Crispin, Max, Chang, Veronica T., Harvey, David J., Dwek, Raymond A., Evans, Edward J., Stuart, David I., Jones, E. Yvonne, Lord, Mike (J. Mike), Spooner, Robert A. and Davis, Simon J.. (2009) A human embryonic kidney 293T cell line mutated at the Golgi -mannosidase II locus. Journal of Biological Chemistry . ISSN 0021-9258

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Abstract

Disruption of Golgi -mannosidase II activity can result in type II congenital dyserythropoietic anemia and can induce lupus-like autoimmunity in mice. Here, we isolate a mutant human embryonic kidney (HEK) 293T cell line, called Lec36, that displays sensitivity to ricin that lies between the parental HEK 293T cells, whose secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which only produce oligomannose-type N-linked glycans. The 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 -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, dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi -mannosidase II: a point mutation in one allele mapping to the active site and an in-frame deletion of twelve-nucleotides in the other. Expression of 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 provides a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.

Item Type:	Journal Article
Subjects:	R Medicine > RC Internal medicine Q Science > QR Microbiology
Divisions:	Faculty of Science > Life Sciences (2010- ) > Biological Sciences ( -2010)
Library of Congress Subject Headings (LCSH):	Golgi apparatus, Cell organelles, Anemia, Glycosylation, Cell populations, Genetics
Journal or Publication Title:	Journal of Biological Chemistry
Publisher:	American Society for Biochemistry and Molecular Biology, Inc.
ISSN:	0021-9258
Date:	22 May 2009
Identification Number:	10.1074/jbc.M109.006254
Status:	Peer Reviewed
Access rights to Published version:	Open Access
Funder:	Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC), Fifth Framework Programme (European Commission) (FP5), National Institutes of Health (U.S.) (NIH), Oxford Glycobiology Institute Endowment (OGIE), Wellcome Trust (London, England), Cancer Research Campaign (Great Britain) (CRC), Medical Research Council (Great Britain) (MRC)
Grant number:	5 U01AI 65869 (NIH), QLG2-CT-2002-00988 (FFP),
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URI:	http://wrap.warwick.ac.uk/id/eprint/809

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