Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity.

Abstract

Lec1 CHO cell glycosylation mutants are defective in N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and therefore cannot convert the oligomannosyl intermediate (Man5GlcNAc2Asn) into complex carbohydrates. Lec1A CHO cell mutants have been shown to belong to the same genetic complementation group but exhibit different phenotypic properties. Evidence is presented that lec1A represents a new mutation at the lec1 locus resulting in partial loss of GlcNAc-TI activity. Structural studies of the carbohydrates associated with vesicular stomatitis virus grown in Lec1A cells (Lec1A/VSV) revealed the presence of biantennary and branched complex carbohydrates as well as the processing intermediate Man5GlcNAc2Asn. By contrast, the glycopeptides from virus grown in CHO cells (CHO/VSV) possessed only fully processed complex carbohydrates, whereas those from Lec1/VSV were almost solely of the Man5GlcNAc2Asn intermediate type. Therefore, the Lec1A glycosylation phenotype appears to result from the partial processing of N-linked carbohydrates because of reduced GlcNAc-TI action on membrane glycoproteins. Genetic experiments provided evidence that lec1A is a single mutation affecting GlcNAc-TI activity. Lec1A mutants could be isolated at frequencies of 10(-5) to 10(-6) from unmutagenized CHO cell populations by single-step selection, a rate inconsistent with two mutations. In addition, segregants selected from Lec1A X parental cell hybrid populations expressed only Lec1A or related lectin-resistant phenotypes and did not include any with a Lec1 phenotype. The Lec1A mutant should be of interest for studies on the mechanisms that control carbohydrate processing in animal cells and the effects of reduced GlcNAc-TI activity on the glycosylation, translocation, and compartmentalization of cellular glycoproteins.

Sialylated Complex-type N-Glycans Enhance the Signaling Activity of Soluble Intercellular Adhesion Molecule-1 in Mouse Astrocytes

“…Thus, Lec8 cells generate nongalactosylated and nonsialylated N-and O-glycans (37). The Lec1 mutant has a single insertion mutation that generates an inactive, truncated GlcNAc transferase I and hence stops processing N-glycans at the Man 5 GlcNAc 2 -Asn stage (38,39). Therefore, Lec1 cells lack sialylated and galactosylated N-glycans of the complex type, whereas their O-glycans have the normal amount of sialic acid and galactose.…”

Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N -acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line

“…Preparation of APTS-labeled Man5GlcNAc2. Bovine RNaseB N-glycans [Man (5)(6)(7)(8)(9) GlcNAc 2 ] (30 nmol) were treated with 50 mM APTS (1,000-fold excess) in a reaction mixture (60 l) containing citric acid (0.6 M), NaCNBH 3 (0.5 M), and DMSO (50% vol͞vol). Incubation was for 4 h at 55°C followed by 16 h at 37°C.…”

“…Such cell lines can be prepared by virtue of resistance to lectins and this approach has been used for preparation of Chinese hamster ovary cell lines with deficient Nglycosylation (4). Indeed, one class of Chinese hamster ovary mutants, designated Lec1, was shown to be N-acetylglucosaminyltransferase-I (GnTI) negative (5). Here, we report on the preparation of a ricin-resistant (ricin R ) HEK293 mutant cell line that has been characterized to be GnTI negative.…”

“…Here we describe the application of this cell line in the expression of WT rhodopsin and the constitutively active rhodopsin mutant, E113Q͞E134Q͞M257Y in 3-6 mg of the proteins per liter of cell culture. The N-glycans in both the expressed proteins have been characterized to be homogeneous Man 5 GlcNAc 2 . ¶…”

The epidermal growth factor–like domains of the human EMR2 receptor mediate cell attachment through chondroitin sulfate glycosaminoglycans

“…During sulphation inhibition assays, CHO-K1 cells were cultured for 2 days in Fischer medium supplemented with 10% dialyzed FCS and 10 mM sodium chlorate in the presence or absence of 10 mM MgSO 4 . Lec1 cells were cultured in minimum essential media (MEM)-Alpha with ribonucleosides and deoxyribonucleosides 26,27 and ARH-77 cells transfected with various proteoglycan expression constructs were grown in RPMI-1640 supplemented with 0.3 g/mL G418 (Gibco, Paisley, United Kingdom). 28,29 CHO-K1 cells expressing transmembrane EMR2 1-5 and EMR2 1,2,5 were selected in media containing G418 (1 mg/mL) for 10 days and further enriched by flow cytometric sorting using EMR2 stalk-specific 2A1 monoclonal antibody (mAb).…”