Effects of N-butyldeoxynojirimycin and the Lec3.2.8.1 mutant phenotype on N-glycan processing in Chinese hamster ovary cells: Application to glycoprotein crystallization

Heterologous gene expression in either (1) the glycosylation-defective, mut ant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin facilitates the trimm ing of N-linked glycans of glycoproteins to single N-acetylglucosamine (Glc NAc) residues with endoglycosidase H (endo Hi). Both approaches are somewha t inefficient, however, with as little as 12% of the total protein being re ndered fully endo H-sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccha ride processing are essentially additive, thereby allowing the production o f glycoproteins that are essentially completely endo H-sensitive. The prepa ration of a soluble chimeric form of CD58, the ligand of the human T-cell s urface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is u nstable at low pH. The endo H-treated chimera produced crystals of space gr oup P3(1)21 or P3(2)21, and unit cell dimensions a = b = 116.4 Angstrom, c = 51.4 Angstrom alpha = beta = 90 degrees, gamma = 120 degrees, that diffra ct to a maximum resolution of 1.8 Angstrom.