title: Engineering novel Lec1 glycosylation mutants in CHO-DUKX cells: molecular insights and effector modulation of N-acetylglucosaminyltransferase I.
abstract: Many secreted or cell surface proteins are post-translationally modified by carbohydrate chains which are a primary source of heterogeneity. The Lec1 mutant, which is defective in Golgi N-acetylglucosaminyltransferase I (GnTI) activity, produces relatively homogeneous Man(5) GlcNAc(2) glycan modifications, and is widely used for various applications. To facilitate the investigation of GnTI, its Man5 glycan endproduct, and the impact of Man5 on effector function, the present study has established several novel Lec1 mutants in dhfr(-) CHO-DUKX cells through chemical mutagenesis and lectin selection. A total of nine clonal lines exhibiting the Lec1-phenotype are characterized, six of which harbor non-sense mutations leading to a truncated GnTI, and three (R415K, D291N, and P138L) of which are novel loss-of-function sense mutations. Analysis of the rabbit GnTI structure (Unligil et al., 2000) indicates that D291 is the proposed catalytic base and R415 is a crucial residue in forming the substrate binding pocket, whereas P138 is key to maintaining two β strands in proximity to the substrate binding pocket. Computational modeling reveals that the oligomannose glycan backbone of a glycoprotein (the acceptor substrate) fits nicely into the unoccupied channel of the substrate binding pocket partly through hydrogen bonding with R415 and D291. This finding is consistent with the ordered sequential Bi Bi kinetic mechanism suggested for GnTI, in which binding of UDP-GlcNAc (the donor substrate)/Mn(2+) induces conformational changes that promote acceptor binding. When an anti-human CD20 antibody protein is stably expressed in one CHO-DUKX-Lec1 line, it is confirmed that N-glycans are predominantly Man(5) GlcNAc(2) and they do not contain an α1,6-fucose linked to the innermost GlcNAc. Furthermore, this Man(5) GlcNAc(2) modified antibody exhibits a significantly increased ADCC activity than the wild-type protein, while displaying a lower CDC activity. The data support the hypothesis that modulating GnTI activity can influence antibody effector functions for proteins with an IgG1 immunoglobulin Fc domain.
authors.affiliation: Global BioTherapeutics Technologies, Pfizer BioTherapeutic Research and Development, 87 Cambridge Park Drive, Cambridge, Massachusetts 02140, USA. xiaotian.zhong@pfizer.com
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Zhong
authors.forename: Cecilia
authors.initials: C
authors.lastname: Cooley
authors.forename: Nilufer
authors.initials: N
authors.lastname: Seth
authors.forename: Z Sean
authors.initials: ZS
authors.lastname: Juo
authors.forename: Ella
authors.initials: E
authors.lastname: Presman
authors.forename: Nicole
authors.initials: N
authors.lastname: Resendes
authors.forename: Ravi
authors.initials: R
authors.lastname: Kumar
authors.forename: Martin
authors.initials: M
authors.lastname: Allen
authors.forename: Lidia
authors.initials: L
authors.lastname: Mosyak
authors.forename: Mark
authors.initials: M
authors.lastname: Stahl
authors.forename: Will
authors.initials: W
authors.lastname: Somers
authors.forename: Ronald
authors.initials: R
authors.lastname: Kriz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002999:Clone Cells; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009844:Oligosaccharides / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D007074:Immunoglobulin G; D009844:Oligosaccharides; D011994:Recombinant Proteins; C058642:mannosyl(5)-N-acetyl(2)-glucose; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I