Terminal glycosylation in cystic fibrosis.

Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade. Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression. CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells. The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR. Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations. Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions. In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates. We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF.

Gluconoylated and glycosylated polylysines as vectors for gene transfer into cystic fibrosis airway epithelial cells.

To provide an alternative to viral vectors for the transfer of genes into airway epithelial cells in cystic fibrosis (CF), a novel set of substituted polylysines were employed. Polylysine was partially neutralized by blocking a number of positively charged residues with gluconoyl groups. In addition, polylysine was substituted with sugar residues on a specified number of amino groups. Using the gluconoylated polylysine as vector, the pCMVLuc plasmid gave high expression of the reporter gene luciferase in immortalized CF/T43 cells. The luciferase activity was 75-fold greater in the presence of 100 microM chloroquine. Luciferase gene expression persisted at high levels for up to at least 120 hr following transfection. Glycosylated polylysines/pCMVLuc complexes were compared to the gluconoylated polylysine/pCMVLuc complex and beta-Gal-, alpha-Glc-, and Lac-substituted polylysines gave 320%, 300%, and 290%, respectively, higher expression of the reporter gene luciferase. Luciferase expression ranged from 35 to 2 ng of luciferase per milligram of cell protein in the order: beta-Gal = alpha-Glc = Lac > alpha-Gal = Rha = Man > beta-GalNAc > alpha-GalNAc = alpha-Fuc, suggesting that the transfection efficiency is sugar dependent. Most importantly, in primary cultures of both CF and non-CF airway epithelial cells grown from tracheal tissue explants, lactosylated polylysine gave uniformly high expression of luciferase. The glycosylated polylysines provide an attractive nonviral approach for the transfer of genes into airway epithelial cells.

High-efficiency transfer of cystic fibrosis transmembrane conductance regulator cDNA into cystic fibrosis airway cells in culture using lactosylated polylysine as a vector.

To find more efficient vectors for the transfer of CFTR cDNA, lactosylated polylysine was explored for transfer into airway epithelial cells in primary culture. The efficacy and high efficiency of transfection were shown by several criteria: expression of both mRNA and protein for CFTR and the functional correction of the Cl- channel activity. Using specific combinations of agents to enhance the transfection, an efficiency of 90% was obtained as detected by in situ hybridization with digoxigenin-labeled probes generated against exon 14 of CFTR. The highest efficiency was observed by adding E5CA peptide (10 microg) and 5% glycerol to the transfection mixture. The degree of transfection could be controlled by the enhancing agents, thus modulating the efficiency of transfection. The highest level of transfection efficiency is equivalent to that reported for viral vectors. None of the agents or their combinations in the concentrations used were cytotoxic to the primary cells. Antibody pAb3145 was used to detect the expression of the CFTR protein in the cells. When an N-terminal GFP-CFTR fusion gene was used to transfect the CF cells a functional correction of the CFTR Cl- channel was detected by patch-clamp electrophysiology. The high efficiency of CFTR gene transfer with lactosylated polylysine leads to the conclusion that lactosylated polylysine is a promising vector to transfer the CFTR gene into human airway cells in culture.

Body composition of children with cystic fibrosis with mild clinical manifestations compared with normal children.

Because growth and anthropometric measurements are frequently below normal in patients with cystic fibrosis (CF), the body composition of 23 children (6-9 y of age) who had mild manifestations of CF was examined and compared with a control group of 24 subjects similar in age, sex, and weight. Skinfold measurements, total body water (TBW) by deuterium oxide dilution, and total body electroconductivity (TOBEC) were measured. Skinfold and TBW measurements demonstrated that fat mass and fat-free mass did not differ significantly between the two groups. No significant difference in percent body fat was found by using the skinfold equations of Slaughter et al (13.8% vs 15.3%), although percent body fat (TBW method) differed between the groups (P < 0.05). TOBEC measurements (CF, n = 14) were not used in the group comparison. The majority of the methods demonstrated that the CF group achieved normal growth and body composition, with a possible trend of fat depletion.

