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, mutant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin facilitates the trimming of N-linked glycans of glycoproteins to single N-acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the total protein being rendered fully endo H-sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccharide processing are essentially additive, thereby allowing the production of glycoproteins that are essentially completely endo H-sensitive. The preparation of a soluble chimeric form of CD58, the ligand of the human T-cell surface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is unstable at low pH. The endo H-treated chimera produced crystals of space group P3(1)21 or P3(2)21, and unit cell dimensions a = b = 116.4 A, c = 51.4 A alpha = beta = 90 degrees , gamma = 120 degrees , that diffract to a maximum resolution of 1.8 A.
Butters
T D
TD
Glycobiology Institute, Department of Biochemistry, The University of Oxford, United Kingdom.
Sparks
L M
LM
Harlos
K
K
Ikemizu
S
S
Stuart
D I
DI
Jones
E Y
EY
Davis
S J
SJ
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003460:Crystallization; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D010641:Phenotype; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D015703:Antigens, CD; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D017485:1-Deoxynojirimycin; C059896:miglustat