Contrary to received wisdom, the mutant gene implicated in 70percent of cystic fibrosis (CF) patients is no less functional thanits wild-type intact counterpart. Rather, the nascent mutant'sshortcoming is failure to report for duty at its action station inthe cell membrane.
This controversial finding is reported in the April issue ofNature Genetics, out Thursday, by cell biologist Christine Bearof the Research Institute, Hospital for Sick Children, Toronto.Her report is titled, "The cystic fibrosis mutation does notinfluence the chloride channel activity of CFTR."
The mutant cystic fibrosis transmembrane conductanceregulator gene (CFTR) is arrested deep inside the cell, where itis synthesized. Thus it misses the intracellular transport to itsdestination on the plasma membrane to do its job of transitingchloride ions in and out of the cell.
Instead, the aberrant protein, whose gene lacks three basepairs that encode the amino acid phenylalanine, remainstrapped in the endoplasmic reticulum (ER), or nearby Golgiapparatus, and is eventually degraded.
The absence of such chloride channels in the respiratoryepithelial cells of cystic fibrosis (CF) victims is believed toexplain the life-threatening buildup of infection-prone mucusin their lungs. CF has been described as the most common fatalgenetic disease in humans.
Current assumptions that the phenylalanine-minus CF geneproduct is dysfunctional as well as mislocated, Bear's paperstates, are based on testing it in Xenopus laevis, Vero and 3T3fibroblast cells. All of these have biosynthetic quality controlmechanisms that weed out defective proteins by subjectingthem to biosynthetic arrest -- such as halting their progressionto the plasma membrane.
Bear, in contrast, used the baculovirus armyworm insect cellexpression system, which lacks this quality-control step. Shefound that "the mutant protein does not appear to undergobiosynthetic arrest in these cells and is expressed at high levelscomparable to those of the wild-type protein."
If this is so, then gene therapy should focus not only onrepairing the presumed aberration, but on hustling the proteinfrom ER to membrane. As she told BioWorld: "There doesn'tseem to be any difference in the chloride-channel activity ofthe wild-type and mutant versions of the protein. And thatsuggests if you are successful in developing strategies forescorting the protein to the cell surface, then you would havenormal activity."
Bear herself is not pursuing the escort ploy, but ratheranalyzing some of the myriad, statistically minor gene defects,which account for most of the remaining 30 percent of CFpatients.
In a nearby laboratory at Toronto's Hospital for Sick Children,CF research pioneer John Riordan is doing just that. He toldBioWorld that his work is aimed at understanding thechaperoning process. "The escort concept is a reasonable one, areally well-focused opportunity to approach this area from atherapeutic angle, but I don't think the science is quite at thatstage just yet."
Riordan finds Bear's escort suggestion "a new area of endeavor,but not totally harebrained." Molecular geneticist John Teem,who did a post-doc with Riordan before moving to a lab of hisown at the University of Iowa, would agree.
He is making hybrid protein transporters between CF genesand yeast sequences, trying to reconstruct the CF defects -- offunction and mislocalization. "You can assay mutations in yeastas a growth phenotype," he explained, "instead of inmammalian cells as a chloride efflux defect." When he finds asuppresser mutation in the yeast construct, "which restorestransport of, say, the mutant protein to the plasma membrane,then my work feeds back to the human system."
Teem's system is far from human therapy, but "it does providea possible means to screen for drugs that might correct amislocalization defect."
He doubt's Bear's conclusion that the mutant gene's defect isone of localization, not function, because in his yeast system hehas been able to cause such mutations to revert to full function.
Molecular geneticist Seng Cheng discovered the phenylalanine-deleted mutant's cell-trafficking problem at Genzyme Corp.three years ago. Apropos Bear's finding that the mutant'shangup is due to biosynthetic arrest rather than dysfunction,he told BioWorld, "If Christine says that's the case, there's avery good chance she is correct."
Her proposal for intracellular escort therapy, Cheng added, "isnot a new suggestion. But I think its significance is that, if she'scorrect, the statement now holds very tightly, and a lot ofpeople would say that the approach should proceed with vigor."
A number of companies are pursuing the escort ploy, Chengsaid. "How they're doing it, I really don't know. We're exploringthat avenue here at Genzyme, too, but it's not our main focus."
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.