Isolation of new CHO cell mutants defective in CMP-sialic acid biosynthesis and transport.
Sialic acid is a sugar typically found at the N-glycan termini of glycoproteins in mammalian cells. Lec3 CHO cell mutants are deficient in epimerase activity, due to a defect in the gene that encodes a bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sialic acid modification on the cell surface is partially affected in these cells. We have mutagenized Lec3 CHO cells and isolated six mutants (termed C2m) deficient in the cell surface expression of polysialic acid (PSA). Mutant C2m9 was partially defective in expression of cell-surface PSA and wheat germ agglutinin (WGA) binding, while in the other five mutants, both cell-surface PSA and WGA binding were undetectable. PSA expression was restored by complementation with the gene encoding the CMP-sialic acid transporter (CST), indicating that CST mutations were responsible for the phenotypes of the C2m cells. We characterized the CST mutations in these cells by Northern blotting and RT-PCR. C2m9 and C2m45 carried missense mutations resulting in glycine to glutamate substitutions at amino acids 217 (G217E) and 256 (G256E), respectively. C2m13, C2m39 and C2m31 had nonsense mutations that resulted in decreased CST mRNA stability, and C2m34 carried a putative splice site mutation. PSA and CD15s expression in CST-deficient Lec2 cells were partially rescued by G217E CST, but not by G256E CST, although both proteins were expressed at similar levels, and localized to the Golgi. These results indicate that the novel missense mutations isolated in this study affect CST activity.
Shin
Dong-Jun
DJ
Genomic Research Center for Enteropathogenic Bacteria and Department of Microbiology, Chonnam National University Medical School, Gwangju 501-746, Korea.
Kang
Ji Young
JY
Kim
Youn Uck
YU
Yoon
Joong Sik
JS
Choy
Hyon E
HE
Maeda
Yusuke
Y
Kinoshita
Taroh
T
Hong
Yeongjin
Y
D000818:Animals; D001692:Biological Transport; D015152:Blotting, Northern; D016466:CHO Cells; D002238:Carbohydrate Epimerases / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000378:metabolism; D020125:Mutation, Missense; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D033704:Nucleotide Transport Proteins / Q000235:genetics; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012794:Sialic Acids / Q000378:metabolism
D008562:Membrane Glycoproteins; D019006:Neural Cell Adhesion Molecules; D033704:Nucleotide Transport Proteins; D012794:Sialic Acids; C021319:polysialic acid; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase