scholarly journals Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods

2004 ◽  
Vol 378 (1) ◽  
pp. 201-206 ◽  
Author(s):  
Wei CAO ◽  
Larry H. MATHERLY
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Transmembrane Domains ◽  
Membrane Topology ◽  
Cysteine Residues ◽  
Reduced Folate Carrier ◽  
Cell Permeabilization ◽  
Topology Structure ◽  
Ovary Cells ◽  
Pre Treatment

The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting TMDs 6 and 7. Previous studies using haemagglutinin epitope insertion and scanning glycosylation mutagenesis methods verified portions of the predicted topology model, including TMDs 1–8 and the N- and C-termini of hRFC. However, the topology structure for TMDs 9–12 remains controversial. To further determine the membrane topology of the hRFC protein, single cysteine residues were introduced into the predicted extracellular or cytoplasmic loops of a fully functional cysteine-less hRFC expressed in transport impaired MtxRIIOuaR2-4 Chinese hamster ovary cells. The membrane orientations of the substituted cysteines were determined by treatments with the thiol reagents 3-(N-maleimidylpropionyl)-biocytin (biotin maleimide) and 4-acetamido-4´maleimidylstilbene-2,2´-disulphonic acid (stilbenedisulphonate maleimide; SM) or N-ethylmaleimide, combined with the cell-permeabilizing reagent SLO (streptolysin O). We found that cysteine residues placed in the predicted extracellular loops between TMDs 7 and 8 (position 301), 9 and 10 (360), and 11 and 12 (429) could be biotinylated with 200 µM biotin maleimide, and labelling could be blocked with SM. However, biotinylation of cysteines placed in the predicted intracellular loops between TMDs 8 and 9 (position 332) and TMDs 10 and 11 (position 388) was only detected after cell permeabilization with SLO and was abolished by pre-treatment with N-ethylmaleimide. These results strongly support a 12-TMD topology structure for the hRFC protein.

Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane

Blood ◽  
2003 ◽  
Vol 102 (5) ◽  
pp. 1884-1892 ◽  
Author(s):  
John R. Forbes ◽  
Philippe Gros
Keyword(s):  
Plasma Membrane ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Topology ◽  
Co2 Uptake ◽  
Ovary Cells ◽  
Susceptibility To Infection ◽  
Transport Studies ◽  
Phagosomal Membrane ◽  
Iron Manganese

AbstractMutations in the Nramp1 gene (Slc11a1) cause susceptibility to infection by intracellular pathogens. The Nramp1 protein is expressed at the phagosomal membrane of macrophages and neutrophils and is a paralog of the Nramp2 (Slc11a2) iron transporter. The Nramp1 transport mechanism at the phagosomal membrane has remained controversial. An Nramp1 protein modified by insertion of a hemagglutinin epitope into the predicted TM7/8 loop was expressed at the plasma membrane of Chinese hamster ovary cells as demonstrated by immunofluorescence and surface biotinylation. Experiments in Nramp1HA transfectants using the metal-sensitive fluorophors calcein and Fura2 showed that Nramp1HA can mediate Fe2+, Mn2+, and Co2+ uptake. Similar results were obtained in transport studies using radioisotopic 55Fe2+ and 54Mn2+. Nramp1HA transport was dependent on time, temperature, and acidic pH, occurring down the proton gradient. These experiments suggest that Nramp1HA may be a more efficient transporter of Mn2+ compared to Fe2+ and a more efficient Mn2+ transporter than Nramp2HA. The membrane topology and transport properties of Nramp1HA and Nramp2HA were indistinguishable, suggesting that Nramp1 divalent-metal transport at the phagosomal membrane is mechanistically similar to that of Nramp2 at the membrane of acidified endosomes. These results clarify the mechanism by which Nramp1 contributes to phagocyte defenses against infections.

scholarly journals Human Acyl-Coenzyme A:Cholesterol Acyltransferase Expressed in Chinese Hamster Ovary Cells: Membrane Topology and Active Site Location

