Mutants of the CMP-sialic Acid Transporter Causing the Lec2 Phenotype

Eckhardt, Matthias; Gotza, Birgit; Gerardy‐Schahn, Rita

doi:10.1074/jbc.273.32.20189

Cited by 104 publications

(90 citation statements)

References 33 publications

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“…Additional independent Lec8 mutants listed in Table I were selected for lectin resistance and shown by complementation in somatic cell hybrids to belong to the Lec8 complementation group. 6B2 cells belong to complementation group Lec2 and were characterized previously (21). CHO K1 cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 (Life Technologies, Inc.) supplemented with 5% fetal calf serum, 1 mM sodium pyruvate, penicillin (50 units/ml), and streptomycin (50 g/ml).…”

Section: Methodsmentioning

confidence: 99%

“…Transient Transfections-Recipient cells for transient transfections were the Lec8 subclone Lec4.8.7A and the Lec2 subclone 6B2 (21). Expression was monitored by Western analysis, and UGT function was measured by the ability to produce sialylated NCAM detected by anti-PSA antibody (22).…”

Section: Reverse Transcription Polymerase Chain Reaction (Rt-pcr)-1 Gmentioning

confidence: 99%

“…The nonapeptide underlined in the FLAGsequence is recognized by the anti-FLAG mAb M5. The construction of the FLAG-tagged murine CST has been described previously (21).…”

Section: Udp-galactose Transporter Mutationsmentioning

confidence: 99%

“…Such an analysis in CHO Lec2 cells (defective in Golgi transport of CMP-sialic acid) and in murine Had-1 cells identified two highly conserved amino acids (Gly 189 in the hamster CST and Gly 178 in the murine UGT) that are likely to participate in the folding of active transporters (11,21).…”

mentioning

confidence: 99%

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Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

Oelmann

Stanley

Gerardy‐Schahn

2001

Journal of Biological Chemistry

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Nucleotide-sugar transporters (NSTs) are critical components of glycosylation pathways in eukaryotes. The identification of structural elements that are involved in NST functions provides an important task. Chinese hamster ovary glycosylation mutants defective in nucleotide-sugar transport provide access to inactive transporters that can define such structure/function relationships. In this study, we have cloned the hamster UDP-galactose transporter (UGT) and identified defects in UGT gene transcripts from nine independent Chinese hamster ovary mutants that belong to the Lec8 complementation group. Reverse transcription polymerase chain reaction with primers that span the UGT open reading frame showed that three Lec8 mutants express a full-length open reading frame, while six Lec8 mutants predominantly express truncated UGT gene transcripts. Sequencing identified different single or triplet nucleotide changes in full-length UGT transcripts from three of the mutants. These mutations translate into three different amino acid changes at positions that are highly conserved in all the known mammalian NSTs. Transfection of a cDNA encoding either of the mutations ⌬serine 213 or G281D failed to correct the UDP-galactose transport defect in Lec8 transfectants. Most importantly, introducing these same mutations into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter. Thus, identifying point mutations that inactivate UGT in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CST, the two most divergent mammalian NSTs.

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Section: Methodsmentioning

confidence: 99%

Section: Reverse Transcription Polymerase Chain Reaction (Rt-pcr)-1 Gmentioning

confidence: 99%

“…The nonapeptide underlined in the FLAGsequence is recognized by the anti-FLAG mAb M5. The construction of the FLAG-tagged murine CST has been described previously (21).…”

Section: Udp-galactose Transporter Mutationsmentioning

confidence: 99%

mentioning

confidence: 99%

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Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

Oelmann

Stanley

Gerardy‐Schahn

2001

Journal of Biological Chemistry

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“…sICAM-1 was expressed in stably transfected Lec2, Lec8, and Lec1 glycosylation mutants of CHO cells (Table I). The Lec2 mutant has a deletion mutation in the CMP-sialic acid transporter resulting in reduced targeting of the trans- porter to the Golgi (34). This defect in CMP-sialic acid transport results in N-and O-glycans with a greater than 90% decrease in sialic acid content (35).…”

Section: Sialylation and Galactosylation Of Sicam-1 Expressed In Cho mentioning

confidence: 99%

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

Otto

Schürpf

Folkers

et al. 2004

Journal of Biological Chemistry

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Intercellular adhesion molecule-1 (ICAM-1) occurs as both a membrane and a soluble, secreted glycoprotein (sICAM-1). ICAM-1 on endothelial cells mediates leukocyte adhesion by binding to leukocyte function associated antigen-1 (LFA-1) and macrophage antigen-1 (Mac-1). Recombinant mouse sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2) in mouse astrocytes by a novel LFA-1-and Mac-1-independent mechanism. Here we showed that N-glycan structures of sI-CAM-1 influence its ability to induce MIP-2 production. sICAM-1 expressed in Chinese hamster ovary (CHO) cells was a more potent inducer of MIP-2 production than sI-CAM-1 expressed in HEK 293 cells, suggesting that posttranslational modification of sICAM-1 could influence its signaling activity. To explore the roles of glycosylation in sICAM-1 activity, we expressed sICAM-1 in mutant CHO cell lines differing in glycosylation, including Lec2, Lec8, and Lec1 as well as in CHO cells cultured in the presence of the ␣-mannosidase-I inhibitor kifunensine. Signaling activity of sICAM-1 lacking sialic acid was reduced 3-fold compared with sICAM-1 from CHO cells. The activity of sICAM-1 lacking both sialic acid and galactose was reduced 12-fold, whereas the activity of sICAM-1 carrying only high mannose-type N-glycans was reduced 12-26-fold. sICAM-1 glycoforms carrying truncated glycans retained full ability to bind to LFA-1 on leukocytes. Thus, sialylated and galactosylated complex-type N-glycans strongly enhanced the ability of sICAM-1 to induce MIP-2 production in astrocytes but did not alter its binding to LFA-1 on leukocytes. Glycosylation could therefore serve as a means to regulate specifically the signaling function of sICAM-1 in vivo.

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Rapid Assays for Lectin Toxicity and Binding Changes that Reflect Altered Glycosylation in Mammalian Cells

Stanley

Sundaram

2014

CP Chemical Biology

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Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain‐of‐function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large‐volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan‐binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss‐of‐function or gain‐of‐function mutation in a specific gene involved in glycan synthesis. Curr. Protoc. Chem. Biol. 6:117‐133 © 2014 by John Wiley & Sons, Inc.

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Mutants of the CMP-sialic Acid Transporter Causing the Lec2 Phenotype

Cited by 104 publications

References 33 publications

Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

Point Mutations Identified in Lec8 Chinese Hamster Ovary Glycosylation Mutants That Inactivate Both the UDP-galactose and CMP-sialic Acid Transporters

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

Rapid Assays for Lectin Toxicity and Binding Changes that Reflect Altered Glycosylation in Mammalian Cells

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