Prospective evaluation of growth, nutritional status, and body composition in children with cystic fibrosis.

BACKGROUND: Several cross-sectional studies have shown improvement in the growth of children with cystic fibrosis (CF) because of increased awareness of and more comprehensive care of their special nutritional needs. However, longitudinal data on the nutritional status of these children are rare. OBJECTIVE: The objective was to compare changes in growth, body composition, and nutritional status between children with and without CF. DESIGN: This was a prospective 3-y cohort study of 25 children aged 5-10 y with CF, mild pulmonary disease, and pancreatic insufficiency and of 26 healthy control children. Three methods were used to assess body composition: measurements of skinfold thickness, total body water by deuterium oxide, and total-body electrical conductivity. Growth and body-composition changes over time were analyzed by a longitudinal mixed-effects model. RESULTS: Over the 3 y of the study, the statural growth of the boys with CF was slower than that of the control subjects (P = 0.004). The same divergence over time between the boys with and without CF was observed for fat-free mass assessed by skinfold-thickness measurements and total body water (P = 0.008 and 0.02, respectively) and for fat mass assessed by skinfold-thickness measurements and total-body electrical conductivity (P = 0.009 and 0.001, respectively). The differences in the pattern of changes in growth and body composition were less striking for girls. CONCLUSIONS: Despite comprehensive care, the growth of boys with CF was impaired on the basis of height, fat-free mass, and fat mass, when observed longitudinally. Caution should be used when interpreting cross-sectional measurements because they often do not detect suboptimal growth.

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.

Dietary intake of healthy children with cystic fibrosis compared with normal control children.

Because a low calorie intake may result in growth failure and malnutrition in patients with cystic fibrosis (CF), the dietary and energy intakes of 22 children with CF who had mild lung disease and excellent Shwachman scores were examined and compared with those of 23 normal control subjects similar in age (5 to 10 years), gender, and weight. The children with CF consumed significantly more calories than the control subjects. Calorie intake was 111 +/- 19.9% (mean +/- SD) of estimated requirements (World Health Organization recommendations) for the CF group and 97 +/- 18.7% for the control group (P = .014). Calories consumed per kilogram of body weight were 117% of World Health Organization (CF) vs 97% (control) (P = .009). Calorie intake compared to the 1989 Recommended Dietary Allowance was 106 +/- 20.6% for the CF group vs 93 +/- 19.1% for the control group. Fat consumed as a percent of total calories was similar: 33.5% (CF) vs 32.2% (control). All children with CF had pancreatic insufficiency and, on average, consumed a large number of pancreatic enzyme supplements, resulting in dietary fat absorption of 86 +/- 12%. It is concluded that these children with CF were able to maintain normal growth and energy stores on a diet that was relatively high in calories compared to control subjects and moderate in percent fat, along with an aggressive pancreatic enzyme supplement regimen.

A prospective study of body composition changes in children with cystic fibrosis.

Recent cross-sectional studies of children with cystic fibrosis (CF) have shown an improvement in their growth with improved nutritional status, but there are only a few longitudinal studies in this population. A four-year prospective study was conducted in 25 children with CF and 26 controls, ages 5 to 10 years at baseline, to describe changes in body composition using three independent methods of assessment: skinfold thickness, total body water by deuterium dilution, and total body electrical conductivity (TOBEC). The statural growth of the boys with CF was slower than that of the control boys. Using different methods, the fat-free mass and fat-mass increases were shown to be slower in boys with CF than in controls. These differences were less significant in girls. Despite comprehensive care, the growth of boys with CF may still not be optimal, as observed longitudinally.

Abnormal distribution of alpha-L-fucosidase in cystic fibrosis: decreased activity in serum.

The activity of alpha-L-fucosidase is decreased in the serum of cystic fibrosis patients when compared to age-matched controls. This result, combined with the elevated activity in skin fibroblasts, supports the concept of an abnormal intracellular and extracellular distribution of alpha-L-fucosidase in cystic fibrosis.

