Homogeneously N‐glycosylated proteins derived from the GlycoDelete HEK293 cell line enable diffraction‐quality crystallogenesis. Issue 12 (2nd December 2020) - Kozak, Sandra
Abstract : Structural studies of glycoproteins are often complicated by glycan complexity. Here, it is shown that the GlycoDelete HEK293 cell line engineered to produce glycan stumps produces homogeneous glycoproteins that are ideal for crystallogenesis and other structural studies. Abstract : Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N‐linked glycosylation, a key post‐translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N‐linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X‐ray crystallography or other methods. In particular, obtaining diffraction‐quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N‐glycans as short glycan stumps comprising N ‐acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell‐adhesion molecule and colony‐stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N ‐glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X‐ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL‐12B is shown to lead to N ‐glycosylation featuring an immature glycan in diffraction‐quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go‐to option for the production of homogeneous glycoproteins and their complexes for structural studies by X‐ray crystallography and cryo‐electron microscopy.