The Golgi CMP-sialic acid transporter: A new CHO mutant provides functional insights.
A CHO mutant line, MAR-11, was isolated using a cytotoxic lectin, Maackia amurensis agglutinin (MAA). This mutant has decreased levels of cell surface sialic acid relative to both wild-type CHO-K1 and Lec2 mutant CHO cells. The CMP-sialic acid transporter (CMP-SAT) gene in the MAR-11 mutant cell has a C-T mutation that results in a premature stop codon. As a result, MAR-11 cells express a truncated version of CMP-SAT which contains only 100 amino acids rather than the normal CMP-SAT which contains 336 amino acids. Biochemical analyses indicate that recombinant interferon-gamma (IFN-gamma) produced by the mutant cells lack sialic acid. Using MAR-11 as host cells, an EPO/IEF assay for the structure-function study of CMP-SAT was developed. This assay seems more sensitive than previous assays that were used to analyze sialylation in Lec2 cells. Cotransfection of constructs that express CMP-SAT into MAR-11 cells completely converted the recombinant EPO to a sialylation pattern that is similar to the EPO produced by the wild-type CHO cells. Using this assay, we showed that CMP-SAT lacking C-terminal 18 amino acids from the cytosolic tail was able to allow high levels of EPO sialylation. Substitution of the Gly residues with Ile in three different transmembrane domains of CMP-SAT resulted in dramatic decreases in transporter's activity. The CMP-SAT only lost partial activity if the same Gly residues were substituted with Ala, suggesting that the lack of side chain in Gly residues in the transmembrane domains is essential for transport activity.
Lim
Sing Fee
SF
Bioprocessing Technology Institute, Agency for Science, Technology and Research, Centros, Singapore.
Lee
May May
MM
Zhang
Peiqing
P
Song
Zhiwei
Z
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004921:Erythropoietin / Q000235:genetics / Q000378:metabolism; D037161:Galectins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D037161:Galectins; D027361:Organic Anion Transporters; D011994:Recombinant Proteins; D027981:Symporters; C421224:sialic acid transport proteins; D004921:Erythropoietin; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D007371:Interferon-gamma