The cystic fibrosis mutation (delta F508) does not influence the chloride channel activity of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated Cl- channel. In most mammalian cells, the functional consequences of the most common CF mutation, delta F508-CFTR, cannot be assessed as the mutant protein undergoes biosynthetic arrest. However, function can be studied in the baculovirus-insect cell expression system where delta F508-CFTR does not appear to undergo such arrest. Our results show that phosphorylation-regulated Cl- channel activity of delta F508-CFTR is similar to that of wild-type CFTR. This observation was confirmed in comparative studies of purified delta F508-CFTR and CFTR reconstituted in planar lipid bilayers. Therefore, we suggest that this common mutation does not result in a significant alteration in CFTR function.

The amino acid conjugate formed by the interaction of the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) with band 3 protein from human red blood cell membranes.

The specific anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and its reduced analog (H2DIDS), when irreversibly bound to band 3 protein of the red blood cell membrane, form amino acid conjugates through interaction with the epsilon-amino group of a particular lysine residue. The specific residue is located in a transmembrane segment of band 3 protein and appears to be a close neighbor of the transport site.

The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red blood cell membrane.

The binding site for 4,4'-diisothiocyano-2,2'-stilbenedi sulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000 daltons by CNBr. The C-terminus of the segment is located in the 7000 daltons by the N-terminus in one of the 4000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by N-bromosuccinimide into two fragments of 2000 daltons. The binding site for 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.

Pepsin cleavage of band 3 produces its membrane-crossing domains.

After prolonged treatment of red-cell ghosts with pepsin followed by SDS-urea-acrylamide gel electrophoresis of the membrane peptide fraction, a heavily stained band representing peptides of about 4 kDa (with traces of higher molecular weights) was found. If the cells were first labelled with the disulfonic stilbene, DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) or with N-ethylmaleimide, probes that react with specific sites in Band 3 the anion transport protein, both agents were largely located in the 4 kDA band. With less intensive pepsin treatment, Stained bands of about 17, 12 and 8 kDa were also visible, and DIDS labelling was associated with these higher molecular weight peptides. The 4 kDa band apparently contains at least five or six different peptides. A single peptide containing the DIDS-binding site was separated from others in the band by ion-exchange chromatography. The location of the DIDS-peptide in the primary structure of Band 3 was determined by matching the known location of DIDS and of a methionine residue cleavable by cyanogen bromide. It is concluded that two additional 4 kDA peptides are labelled with N-ethylmaleimide. Because the location of the N-ethylmaleimide-binding sites are known, these two peptides could also be mapped in the primary structure of Band 3. The findings are consistent with the suggestion that pepsin can digest those portions of Band 3 (and probably of other intrinsic peptides) that are exposed on either side of the membrane, leaving only those domains that cross the bilayer. For Band 3, the data are consistent with a structure containing five crossing strands per monomer, each crossing strand being about 4 kDa.

Assessment of the efficacy of in vivo CFTR protein replacement therapy in CF mice.

Cystic Fibrosis (CF) is caused by mutations in the CF gene that lead, for the most part, to mislocalization of the protein product, the cystic fibrosis transmembrane conductance regulatory (CFTR). CFTR is a chloride channel normally situated in the apical membrane of epithelial cells where it contributes to transepithelial ion transport. In this study we demonstrated the feasibility of in vivo transfer of purified CFTR protein via phospholipid liposomes into the apical membrane of nasal epithelia of CFTR knockout mice. Membrane incorporation of immunogold-labeled CFTR could be visualized by electron microscopy and correction of CF-related defects in ion transport measured by nasal potential difference (PD) measurements in about one-third of the animals treated. Although these initial results are promising, effectiveness of this therapeutic approach appears to be limited by the inefficient incorporation of CFTR into the apical epithelial cell membrane.

Antigen protection of monoclonal antibodies undergoing labelling.

The effectiveness of a methodology designed to protect the antigen binding capacity of monoclonal antibodies undergoing labelling with a number of reagents was examined. The antigen binding sites of monoclonal antibodies were protected by complexing them with their antigen. Chemical modification with 6 mM of the water soluble Bolton-Hunter reagent of site protected monoclonal antibodies to glucoamylase resulted in antibodies that could tolerate a four-fold increase in reagent incorporation, without any loss of antigen binding capacity. Iodination of these antibodies (modified under site protected conditions) yielded over 70% increase in radioactivity incorporated in the active antibody fraction, compared with the incorporation into unprotected antibodies. Site protected labeling was found to be effective in retaining the antigen binding capacity of monoclonal antibodies modified with all reagents tested with the exception of chloramine-T.

