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Papers In Press, published online ahead of print February 2, 2001
Cell Biology, Albert Einstein College Medicine, New York, NY 10461
Corresponding Author: stanley{at}aecom.yu.edu
Six
J. Biol. Chem, 10.1074/jbc.M010046200
Submitted on November 3, 2000
Revised on January 31, 2001
Accepted on February 2, 2001
CHO Cells May Express Six
4Galactosyltransferases. Consequences of the Loss of Functional 4GalT-1, 4GalT-6 or Both in CHO Glycosylation Mutants
4GalT genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat-2 line of Chinese hamster ovary cells (Gat-2 CHO). Two independent mutants termed Pro-5Lec20 and Gat-2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro-5Lec20 were proportionately less ricin-bound than similar species from parent CHO cells and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Transfection of a bovine 4GalT-1 cDNA reverted the lectin resistance phenotype and increased the 4GalT activity of Pro-5Lec20 cells. Northern blot analysis revealed a severe reduction in 4GalT-1 transcripts in Pro-5Lec20 cells. The Gat-2Lec20 mutant expressed 4GalT-1 transcripts of reduced size due to a 311 bp deletion in the 4GalT-1 gene coding region. Northern analysis with probes from the remaining five 4GalT genes revealed that Gat-2 CHO and Gat-2Lec20 cells express all six 4GalT genes. Unexpectedly, the 4GalT-6 gene is not expressed in either Pro-5 or Pro-5Lec20 cells. Thus in addition to a deficiency in 4GalT-1, Pro-5Lec20 cells lack 4GalT-6. Nevertheless, MALDI-TOF mass spectral data of N-glycans released from cellular glycoproteins showed that both the 4GalT-1- (Gat-2Lec20) and 4GalT-1-/4GalT-6- (Pro-5Lec20) mutants have a similar Gal deficiency affecting neutral and sialylated bi-, tri-, and tetra-antennary 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 to investigate roles for 4GalT-1 and 4GalT-6 in generating specific glycan ligands.
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