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Glycobiology Advance Access published online on August 25, 2004
Glycobiology, doi:10.1093/glycob/cwh136
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Received May 28, 2004
Revised August 9, 2004
Accepted August 18, 2004

ORIGINAL ARTICLES

Molecular analysis of three gain-of-function CHO mutants that add the bisecting GlcNAc to N-glycans

Pamela Stanley 1*, Subha Sundaram 1, Jian Tang 1, Shaolin Shi 1

1 Dept. Cell Biology, Albert Einstein College Medicine, New York, 10461

* To whom correspondence should be addressed. E-mail: stanley{at}aecom.yu.edu.


  Abstract

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. Here we show that three CHO mutants termed LEC10, LEC10A and LEC10B arose due to transcriptional activation of the quiescent CHO Mgat3 gene. They each express Mgat3 gene transcripts of ~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 that is 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 mass spectrometry 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.

Keywords: N-acetylglucosaminyltransferase III; LEC10 mutants; complex bisected N-glycans; MALDI-TOF mass spectrometry.
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S. K. Patnaik, B. Potvin, S. Carlsson, D. Sturm, H. Leffler, and P. Stanley
Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells
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