Stabilization of enzymes by their specific antibodies.

In nature, increased stability of enzymes has often been found to be associated with noncovalent protein-protein interactions. Specific antibodies should be suitable for this purpose. To test this hypothesis, we used a number of model enzymes, complexed them with their specific antibodies, and exposed them and the free enzymes to low and high temperature, lyophilization, oxidation, and alcohol. The retained activity of the antibody-complexed enzymes was substantially, and in some cases dramatically, higher. In general mechanistic terms, stabilization may have been accomplished either by noncovalent antibody crosslinking of discontinuous oligopeptide chains on the surface of the enzyme, thereby increasing resistance to unfolding of the enzyme, or by physical shielding by the antibodies of vulnerable sites on the surface of the enzyme.

Engineering resistance to trypsin inactivation into L-asparaginase through the production of a chimeric protein between the enzyme and a protective single-chain antibody.

We have demonstrated that a trypsin sensitive enzyme such as L-asparaginase can be rendered trypsin resistant by genetically fusing its gene with that of a single-chain antibody derived from a preselected monoclonal antibody capable of providing protection against trypsin. The chimeric L-asparaginase retained 75% of its original activity upon exposure to trypsin, whereas the native unprotected L-asparaginase control was totally inactivated.

Monoclonal antibodies can protect L-asparaginase against inactivation by trypsin.

We show that a non-inhibitory monoclonal antibody (MAB) can be selected that provides substantial and sustained protection against proteolytic inactivation of L-asparaginase by trypsin. Of six non-inhibitory, high affinity, monoclonal antibodies to L-asparaginase, one afforded approximately 70% protection. Inactivation of L-asparaginase is associated with a single cleavage adjacent to lysine-29 that results in loss of an N-terminal fragment with a calculated MW of 2,647. The protective MAB prevented this trypsin cleavage. The products of gene fusions of "humanized" fragments of such antibodies and L-asparaginase could have increased clinical utility.

The red cell band 3 protein: its role in anion transport.

Studies of anion transport across the red blood cell membrane fall generally into two categories: (1) those concerned with the operational characterization of the transport system, largely by kinetic analysis and inhibitor studies; and (2) those concerned with the structure of band 3, a transmembrane peptide identified as the transport protein. The kinetics are consistent with a ping-pong model in which positively charged anion-binding sites can alternate between exposure to the inside and outside compartments but can only shift one position to the other when occupied by an anion. The structural studies on band 3 indicate that only 60% of the peptide is essential for transport. That particular portion is in the form of a dimer consisting of an assembly of membrane-crossing strands (each monomer appears to cross at least five times). The assembly presents its hydrophobic residues toward the interior of the bilayer, but its hydrophilic residues provide an aqueous core. The transport involves a small conformational change in which an anion-binding site (involving positively charged residues) can alternate between positions that are topologically in and topologically out.

The location of a chymotrypsin cleavage site and of other sites in the primary structure of the 17,000-dalton transmembrane segment of band 3, the anion transport protein of red cell.

A 17,000-dalton transmembrane segment of band 3 protein is further cleaved by chymotrypsin treatment of red blood cell ghosts to 15,000 daltons. The location of this particular chymotrypsin cleavage site was determined by comparing the fragmentation pattern of the 17,000- and 15,000-dalton peptides using cyanogen bromide (CNBr). Each peptide is cleaved at its two methionine residues into three fragments. For each peptide two of the fragments are the same size, 7000 and 4000 daltons, the latter containing, in each case, the binding site of the anion transport inhibitor 4,4'-diisothiocyano-2,2' disulfonic acid (DIDS). The third fragment is 2000 daltons larger in the case of the 17,000-dalton peptide (6000 compared to 4000 daltons). These findings indicate that the chymotrypsin cleavage site is located at the cytoplasmic side of the membrane, 2000 daltons from the N-terminus of the 17,000-dalton peptide. This information allows the mapping of a number of defined sites of the 15,000-dalton segment within the primary structure of band 3. These sites support the suggestion that this peptide segment is folded within the bilayer.

