel BGAL open reading frames from N. benthamiana leaf material and characterized the biochemical activity of these enzymes. We identified different BGAL enzymes responsible for the cleavage of antennary β1,4-galactose residues of N-glycans on the model glycoprotein kappa-5. Furthermore, we targeted undesired BGAL activity with co-infiltration of a BGAL inhibitor. The identification of additional BGAL enzymes as major targets for efficient engineering of galactose-containing glycans will enable a targeted genome editing approach to reduce undesired processing of these glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made galactosylated glycans in plants.