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J. Biol. Chem., Vol. 276, Issue 17, 13924-13934, April 27, 2001

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Chinese Hamster Ovary (CHO) Cells May Express Six 4-Galactosyltransferases (4GalTs)
CONSEQUENCES OF THE LOSS OF FUNCTIONAL 4GalT-1, 4GalT-6, OR BOTH IN CHO GLYCOSYLATION MUTANTS^*

JaeHoon Lee, Subha Sundaram, Nancy L. Shaper^§, T. Shantha Raju^¶, and Pamela Stanley

From the  Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461, the ^§ Oncology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, and ^¶ Analytical Chemistry, Genentech Inc., South San Francisco, California 94080

Six 4-galactosyltransferase (4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat2 line of Chinese hamster ovary cells (Gat2 CHO). Two independent mutants termed Pro5Lec20 and Gat2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in 4GalT-1 transcripts in Pro5Lec20 cells. The Gat2Lec20 mutant expressed 4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the 4GalT-1 gene coding region. Northern analysis with probes from the remaining five 4GalT genes revealed that Gat2 CHO and Gat2Lec20 cells express all six 4GalT genes. Unexpectedly, the 4GalT-6 gene is not expressed in either Pro5 or Pro5Lec20 cells. Thus, in addition to a deficiency in 4GalT-1, Pro5Lec20 cells lack 4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the 4GalT-1 (Gat2Lec20) and 4GalT-1/4GalT-6 (Pro5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, 4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. 4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different 4GalTs. They may be used in glycosylation engineering and in investigating roles for 4GalT-1 and 4GalT-6 in generating specific glycan ligands.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF318896.

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