title: A human embryonic kidney 293T cell line mutated at the Golgi alpha-mannosidase II locus.
abstract: Disruption of Golgi alpha-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi alpha-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi alpha-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Headington, Oxford OX3 7BN, United Kingdom.
authors.forename: Max
authors.initials: M
authors.lastname: Crispin
authors.forename: Veronica T
authors.initials: VT
authors.lastname: Chang
authors.forename: David J
authors.initials: DJ
authors.lastname: Harvey
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Evans
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.forename: E Yvonne
authors.initials: EY
authors.lastname: Jones
authors.forename: J Michael
authors.initials: JM
authors.lastname: Lord
authors.forename: Robert A
authors.initials: RA
authors.lastname: Spooner
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Davis
mesh_terms: D000483:Alleles; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D017422:Sequence Analysis, DNA; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D043323:alpha-Mannosidase / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D009711:Nucleotides; D009841:Oligonucleotides; D011134:Polysaccharides; D043323:alpha-Mannosidase