Isolation of Chinese hamster ovary cell lines producing Man3GlcNAc2 asparagine-linked glycans

Zeng, Yucheng; Lehrman, Mark A.

doi:10.1016/0003-2697(91)90020-t

Cited by 24 publications

(20 citation statements)

References 19 publications

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“…3 H-Labeled LLOs were extracted with organic solvent, fractionated by HPLC, and detected as described previously (6,19). The proteinaceous pellet remaining after organic extraction was digested with N-glycanase (Calbiochem catalogue number 362185) as directed by the manufacturer, and the released N-glycans were analyzed by HPLC as described (22). In some experiments the amounts of [ 3 H]mannose were varied, or 0.1 mM unlabeled D-mannose was included during the 20-min labeling period.…”

Section: Analysis Of [ 3 H]mannose-labeled Llos and N-linkedmentioning

confidence: 99%

Metformin-stimulated Mannose Transport in Dermal Fibroblasts

Shang

Lehrman

2004

Journal of Biological Chemistry

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The biguanide drug metformin stimulates AMP-activated protein kinase, a master regulator of cellular energy metabolism, and has antihyperglycemic activity due to attenuation of gluconeogenesis in hepatocytes and 2-fold stimulation of glucose transport by skeletal muscle. Here we identify a metformin-stimulated D-mannose transport (MSMT) activity in dermal fibroblasts. MSMT increased mannose uptake 1.8-fold and had greater affinity for mannose than basal mannose transport activity. It was attributed to robust stimulation of a transporter expressed weakly in untreated cells. MSMT was not explained by greater glucose transporter activity because metformin unexpectedly decreased transport of 2-deoxy-D-glucose and 3-O-methyl-D-glucose by fibroblasts. Effective inhibitors of MSMT retained specificity for the 3-, 4-, and 6-OH groups of the mannose ring but not the 2-OH group. Thus, MSMT could be strongly inhibited by glucose and 2-deoxy-D-glucose even though the latter was not a good transport substrate. MSMT was significant because in the presence of 2.5 M mannose, metformin corrected experimentally induced deficiencies in the synthesis of glucose 3 mannose 9 GlcNAc 2 -P-Pdolichol and N-linked glycosylation. MSMT was also identified in congenital disorder of glycosylation types Ia and Ib fibroblasts, and metformin acted synergistically with 100 M mannose to correct lipid-linked oligosaccharide synthesis and N-glycosylation in the Ia cells. In conclusion, metformin activates a novel fibroblast mannose-selective transport system. This suggests that AMP-activated protein kinase may be a regulator of mannose metabolism and implies a therapy for congenital disorders of glycosylation-Ia.

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Section: Analysis Of [ 3 H]mannose-labeled Llos and N-linkedmentioning

confidence: 99%

Metformin-stimulated Mannose Transport in Dermal Fibroblasts

Shang

Lehrman

2004

Journal of Biological Chemistry

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“…The LLO fractions of intact and permeablized cells were recovered, treated with mild acid to hydrolyze the pyrophosphate linkages between the dolichol and oligosaccharide moieties, and analyzed by HPLC as described (Turco, 1981;Zeng and Lehrman, 1991), except that final cleanup of reduced oligosaccharides was achieved by treatments with Dowex 50WX8-200 (hydrogen form) followed by Dowex AG1-X8 (formate form). In figures showing HPLC profiles, the positions of the following tritium-labeled standards [obtained in the general manner described (Zeng and Lehrman, 1991) ]MPC, and mannose-␤-1-P-nerol (MPN) were prepared as described (Rush et al, 1993) …”

Section: In Vitro C-mannosyltransferase Assaymentioning

confidence: 99%

Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

Anand

Rush

Ray

et al. 2001

MBoC

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The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-␤-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

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“…Since the action of specific glycosyltransferases generates the observed variety, questions of glycan function can be addressed by inactivating genes that code for these enzymes (2)(3)(4)(5)(6). Interestingly, glycosylationdefective cell mutants with a highly simplified spectrum of surface glycans grow normally in culture (7)(8)(9)(10). Thus, Chinese hamster ovary (CHO) cells lacking N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101) and synthesizing only oligomannosyl carbohydrates (Man5_9GlcNAc2Asn) at Nglycan sites, suffer no apparent consequences (8,9).…”

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confidence: 99%

Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.

Ioffe

Liu

Stanley³

1996

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

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Mice lacking the complex subset ofN-glycans due to inactivation of the Mgatl gene die at mid-gestation, making it difficult to identify specific biological functions for this class of cell surface carbohydrates. To circumvent this embryonic lethality and to uncover tissue-specific functions for complex N-glycans, WW6 embryonic stem cells with inactivated Mgatl alleles were tracked in chimeric embryos. The Mgatl gene encodes N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101), the transferase that initiates the synthesis of complex N-glycans. WW6 cells carry an inert .8-globin transgene that allows their identification in chimeras by DNA-DNA in situ hybridization. Independent MgatP -' and Mgatl +1-mutant WW6 isolates contributed like parent WW6 cells to the tissues of embryonic day (E) 10.5 to E16.5 chimeras. However, a cell type-specific difference was observed in lung. Homozygous null Mgatl -l WW6 cells did not contribute to the epithelial layer in more than 999o bronchi. This deficiency was corrected by transfection of a Mgatl transgene. Interestingly, heterozygous Mgatl+'-WW6 cells were also deficient in populating the layer of bronchial epithelium. Furthermore, examination of lung bud in E9.5

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Isolation of Chinese hamster ovary cell lines producing Man3GlcNAc2 asparagine-linked glycans

Cited by 24 publications

References 19 publications

Metformin-stimulated Mannose Transport in Dermal Fibroblasts

Metformin-stimulated Mannose Transport in Dermal Fibroblasts

Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.

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