2003 ◽  
Vol 14 (6) ◽  
pp. 2447-2460 ◽  
Author(s):  
Song Lin ◽  
Xiaohui Lu ◽  
Catherine C.Y. Chang ◽  
Ta-Yuan Chang
Keyword(s):  
Chinese Hamster Ovary ◽  
Active Site ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Protein Band ◽  
Membrane Topology ◽  
Site Location ◽  
Ovary Cells ◽  
Membrane Bound ◽  
Cytoplasmic Side

Acyl-CoA:cholesterol acyltransferase (ACAT) is a membrane-bound enzyme that produces cholesteryl esters intracellularly. Two ACAT genes (ACAT1 and ACAT2) have been identified. The expression of ACAT1 is ubiquitous, whereas that of ACAT2 is tissue restricted. Previous research indicates that ACAT1 may contain seven transmembrane domains (TMDs). To study ACAT2 topology, we inserted two different antigenic tags (hemagglutinin, monoclonal antibody Mab1) at various hydrophilic regions flanking each of its predicted TMDs, and expressed the recombinant proteins in mutant Chinese hamster ovary cells lacking endogenous ACAT. Each tagged ACAT2 was expressed in the endoplasmic reticulum as a single undegraded protein band and was at least partially active enzymatically. We then used cytoimmunofluorescence and protease protection assays to monitor the sidedness of the hemagglutinin and Mab1 tags along the ER membranes. The results indicated that ACAT2 contains only two detectable TMDs, located near the N terminal region. We also show that a conserved serine (S245), a candidate active site residue, is not essential for ACAT catalysis. Instead, a conserved histidine (H434) present within a hydrophobic peptide segment, may be essential for ACAT catalysis. H434 may be located at the cytoplasmic side of the membrane.

scholarly journals Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods

2003 ◽  
Vol 374 (1) ◽  
pp. 27-36 ◽  
Author(s):  
Wei CAO ◽  
Larry H. MATHERLY
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Substrate Binding ◽  
Chinese Hamster ◽  
Water Soluble ◽  
Cysteine Residues ◽  
Reduced Folate Carrier ◽  
Cysteine Scanning Mutagenesis

The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or flanking this region. An hRFC mutant devoid of cysteine residues was prepared by deleting the C-terminal 56 amino acids, including four cysteine residues, and mutagenizing the remaining cysteine residues to serine residues. A fully functional cysteine-less hRFC protein was expressed in transport-impaired MtxRIIOuaR2-4 Chinese-hamster ovary cells. To explore the role of residues in or flanking TMD1 in transport, all 24 amino acids from Trp25 to Ile48 of hRFC were mutated individually to cysteine residues, and the mutant hRFCs were transfected into MtxRIIOuaR2-4 cells. All of the 24 cysteine mutants were expressed and, with the exception of R42C (Arg42→Cys), were capable of mediating methotrexate uptake above the low level in MtxRIIOuaR2-4 cells. We found that by treating the transfected cells with the small, water-soluble, thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulphonate, methotrexate transport by several of the cysteine-substituted hRFC mutants was significantly inhibited, including Q40C, G44C, E45C and I48C. Sodium (2-sulphonatoethyl) methanethiosulphonate transport inhibition of the Q40C, G44C and I48C mutants was protected by leucovorin [(6R,S)-5-formyltetrahydrofolate], indicating that these residues lie at or near a substrate-binding site. Using surface-labelling reagents [N-biotinylaminoethyl methanethiosulphonate and 3-(N-maleimidylpropionyl)biocytin, combined with 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid] with cysteine mutants from positions 37–48, the extracellular TMD1 boundary was found to lie between residues 39 and 40, and amino acids 44–46 and 48 were localized to the TMD1 exofacial loop. Collectively, our results imply that amino acids 40, 44, 48 and, possibly, 42 serve important roles in hRFC transport, albeit not as structural components of the putative transmembrane channel for folate substrates.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Malignancy-related characteristics of wild type and drug-resistant chinese hamster ovary cells

Pathology ◽  
1993 ◽  
Vol 25 (3) ◽  
pp. 268-276 ◽  
Author(s):  
Wanda B. Mackinnon ◽  
Marlen Dyne ◽  
Rebecca Hancock ◽  
Carolyn E. Mountford ◽  
Adrienne J. Grant ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Drug Resistant ◽  
Wild Type ◽  
Ovary Cells

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

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