Intrinsic segments of band 3 that are associated with anion transport across red blood cell membranes.

After treatment of red cell ghosts with chymotrypsin, the predominant intrinsic peptides remaining in the membrane fraction are 15,000 and 9,000 daltons mol wt. After partial extraction with Triton X-100, the residual membrane vesicles have almost no other stained peptides and such vesicles are reported to carry out anion transport activities sensitive to specific inhibitors. In vesicles derived from cells treated with DIDS(4,4'-diisothiocyano-2,2'-stilbene disulfonic acid), an irreversible inhibitor of anion transport that is highly localized in an abundant intrinsic protein known as band 3, the probe is largely recovered in the 15,000 dalton peptide. The part of band 3 from which it is derived is a previously reported 17,000 transmembrane segment (Steck, T.L., Ramos, R., Strapazon, E., 1976, Biochemistry 15:1154). The 9,000-dalton peptide is present in the vesicles in a one-to-one mole ratio with the 15,000-dalton peptide, suggesting that both are derived from the same protein. This conclusion is supported by the finding that the 35,000-dalton C-terminal end of band 3, derived by chymotrypsin treatment of cells, is further proteolysed if the cells are converted to ghosts and its disappearance coincides with the appearance of the 9,000-dalton fragment. Evidence is presented that the 9,000-dalton fragment crosses the bilayer and that it is closely associated with the 15,000-dalton peptide.

The sulfhydryl groups of the 35,000-dalton C-terminal segment of band 3 are located in a 9000-dalton fragment produced by chymotrypsin treatment of red cell ghosts.

Five sulfhydryl groups of band 3, the anion-transport protein of the red blood cell membrane, can be labeled by N-ethylmaleimide (NEM). Two of these are located in a 35,000-dalton, C-terminal segment produced by chymotrypsin treatment of cells. Extensive treatment of unsealed ghosts with chymotrypsin results in the disappearance of the 35,000-dalton segment, but its two NEM-binding sites area preserved in a 9000-dalton peptide. The latter must therefore be a proteolytic product of the larger segment. Labeling of sulfhydryl groups of band 3 by an impermeant analog of NEM occurs in inside-out, but not in right-side-out vesicles derived from red cell ghosts, supporting the conclusion that NEM-reactive sulfhydryl groups, including those in the 35,000- and 9000-dalton segments, are exposed at the cytoplasmic face of the membrane. These findings support the conclusion that the 35,000-dalton segment crosses the bilayer, and suggest that the 9000-dalton segment may be a membrane-crossing portion of the 35,000-dalton segment.

Purified cystic fibrosis transmembrane conductance regulator (CFTR) does not function as an ATP channel.

The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR). Previously, we provided definitive evidence that CFTR functions as a phosphorylation-regulated chloride channel in our planar lipid bilayer studies of the purified, reconstituted protein. Recent patch-clamp studies have lead to the suggestion that CFTR may also be capable of conducting ATP or inducing this function in neighboring channels. In the present study, we assessed the ATP channel activity of purified CFTR and found that the purified protein does not function as an ATP channel in planar bilayer studies of single channel activity nor in ATP flux measurements in proteoliposomes. Hence, CFTR does not possess intrinsic ATP channel activity and its putative role in cellular ATP transport may be indirect.

Novel method for evaluation of the oligomeric structure of membrane proteins.

Assessment of the quaternary structure of membrane proteins by PAGE has been problematic owing to their relatively poor solubility in non-dissociative detergents. Here we report that several membrane proteins can be readily solubilized in their native quaternary structure with the use of the detergent perfluoro-octanoic acid (PFO). Further, PFO can be used with PAGE, thereby providing a novel, accessible tool with which to assess the molecular mass of homo-multimeric protein complexes.

Coupling of ATP hydrolysis with channel gating by purified, reconstituted CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel situated on the apical membrane of epithelial cells. Our recent studies of purified, reconstituted CFTR revealed that it also functions as an ATPase and that there may be coupling between ATP hydrolysis and channel gating. Both the ATP turnover rate and channel gating are slow, in the range of 0.2 to 1 s(-1), and both activities are suppressed in a disease-causing mutation situated in a putative nucleotide binding motif. Our future studies using purified protein will be directed toward understanding the structural basis and mechanism for coupling between hydrolysis and channel function.