High-frequency transfection of CHO cells using polybrene

135

112

The three deletion variants were incapable of binding ALS; but of the site-specific variants, the MDGEA mutant bound ALS with 90% lower affinity (K a ‫؍‬ 2.5 ؎ 0.9 liters/nmol) than seen for rhIGF-BP-3 (K a ‫؍‬ 24.3 ؎ 5.2 liters/nmol), whereas the RGD mutation had no effect on ALS affinity (K a ‫؍‬ 21.7 ؎ 4.5 liters/nmol). The ability of IGFBP-3 to associate with the cell surface was lost in variants lacking residues 185-264 and in the 228 KGRKR 3 MDGEA mutant. We conclude that residues 228 -232 of IGFBP-3 are essential for cell association and are required for normal ALS binding affinity.

Section: Methodsmentioning

confidence: 99%

Structural Determinants of Ligand and Cell Surface Binding of Insulin-like Growth Factor-binding Protein-3

Firth

Ganeshprasad

Baxter

1998

135

112

“…Generation of ␤4GalT-1 Transfectants-Different amounts of plasmid pSVL DNA containing a bovine ␤4GalT-1 cDNA (a generous gift of Dr. Joel H. Shaper) were mixed with pSV2neo DNA (5 g) separately and transfected into Pro Ϫ 5Lec20 cells using the Polybrene method described previously (28). Transfectants were selected for resistance to G418 (1.5 mg/ml active weight).…”

Section: Matrix-assisted Laser Desorption/ionization Time-of-flight Mmentioning

confidence: 99%

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

Lee

Sundaram

Shaper

et al. 2001

Self Cite

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.

“…After addition of ␣-medium containing 20% fetal bovine serum to each well, the cells were incubated a further 18 h prior to immunofluorescence labeling and microscopy. Stable transfectants of MtxRII 5-3 cells were obtained using the Polybrene procedure described previously (44). Stable clones of the HA insertion constructs in either the 5Ј⌬RFC or 5Ј⌬RFC-EGFP background (described below) were selected and maintained in folic acid-free medium containing 10% dialyzed fetal bovine serum and 2 nM folinic acid (45).…”

Section: Chemicals-restrictionmentioning

confidence: 99%

Topological and Functional Analysis of the Human Reduced Folate Carrier by Hemagglutinin Epitope Insertion

Ferguson¹,

Flintoff

1999

The membrane topology of the human reduced folate carrier protein (591 amino acids) was assessed by single insertions of the hemagglutinin epitope into nine sites of the protein. Reduced folate carrier-deficient Chinese hamster ovary cells expressing each of these constructs were probed with anti-hemagglutinin epitope monoclonal antibodies to assess whether the insertion was exposed to the external environment or to the cytoplasm. The results are consistent with the 12-transmembrane topology predicted for this protein. The hemagglutinin epitope insertion mutants were also tested for their effects on the function of the reduced folate carrier. For these studies, each of the constructs had a carboxyl-terminal fusion of the enhanced green fluorescent protein to monitor and quantitate expression. Insertions into the external loop between transmembrane regions 7 and 8 (Pro-297), the cytoplasmic loop between transmembrane regions 6 and 7 (Ser-225), and near the cytoplasmic amino and carboxyl termini (Pro-20 and Gly-492, respectively) had minor effects on methotrexate binding and uptake. The insertion into the cytoplasmic loop between transmembrane regions 10 and 11 (Gln-385) greatly reduced both binding and uptake of methotrexate, whereas the insertion into the external loop between transmembrane regions 11 and 12 (Pro-427) selectively interfered with uptake but not binding.Mammalian cells require reduced folates for several biochemical pathways involving purine, pyrimidine, and amino acid metabolism. Since mammals cannot synthesize folates, these compounds must be obtained in the diet and imported by cells via either the glycosylphosphatidylinositol-linked folate receptor (1-5) or the reduced folate carrier (RFC), 1 an integral membrane protein (6 -9). A third, low pH-dependent component for folate transport has been reported (10, 11), but its involvement in folate delivery is not clearly understood. The RFC is the main route for transport of 5-methyltetrahydrofolate, the major circulating folate in the bloodstream, 5-formyltetrahydrofolate (folinic acid), and the folate analog, methotrexate (Mtx) (9,(12)(13)(14).The rfc gene has been cloned from hamster (15), human (16 -19), murine (20), and rat 2 sources. The identity of the rfc gene was verified by cDNA transfections that restored Mtx sensitivity to RFC-deficient cells (15)(16)(17)(18)(19)(20). The human RFC protein is 591 amino acids as deduced from its cDNA sequence, and the hydropathy profile predicts 12 transmembrane-spanning (TM) domains with the amino and carboxyl termini located in the cytoplasm. Recently, the cytoplasmic location of the carboxyl-terminal tail has been confirmed for the human RFC (22). The mouse and hamster homologues (518 amino acids each) have significantly shorter carboxyl-terminal tails than the human RFC (15-20), although all three RFCs share about 68% identical or similar amino acid residues. The human RFC is glycosylated at an asparagine residue in the first extracellular loop (9,22,23), and while the hamster RFC contains the conserv...