Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro

John Marshall; Shaona Fang; Lynda S Ostedgaard; Catherine R O’Riordan; Dawn Ferrara; Jane F Amara; Henry  Hoppe IV; Ronald K Scheule; Michael J Welsh; Alan E Smith; Seng H Cheng

doi:10.1016/S0021-9258(17)42037-0

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Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro

Journal article

Open access

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Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro

John Marshall, Shaona Fang, Lynda S Ostedgaard, Catherine R O’Riordan, Dawn Ferrara, Jane F Amara, Henry Hoppe IV, Ronald K Scheule, Michael J Welsh, Alan E Smith, …

The Journal of biological chemistry, Vol.269(4), pp.2987-2995

1994

DOI: 10.1016/S0021-9258(17)42037-0

PMID: 7507932

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Abstract

We have generated several clones of Chinese hamster ovary, mouse epitheloid C127, and pig kidney epithelial LLCPK1 cells producing high levels of functional recombinant human cystic fibrosis transmembrane conductance regulator (CFTR). Processing of CFTR to the mature and fully glycosylated form in these cells is inefficient with only approximately 40% of all newly synthesized CFTR being converted to the mature form. Furthermore, expression of the most frequent mutant allele of the cystic fibrosis (CF) gene, the delta F508 mutant in these epithelial cells, indicated that it is biosynthetically arrested at the endoplasmic reticulum and fails to traffic to the plasma membrane. Using a combination of CFTR mutants and monoclonal antibodies, all the detectable recombinant CFTR in these cells was determined at least under the conditions used, to be present as a monomer. To demonstrate the feasibility of protein replacement therapy, we were able to effect the physical transfer of functional recombinant CFTR produced in Chinese hamster ovary cells to the plasma membranes of Ha3b fibroblasts, a cell line devoid of cAMP-stimulated chloride channels. Transfer of CFTR was mediated by the hemagglutinin viral fusion protein of influenza virus present on the Ha3b cells. Efficiency of transfer was up to 25% of the target cells, and CFTR chloride channel activity was detectable for up to 12 h post-fusion. Therefore, with the development of an appropriate formulation of fusogenic proteoliposome or virosome containing reconstituted purified CFTR, it should be feasible to introduce functional CFTR into CF-affected cells.

Fundamental and applied biological sciences. Psychology

Biological and medical sciences

Molecular and cellular biology

Cell structures and functions

Cell membranes. Ionic channels. Membrane pores

Details

Title: Subtitle: Stoichiometry of recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro
Creators: John Marshall - Genzyme Corp., Framingham MA 01701, United States
Shaona Fang - Genzyme Corp., Framingham MA 01701, United States
Lynda S Ostedgaard - Genzyme Corp., Framingham MA 01701, United States
Catherine R O’Riordan - Genzyme Corp., Framingham MA 01701, United States
Dawn Ferrara - Genzyme Corp., Framingham MA 01701, United States
Jane F Amara - Genzyme Corp., Framingham MA 01701, United States
Henry Hoppe IV - Genzyme Corp., Framingham MA 01701, United States
Ronald K Scheule - Genzyme Corp., Framingham MA 01701, United States
Michael J Welsh - Genzyme Corp., Framingham MA 01701, United States
Alan E Smith - Genzyme Corp., Framingham MA 01701, United States
Seng H Cheng - Genzyme Corp., Framingham MA 01701, United States
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.269(4), pp.2987-2995
DOI: 10.1016/S0021-9258(17)42037-0
PMID: 7507932
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology; Bethesda, MD
Language: English
Date published: 1994
Academic Unit: Neurology; Molecular Physiology and Biophysics; Pulmonary, Critical Care, and Occupational Medicine; Fraternal Order of Eagles Diabetes Research Center; Neurosurgery; Internal Medicine
Record Identifier: 9984020717202771

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