Independent Lec1A CHO glycosylation mutants arise from point mutations in N-acetylglucosaminyltransferase I that reduce affinity for both substrates. Molecular consequences based on the crystal structure of GlcNAc-TI.
A key enzyme in regulating the maturation of N-linked glycans is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC 2.4.1.101). Lec1 CHO cells lack GlcNAc-TI activity and synthesize only the oligomannosyl class of N-glycans. By contrast, Lec1A CHO mutants have weak GlcNAc-TI activity due to the reduced affinity of GlcNAc-TI for both the UDP-GlcNAc and Man(5)GlcNAc(2)Asn substrates. Lec1A CHO mutants synthesize hybrid and complex N-glycans, albeit in reduced amounts compared to parental CHO cells. In this paper, we identify two point mutations that gave rise to the Lec1A phenotype in three independent Lec1A CHO mutants. The G634A mutation in Lec1A.2C converts an aspartic acid to an asparagine at amino acid 212, disrupting a conserved DXD motif (E(211)DD(213) in all GlcNAc-TIs) that makes critical interactions with bound UDP-GlcNAc and Mn(2+) ion in rabbit GlcNAc-TI. The C907T mutation in Lec1A.3E and Lec1A.5J converts an arginine conserved in all GlcNAc-TIs to a tryptophan at amino acid 303, altering interactions that are important in stabilizing a critical structural element in rabbit GlcNAc-TI. Correction of each mutation by site-directed mutagenesis restored their GlcNAc-TI activity and lectin binding properties to parental levels. The effect of the two amino acid changes on GlcNAc-TI catalysis is discussed in relation to the crystal structure of rabbit GlcNAc-TI complexed with manganese and UDP-GlcNAc.
Chen
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W
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Unligil
U M
UM
Rini
J M
JM
Stanley
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P
D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D016466:CHO Cells; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D011817:Rabbits; D013379:Substrate Specificity / Q000235:genetics; D014158:Transcription, Genetic
D001224:Aspartic Acid; D001120:Arginine; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I