A mutation causing a reduced level of expression of six beta4-galactosyltransferase genes is the basis of the Lec19 CHO glycosylation mutant.
To identify factors required for the synthesis of complex glycans, we have isolated Chinese hamster ovary (CHO) cell mutants resistant to plant lectins. We previously identified Lec19 CHO cells as resistant to the Gal-binding lectins ricin, abrin, and modeccin and hypersensitive to the toxicity of other lectins that bind Gal, including L-PHA and E-PHA. Here we show that Lec19 cell extracts have a decreased ability to transfer Gal to simple sugar, oligosaccharide, and glycopeptide acceptors, particularly to biantennary, GlcNAc-terminated acceptors. Ricin(II)-agarose lectin affinity chromatography, oligomapping, and monosaccharide analyses provided evidence that Lec19 N-glycans have fewer Gal residues than CHO N-glycans. MALDI-TOF mass spectra of N-glycans released from Lec19 cell glycoproteins by peptide N-glycanase F revealed species with the predicted masses of neutral N-glycans with few Gal residues. Such truncated species are essentially absent from CHO cell glycoproteins. However, the complement of fully galactosylated or sialylated bi-, tri-, and tetra-antennary N-glycans was largely equivalent in Lec19 and CHO cells. In addition, the coding region sequences of the beta4GalT-1, -T-2, -T-3, -T-4, -T-5, and -T-6 genes were identical in CHO and Lec19 cells. However, Northern analyses revealed an approximately 2-4-fold reduction in the level of transcripts of all six beta4GalT genes in Lec19 cells. Since the recessive Lec19 phenotype is the result of a loss-of-function mutation, the combined data predict the existence of a trans-acting regulator of the steady-state level of transcripts that derive from these six mammalian beta4GalT genes.
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
JaeHoon
J
Lee
Sung-Hae
SH
Park
Subha
S
Sundaram
T Shantha
TS
Raju
Nancy L
NL
Shaper
Pamela
P
Stanley
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D017931:DNA Primers; D005700:Galactosyltransferases / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D037102:Lectins / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D037102:Lectins; D005700:Galactosyltransferases