Thomas F. Scanlin

Terminal glycosylation in cystic fibrosis (CF): A review emphasizing the airway epithelial cell

Altered terminal glycosylation, with increased fucosylation and decreased sialylation is a hallmark of the cystic fibrosis (CF) glycosylation phenotype. Oligosaccharides purified from the surface membrane glycoconjugates of CF airway epithelial cells have the Lewis x, selectin ligand in terminal positions. This review is focused on the investigations of the glycoconjugates of the CF airway epithelial cell surface. Two of the major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins which recognize fucose in α-1,3 linkage and asialoglycoconjugates. Therefore, consideration has been given to the possibility that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to both the chronic infection and the robust, but ineffective, inflammatory response in the CF lung. Since the glycosylation phenotype of CF airway epithelial cells have been modulated by the expression of wtCFTR, the hypotheses which have been proposed to relate altered function of CFTR to the regulation of the glycosyltransferases are discussed. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as new approaches to the therapy of CF.

Activity of fucosyltransferases and altered glycosylation in cystic fibrosis airway epithelial cells.

Cystic fibrosis (CF) glycoconjugates have a glycosylation phenotype of increased fucosylation and/or decreased sialylation when compared with non-CF. A major increase in fucosyl residues linked alpha 1,3 to antennary GlcNAc was observed when surface membrane glycoproteins of CF airway epithelial cells were compared to those of non-CF airway cells. Importantly, the increase in the fucosyl residues was reversed with transfection of CF cells with wild type CFTR cDNA under conditions which brought about a functional correction of the Cl(-) channel defect in the CF cells. In contrast, examination of fucosyl residues in alpha 1,2 linkage by a specific alpha 1,2 fucosidase showed that cell surface glycoproteins of the non-CF cells had a higher percentage of fucose in alpha 1,2 linkage than the CF cells. Airway epithelial cells in primary culture had a similar reciprocal relationship of alpha 1,2- and alpha 1,3-fucosylation when CF and non-CF surface membrane glycoconjugates were compared. In striking contrast, the enzyme activity and the mRNA of alpha 1,2 fucosyltransferase did not reflect the difference in glycoconjugates observed between the CF and non-CF cells. We hypothesize that mutated CFTR may cause faulty compartmentalization in the Golgi so that the nascent glycoproteins encounter alpha 1,3FucT before either the sialyl- or alpha 1,2 fucosyltransferases. In subsequent compartments, little or no terminal glycosylation can take place since the sialyl- or alpha 1,2 fucosyltransferases are unable to utilize a substrate, which is fucosylated in alpha 1,3 position on antennary GlcNAc. This hypothesis, if proven correct, could account for the CF glycophenotype.

Terminal glycosylation and disease: Influence on cancer and cystic fibrosis

AbstractTerminal glycosylation has been a recurring theme of the laboratory. In cystic fibrosis (CF), decreased sialic acid and increased fucosyl residues in α1,3 position to antennary N -acetyl glucosamine is the CF glycosylation phenotype. The glycosylation phenotype is reversed by transfection of CF airway cells with wtCFTR. In neuronal cells, polymers of α2,8sialyl residues are prominent in oligodendrocytes and human neuroblastoma. These findings are discussed in relationship to early studies in our laboratories and those of other investigators. The potential extension of these concepts to future clinical therapeutics is presented.

Barriers to adherence to cystic fibrosis infection control guidelines

In 2003, the American Cystic Fibrosis (CF) Foundation published revised, evidence‐based guidelines for infection control. We sought to assess potential barriers to adherence to these guidelines experienced by health care professionals (HCPs) caring for CF patients.

Turnover of the cystic fibrosis transmembrane conductance regulator (CFTR): Slow degradation of wild‐type and ΔF508 CFTR in surface membrane preparations of immortalized airway epithelial cells

The protein product of the cystic fibrosis (CF) gene, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is known to function as an apical chloride channel at the surface of airway epithelial cells. It has been proposed that CFTR has additional intracellular functions and that there is altered processing of mutant forms. In examining these functions we found a stable form of CFTR with slow turnover in surface membrane preparations from CF and non‐CF immortalized airway epithelial cell lines. The methods used to study the turnover of CFTR were pulse/chase experiments utilizing saturation labeling of [35S]Met with chase periods of 5–24 h in the presence of 8 mM Met and cell fractionation techniques. Preparations of morphologically identifiable surface membranes were compared to total cell membrane preparations containing intracellular membranes. Surface membrane CFTR had lower turnover defined by pulse/chase ratios than that of the total cell membrane preparations. Moreover, mutant CFTR was stable in the surface membrane fraction with little degradation even after a 24 h chase, whereas wild‐type CFTR had a higher pulse/chase ratio at 24 h. In the presence of 50 μM castanospermine, which is an inhibitor of processing α‐glucosidases, a more rapid turnover of mutant CFTR was found in the total cell membrane preparation, whereas wild‐type CFTR had a lower response. The results are compatible with a pool of CFTR in or near the surface membranes which has an altered turnover in CF and a glycosylation‐dependent alteration in the processing of mutant CFTR.

