Translocation across golgi vesicle membranes: A CHO glycosylation mutant deficient in CMP-sialic acid transport

Deutscher, Susan L.; Nuwayhid, Naziha Faiz; Stanley, Pamela; Briles, Eve Ida Barak; Hirschberg, Carlos B.

doi:10.1016/0092-8674(84)90007-2

Cited by 258 publications

(178 citation statements)

References 27 publications

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“…The biochemical characterisation (Deutscher et al, 1984) strongly suggested that the gene defective in these cells encodes the Golgi CMP-Sia-Tr. Recently, we were able to confirm this hypothesis by cloning the gene defective in Lec2 cells .…”

Section: Discussionmentioning

confidence: 99%

Molecular Cloning of the Hamster CMP‐sialic Acid Transporter

Eckhardt

Gerardy‐Schahn

1997

European Journal of Biochemistry

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Chinese hamster ovary (CHO) glycosylation mutants of the Lec2 complementation group are unable to express sialylated glycoproteins and glycolipids due to a defect in the Golgi CMP-sialic acid transporter (CMP-Sia-Tr). Using an expression cloning strategy, we isolated a cDNA encoding the hamster CMPSia-Tr which complements the Lec2 phenotype. The deduced amino acid sequence of the cloned cDNA shows 95% identity to the recently cloned murine CMP-Sia-Tr. The expression of a hamster CMP-SiaTr fusion protein with an N-terminal MDYKDDDDK (FLAG) sequence revealed Golgi localisation of the transporter. Amino acid sequence comparison revealed strong similarity (44.6% identity and 19.3 % similarity) of CMP-Sia-Tr to the recently cloned human UDP-galactose transporter (UDP-Gal-Tr). In contrast, sequence similarities to the yeast UDP-N-acetylglucosamine transporter (UDP-GlcNAc-Tr) and the GDP-mannose transporter (GDP-Man-Tr) of Leishmania donovani are restricted to a region encoding the two most C-terminally located transmembrane helices. A computer-based structural analysis of CMPSia-Tr proposes an eight transmembrane helix model with the N-and C-termini located on the cytosolic side of the Golgi membrane.

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Section: Discussionmentioning

confidence: 99%

Molecular Cloning of the Hamster CMP‐sialic Acid Transporter

Eckhardt

Gerardy‐Schahn

1997

European Journal of Biochemistry

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“…The stained cells were subjected to FACS analysis as described above. CHO mutant Lec2 cells lack a functional Golgi CMP-sialic acid transporter; and therefore, sialylation is absent in Lec2 cells (34). The absence of sialylation facilitates core 1 extension because core 1 extension and sialylation compete with each other for the same acceptor, Gal␤133GalNAc␣13 R.…”

Section: Expression Of I Antigen In Hela Cells By Different ␤3glcnactmentioning

confidence: 99%

Extended Core 1 and Core 2 Branched O-Glycans Differentially Modulate Sialyl Lewis x-type L-selectin Ligand Activity

