Molecular analysis of three gain-of-function CHO mutants that add the bisecting GlcNAc to N-glycans.
LEC10 Chinese hamster ovary (CHO) cells are gain-of-function mutants that express N-acetylglucosaminyltransferase III (GlcNAc-TIII), the glycosyltransferase that adds the bisecting GlcNAc to complex N-glycans. LEC10 cells are useful for glycosylation engineering of recombinant glycoproteins, including antibody therapeutics, for defining lectin recognition specificities and for determining biological functions of the bisecting GlcNAc. We show that three CHO mutants, LEC10, LEC10A, and LEC10B, arose due to transcriptional activation of the quiescent CHO Mgat3 gene. They each express Mgat3 gene transcripts of approximately 4.7 kb at different levels (LEC10B > LEC10 > LEC10A). Southern analyses gave a single band in LEC10, LEC10A, and parent CHO DNA with four restriction enzymes but an additional band with three of them in LEC10B genomic DNA, indicative of a duplication event in LEC10B. The deduced amino acid sequence of the Mgat3 gene expressed in each CHO mutant and in parent CHO genomic DNA is identical. However, 5' UTR sequences differ with LEC10 and LEC10B containing a 5' UTR segment of the Atf4 gene downstream of the Mgat3 gene in human and mouse. Somatic cell hybrid analysis indicated that the LEC10B Mgat3 gene was induced by a cis mechanism. LEC10B glycoproteins bound more erythroagglutinin lectin (E-PHA) than LEC10 glycoproteins and MALDI-TOF MS revealed a broad spectrum of complex, bisected N-glycans expressed by the LEC10B mutant. LEC10B is therefore the cell line of choice for producing recombinant glycoproteins carrying bisected N-glycans and for investigating biological functions of the bisecting GlcNAc.
Department of Cell Biology, Albert Einstein stanley@aecom.yu.edu
Pamela
P
Stanley
Subha
S
Sundaram
Jian
J
Tang
Shaolin
S
Shi
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D006023:Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D006023:Glycoproteins; D011134:Polysaccharides; D012333:RNA, Messenger; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine