Abstract
Six beta4-galactosyltransferase (beta4GalT) 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 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 beta4GalT-1 transcripts in Pro(-)5Lec20 cells. The Gat(-)2Lec20 mutant expressed beta4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the beta4GalT-1 gene coding region. Northern analysis with probes from the remaining five beta4GalT genes revealed that Gat(-)2 CHO and Gat(-)2Lec20 cells express all six beta4GalT genes. Unexpectedly, the beta4GalT-6 gene is not expressed in either Pro(-)5 or Pro(-)5Lec20 cells. Thus, in addition to a deficiency in beta4GalT-1, Pro(-)5Lec20 cells lack beta4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the beta4GalT-1(-) (Gat(-)2Lec20) and beta4GalT-1(-)/beta4GalT-6(-) (Pro(-)5Lec20) 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, beta4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. beta4GalT-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 beta4GalTs. They may be used in glycosylation engineering and in investigating roles for beta4GalT-1 and beta4GalT-6 in generating specific glycan ligands.
Highlights
Six 4-galactosyltransferase (4GalT) genes have been cloned from mammalian sources
Radiolabeled biantennary Nglycans synthesized by Pro؊5Lec20 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
We report that ProϪ5 CHO cells lack 4GalT-6 transcripts; and ProϪ5Lec20 mutants derived from ProϪ5 CHO cells lack both 4GalT-1 and 4GalT-6 activities
Summary
Materials—D-[6-3H]Gal (31.5 Ci/mmol), D-[6-3H]GlcN hydrochloride (31.5 Ci/mmol), UDP-[6-3H]GlcNAc (41.60 Ci/nmol), UDP-[6-3H]Gal (10 Ci/mmol), and ConA-Sepharose were from Amersham Pharmacia Biotech. Cell-free extract was prepared in 1.5% Triton X-100 after cell washing, to which glycerol was added to 20% by volume before storage at Ϫ80 °C as described [23]. Desialylated glycopeptides (3.2 mg of phenol-sulfuric acid-positive material) prepared from fetuin by exhaustive Pronase digestion and mild acid treatment (0.01 M HCl, 80 °C, 2 h) were digested with -galactosidases (jack bean, D. pneumoniae, and bovine testis) separately. Glycopeptides were digested with bovine testis -galactosidase in 50 mM sodium citrate/phosphate buffer, pH 4.3, at 37 °C for 48 h with a total of 40 milliunits enzyme, desalted, and freeze-dried (ϳ1.8 mg of phenol-sulfuric acid-positive material based on the standard curve for mannose). The PHA-L-retarded glycopeptides (1.5 mg) were digested with -galactosidase as described above, desalted, and freeze-dried (0.55 mg of phenol-sulfuric acid-positive material). The dried plate was stained by resorcinol/H2SO4 reagent and scanned
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