Mitoma

Petryniak

Hiraoka

et al. 2003

Journal of Biological Chemistry

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It has been established that sialyl Lewis x in core 2 branched O-glycans serves as an E-and P-selectin ligand. Recently, it was discovered that 6-sulfosialyl Lewis x in extended core 1 O-glycans, NeuNAc␣233Gal-␤134(Fuc␣133(sulfo36))GlcNAc␤13 3Gal␤133Gal-NAc␣13 Ser/Thr, functions as an L-selectin ligand in high endothelial venules. Extended core 1 O-glycans can be synthesized when a core 1 extension enzyme is present. In this study, we first show that ␤1,3-N-acetylglucosaminyltransferase-3 (␤3GlcNAcT-3) is almost exclusively responsible for core 1 extension among seven different ␤3GlcNAcTs and thus acts on core 1 O-glycans attached to PSGL-1. We found that transcripts encoding ␤3GlcNAcT-3 were expressed in human neutrophils and lymphocytes but that their levels were lower than those of transcripts encoding core 2 ␤1,6-N-acetylglucosaminyltransferase I (Core2GlcNAcT-I). Neutrophils also expressed transcripts encoding fucosyltransferase VII (FucT-VII) and Core2GlcNAcT-I, whereas lymphocytes expressed only small amounts of transcripts encoding FucT-VII. To determine the roles of sialyl Lewis x in extended core 1 O-glycans, Chinese hamster ovary (CHO) cells were stably transfected to express PSGL-1, FucT-VII, and either ␤3GlcNAcT-3 or Core2GlcNAcT-I. Glycan structural analyses disclosed that PSGL-1 expressed in these transfected cells carried comparable amounts of sialyl Lewis x in extended core 1 and core 2 branched O-glycans. In a rolling assay, CHO cells expressing sialyl Lewis x in extended core 1 O-glycans supported a significant degree of shear-dependent tethering and rolling of neutrophils and lymphocytes, although less than CHO cells expressing sialyl Lewis x in core 2 branched O-glycans. These results indicate that sialyl Lewis x in extended core 1 O-glycans can function as an L-selectin ligand and is potentially involved in neutrophil adhesion on neutrophils bound to activated endothelial cells.Mucin-type O-glycans are unique in having clusters of large numbers of O-glycans. These O-glycans contain N-acetylgalactosamine residues at reducing ends, which are linked to serine or threonine residues in a polypeptide (1). These attached Oglycans can be classified into several different groups according to the core structure (2). In many cells, a structure called core 1, Gal␤133GalNAc, is the major constituent of O-glycans. Core 1 oligosaccharides are converted to core 2 oligosaccharides, Gal␤133(GlcNAc␤136)GalNAc, when core 2 ␤1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT) 1 is present (3, 4). Various ligand carbohydrates can be formed in core 2 branched oligosaccharides. For example, sialyl Lewis x in mucin-type glycoproteins of blood cells can be found in core 2 branched oligosaccharides such as NeuNAc␣233Gal␤134(Fuc␣133)GlcNAc␤136(NeuNAc␣23 3Gal␤133)GalNAc␣13 Ser/Thr (see Fig.

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“…To further validate our system, we acquired the comparative lectin binding profiles of two additional Lec mutants: Lec1, which is deficient in GlcNAcT1, the enzyme necessary for hybrid and complex N-linked glycans; and Lec2, a mutant lacking CMP-sialic acid transport (24,25). Biological replicates of Lec1 clustered very tightly (R ϭ 0.98, n ϭ 9, P Ͻ 0.001) and displayed a marked increase in exposed mannose residues when compared with Pro-5 as reflected by differences in ConA and GNA binding (Fig.…”

Section: Hierarchical Clustering Of Lec Mutants Reveals Subtle Differmentioning

confidence: 99%

A ratiometric lectin microarray approach to analysis of the dynamic mammalian glycome

Pilobello

Slawek²,

Mahal

2007

Proc. Natl. Acad. Sci. U.S.A.

203

185

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Glycosylation creates an intricate and complex code for biological information that plays a role in cell-cell communication, infection, and immunity among many biological events. Dynamic changes in the glycosylation status of cells have been observed in tumor cell metastasis and cell differentiation but have been difficult to analyze because of a lack of high-throughput and facile technologies. Here, we present a method for the rapid evaluation of differences in the glycosylation of heterogeneous mammalian samples using a ratiometric two-color lectin microarray approach. This work represents a significant improvement in glycomics technology and sets the stage for the systematic evaluation of how glycans encode biological information in complex systems.carbohydrate analysis ͉ glycomics C arbohydrates are intricate information-carrying biopolymers of rising interest in the postgenomic age. Dynamic changes in glycosylation are observed in a myriad of key biological events in mammalian systems, including embryogenesis, neuronal development, and tumor cell metastasis. Despite their importance, how-

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Translocation across golgi vesicle membranes: A CHO glycosylation mutant deficient in CMP-sialic acid transport

Cited by 258 publications

References 27 publications

Molecular Cloning of the Hamster CMP‐sialic Acid Transporter

Molecular Cloning of the Hamster CMP‐sialic Acid Transporter

Extended Core 1 and Core 2 Branched O-Glycans Differentially Modulate Sialyl Lewis x-type L-selectin Ligand Activity

A ratiometric lectin microarray approach to analysis of the dynamic mammalian glycome

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