Lactosylated poly-l-lysine targets a potential lactose receptor in cystic fibrosis and non-cystic fibrosis airway epithelial cells

Poly-L-lysine with 40% of the epsilon -amino groups substituted with lactosyl residues facilitated the internalization of lactosylated poly-L-lysine/cDNA complexes into cystic fibrosis (CF) and non-CF airway epithelial cells. It was previously shown that lactosylated poly-L-lysine enhanced the transfer of cDNA into the cell nucleus, resulting in transfection. The cell entry of lactosylated poly-L-lysine/cDNA complexes, however, has not been elucidated and we hypothesized that entry of the complex was by receptor-mediated endocytosis. It is shown here that binding of the vector/cDNA complexes to the cell membrane was inhibited by lactose but not N-acetyl glucosamine. Examination by electron microscopy revealed the complexes in clathrin-coated pits. Furthermore, the complexes colocalized with transferrin during cell entry and were shown in early endosomes. These results demonstrated that lactosylated poly-L-lysine/cDNA complexes enter airway epithelial cells via receptor-mediated endocytosis utilizing lactose-binding receptors, which employ the clathrin-coated pit for internalization. Taken together with the fact that nuclear translocation also is enhanced by lactose, these results demonstrate why lactosylated poly-L-lysine is an excellent vector for transfection of airway epithelial cells. Moreover, other carbohydrates covalently linked to poly-L-lysine for targeting other specific cell types, combined with lactosyl residues, can be designed for the development of other molecular conjugates for gene transfer.

Terminal glycosylation of cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) has a characteristic glycosylation phenotype usually expressed as a decreased ratio of sialic acid to fucose. The glycosylation phenotype was found in CF/T1 airway epithelial cells (ΔF508/ΔF508). When these cells were transfected and were expressing high amounts of wtCFTR, as detected by Western blot analysis and in situ hybridization, the cell membrane glycoconjugates had an increased sialic acid content and decreased fucosyl residues in α1,3/4 linkage to antennary N[emsp4 ]-acetyl glucosamine (Fucα1,3/4GlcNAc). After the expression of wtCFTR decreased, the amount of sialic acid and Fucα1,3/4GlcNAc returned to levels shown by the parent CF cells. Sialic acid was measured by chemical analysis and Fucα1,3/4GlcNAc was detected with a specific α1,3/4 fucosidase. CF and non-CF airway cells in primary culture also had a similar reciprocal relationship between fucosylation and sialylation. It is possible that the glycosylation phenotype is involved in the pathogenesis of CF lung disease by facilitating bacterial colonization and leukocyte recruitment.

Fucosylation in cystic fibrosis airway epithelial cells

Altered glycosylation is a phenotypic characteristic of cystic fibrosis (CF), and some of the alterations are summarized. The lungs are the site of the lethal pathology of the disease. Therefore, two of the characteristics were examined in CF and non-CF immortalized airway epithelial cell lines (AEC). The activity of α-l-fucosidase was elevated (280%) in CF AEC when compared with non-CF AEC, whereas the activities of the other lysosomal enzymes which were examined were similar in both cell types. α-l-Fucosidase activity was transiently increased in the non-CF cells after treatment with Brefeldin A (BFA) for 6 h. Thus BFA caused the normal cells to express a phenotypic characteristic of CF. Glycopeptides from the CF and non-CF AECs metabolically labeled withl-[3H]fucose were examined for binding to lentil lectin-Sepharose. A higher percentage of CF glycopeptides bound to lentil lectin, 43% compared with 23% for non-CF control. In addition a higher percentage of CF glycopeptides were bound tightly to lentil lectin and required 0.2M α-methylmannoside to be eluted. This species of tightly bound glycopeptides increased dramatically to 77% from 46% when the CF AEC were treated with BFA. In contrast, the non-CF cell glycopeptides had a minor decrease in tightly bound glycopeptides to 26% from 33% after BFA treatment. Thus, the CF AEC showed fucosylation alteration observed previously for other CF cells and tissue.

Gene therapy of cystic fibrosis (CF) airways: a review emphasizing targeting with lactose.

Cystic fibrosis is a disease for which a number of Phase I clinical trials of gene therapy have been initiated. Several factors account for the high level of interest in a gene therapy approach to this disease. CF is the most common lethal inherited disease in Caucasian populations. The lung, the organ that is predominantly responsible for the morbidity and mortality in CF patients, is accessible by a non-invasive method, the inhalation of aerosols. The vectors employed in the Phase I trials have included recombinant adenoviruses, adeno-associated viruses and cationic lipids. While there have been some positive results, the success of the vectors until now has been limited by either immunogenicity or low efficiency. A more fundamental obstacle has been the absence of appropriate receptors on the apical surface of airway epithelial cells. Molecular conjugates with carbohydrate substitution to provide targeting offer several potential advantages. Lactosylated polylysine in which 40% of the lysines have been substituted with lactose has been shown to provide a high efficiency of transfection in primary cultures of CF airway epithelial cells. Other important features include a relatively low immunogenicity and cytotoxicity. Most importantly, the lactosylated polylysine was demonstrated to give nuclear localization in CF airway epithelial cells. Until now, most non-viral vectors did not have the capability to provide nuclear localization. These unique qualities provided by the lactosylation of non-viral vectors, such as polylysine may help to advance the development of molecular conjugates sufficiently to warrant their use in future clinical trials for the gene therapy of inherited diseases of the lung.

Non-radioactive detection of the most common mutations in the cystic fibrosis transmembrane conductance regulator gene by multiplex allele-specific polymerase chain reaction

SummaryA rapid, simple, nonradioactive method for detection of four common mutations causing cystic fibrosis (CF) has been developed combining multiplexing with allele-specific polymerase chain reaction amplification. This approach (MASPCR) provides an easy assay for direct genotyping of normal and mutant CF alleles in homozygotes and heterozygotes. The strategy involves multiplex PCR of exons 10, 11, and 21 within the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a single reaction containing three common oligoprimers and either the four normal or four mutant oligos corresponding to the ΔF508, G551D, G542X, and N1303K mutations. Primers are chosen so that the size of the four PCR products differ, thereby facilitating detection on agarose gels following amplification in the same reaction. Patient samples are primed with either four normal or four mutant oligo mixtures, and PCR products run in parallel on gels to detect band presence or absence. This approach provides a simple and potentially automated method for cost-effective population screening.