Chinese hamster ovary (CHO) cells may express six β 4-galactosyltransferases (β4GalTs). Consequences of the loss of functional β4Galt-1, β4GalT-6, or both in CHO glycosylation mutants

Jae Hoon Lee, Subha Sundaram, Nancy L. Shaper, T. Shantha Raju, Pamela Stanley

Research output: Contribution to journalArticlepeer-review

55 Scopus citations

Abstract

Six β4-galactosyltransferase (β4GalT) 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 β4GalT-1 transcripts in Pro-SLec20 cells. The Gat-2Lec20 mutant expressed β4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the β4GalT-1 gene coding region. Northern analysis with probes from the remaining five β4GalT genes revealed that Gat-2 CHO and Gat-2Lec20 cells express all six β4GalT genes. Unexpectedly, the β4GalT-6 gene is not expressed in either Pro-5 or Pro-SLec20 cells. Thus, in addition to a deficiency in β4GalT-1, Pro-SLec20 cells lack β4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the β4GalT-1- (Gat-2Lec20) and β4GalT-1-/β4GalT-6- (Pro-SLec20) 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, β4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. β4GalT-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 β4GalTs. They may be used in glycosylation engineering and in investigating roles for β4GalT-1 and β4GalT-6 in generating specific glycan ligands.

Original languageEnglish (US)
Pages (from-to)13924-13934
Number of pages11
JournalJournal of Biological Chemistry
Volume276
Issue number17
DOIs
StatePublished - Apr 27 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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