Lectin-resistant CHO cells: Selection of seven new mutants resistant to ricin

Stanley, Pamela; Sallustio, Sandra; Krag, Sharon S.; Dunn, Barbara K.

doi:10.1007/bf01233357

Cited by 20 publications

(19 citation statements)

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“…Two independent CHO mutants that were selected for resistance to PHA-E belong to the Lec20 complementation group and behave as loss-of-function mutants in somatic cell hybrids (21). In this study, we show that they are both defective in the ␤4-galactosylation of N-glycans due to independent mutations in the ␤4GalT-1 gene.…”

Section: ␤4-galactosyltransferase (␤4galt)mentioning

confidence: 80%

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Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Lee

Sundaram

Shaper

et al. 2001

Journal of Biological Chemistry

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Six ␤4-galactosyltransferase (␤4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat ؊ 2 line of Chinese hamster ovary cells (Gat ؊ 2 CHO). Two independent mutants termed Pro ؊ 5Lec20 and Gat ؊ 2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary Nglycans synthesized by Pro ؊ 5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in ␤4GalT-1 transcripts in Pro ؊ 5Lec20 cells. The Gat ؊ 2Lec20 mutant expressed ␤4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the ␤4GalT-1 gene coding region. Northern analysis with probes from the remaining five ␤4GalT genes revealed that Gat ؊ 2 CHO and Gat ؊ 2Lec20 cells express all six ␤4GalT genes. Unexpectedly, the ␤4GalT-6 gene is not expressed in either Pro ؊ 5 or Pro ؊ 5Lec20 cells. Thus, in addition to a deficiency in ␤4GalT-1, Pro ؊ 5Lec20 cells lack ␤4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the ␤4GalT-1 ؊ (Gat ؊ 2Lec20) and ␤4GalT-1 ؊ /␤4GalT-6 ؊ (Pro ؊ 5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, ␤4GalT-1 is a key enzyme in the galactosylation of Nglycans, but is not involved in glycolipid synthesis in CHO cells. ␤4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different ␤4GalTs. They may be used in glycosylation engineering and in investigating roles for ␤4GalT-1 and ␤4GalT-6 in generating specific glycan ligands.

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Section: ␤4-galactosyltransferase (␤4galt)mentioning

confidence: 80%

“…Cell Lines and Cell Cultures-Pro Ϫ 5, Gat Ϫ 2, Pro Ϫ 5Lec20 (clone 15C), and Gat Ϫ 2Lec20 (clone 6A) CHO cells were isolated as previously described (21). Cells were grown in suspension at 37°C in complete ␣-medium containing 10% fetal bovine serum.…”

Section: Materials-d-[6-mentioning

confidence: 99%

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Lee

Sundaram

Shaper

et al. 2001

Journal of Biological Chemistry

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“…In the present experiments, since we wished to reduce the possibility of producing cloned cells exhibiting multiple mutations, we avoided mutagens which have been used by others to raise lectin-resistant mutant cell lines [28][29][30]. Nevertheless, the selected cells were at least 7 times more resistant to ricin than parent Caco-2 cells, i.e.…”

Section: Discussionmentioning

confidence: 99%

Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance

Jackman

Ellis

Gray

et al. 1999

Biochemical Journal

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It has been proposed that killing of mammalian cells by ricin requires efficient endocytic delivery to the trans-Golgi network (TGN) prior to retrograde transport to the endoplasmic reticulum and entry to the cytosol. In polarized epithelial cells, an efficient membrane-traffic pathway to the TGN is present from the basolateral but not the apical plasma-membrane domain. Thus one can hypothesize that a ricin-resistant phenotype might be demonstrated by polarized cells that fail to differentiate and thus fail to develop an efficient membrane-traffic pathway from the basolateral plasma membrane to the TGN. We have isolated and studied a ricin-resistant Caco-2 cell clone (Caco-2-RCAr clone 2) which, when grown on plastic, was deficient in differentiation, measured by the development of polarized-cell-surface marker enzymes. The deficiency in differentiation was partially reversed, and ricin sensitivity was restored, when the cells were grown on filter supports. Our data provide the first evidence of a ricin-resistant cell line where resistance is due to the lack of development of polarized cell surfaces. The observed ricin resistance is consistent with the requirement that ricin is delivered to the TGN before its A chain enters the cytosol to mediate cell killing.

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“…Tout d'abord, les études structurales, biochimiques et de biologie cellulaire ont permis de découvrir les rôles attribués à certains N-glycanes spécifiques dans divers « processus » cellulaires. Ainsi, l'étude de lignées mutantes de levures [4] et de cellules de mammifères [5], porteuses d'erreurs métaboliques de la N-glycosylation a fait largement progresser notre compréhension de la biosynthèse et des fonctions des N-glycanes. Puis, à partir de modèles de souris transgéniques, il a été possible de déterminer l'importance de la présence, ou de l'absence, d'une structure glycanique particulière sur la physiologie de l'animal entier.…”

Section: Importance De La N-glycosylationunclassified