Additional fucosyl residues on membrane glycoproteins but not a secreted glycoprotein from cystic fibrosis fibroblasts.

Glycopeptides derived from peripheral membrane glycoproteins of skin fibroblasts of seven patients with cystic fibrosis (CF) had an increase in fucosyl residues when compared with those of seven age, race and sex matched controls (Pediatr Res 1985;19:368-374). To further define these results, the membrane glycopeptides which bound to immobilized lentil lectin and thereby enriched in fucosyl residues linked alpha 1----6 to N-acetylglucosamine attached to asparagine, were Pronase digested, partially purified and examined by 500-MHz 1H-NMR spectroscopy. The CF derived glycopeptides had two different features when compared to those from Controls (1) an increased number of fucosyl residues linked alpha 1----6 to the N-acetylglucosamine attached to asparagine and (2) fucosyl residues linked alpha 1----3 to a branch N-acetylglucosamine. The glycopeptides from both sources were of the di and triantennary type containing sialic acid linked alpha 2----3 and alpha 2----6 to galactose in an approximate molar ratio of 3:2 and 2:1, from CF and Control, respectively. Glycopeptides derived from a glycoprotein, fibronectin, secreted from CF fibroblasts were also examined by 1H-NMR spectroscopy and showed no evidence of fucosyl residues linked alpha 1----3 to branch N-acetylglucosamine and a lesser percentage of core fucose than found in the peripheral membrane glycopeptides. These results define further the altered fucosylation of the CF peripheral membrane glycoproteins.

Characterization of human fibronectin glycopeptides from cystic fibrosis and control skin fibroblasts.

Fibronectins isolated from different species and tissue sources are glycosylated differently. We report here a characterization of the glycopeptides of fibronectin isolated from the culture medium of skin fibroblasts from patients with cystic fibrosis together with age-, race-, and sex-matched control subjects. The characterization of this fibronectin is of special interest because it is derived from: a non-fetal, cellular source; eight different individuals; and cystic fibrosis and control individuals. The fibronectin glycopeptides were purified by gel-permeation chromatography and Con A-Sepharose and were analyzed by anion-exchange chromatography and affinity columns of immobilized 5-hydroxytryptamine and lectins. One half of the glycopeptides of skin fibroblast fibronectin were shown to contain biantennary oligosaccharides which were core-fucosylated and partially sialylated. Although the remaining half was a complex mixture of glycopeptides, there was remarkably little inter-individual variation. No difference between cystic fibrosis and control subjects was discernible by the techniques employed here. Unlike the biantennary glycopeptides of human plasma fibronectin, those from skin fibroblast fibronectin were core-fucosylated and less highly sialylated. However, compared to human cellular fibronectin glycopeptides from fetal sources, those from skin fibroblast fibronectin were both more highly fucosylated and sialylated.

Abnormal sweat electrolytes in a case of celiac disease and a case of psychosocial failure to thrive. Review of other reported causes.

Sweat electrolytes were initially elevated in a child who was diagnosed as having celiac disease and also in one with psychosocial failure to thrive. Subsequent sweat tests were normal after nutritional status of the patients had improved with therapy. The reports of elevated sweat electrolytes in conditions other than cystic fibrosis are discussed. It is emphasized that sweat test methods other than the quantitative pilocarpine iontophoresis method are not reliable. The occurrence of false-positive sweat tests demonstrates the need for care in the interpretation of elevated sweat electrolytes and the necessity of performing repeat quantitative pilocarpine iontophoresis for the establishment of the diagnosis of CF. Celiac disease and malnutrition from other causes may cause sweat electrolytes to be elevated.

Hypoelectrolytemia as a presentation and complication of cystic fibrosis.

We describe three patients with cystic fibrosis (CF) with four episodes of hypoelectrolytemia, two of which were associated with documented metabolic alkalosis. CF should be included in the differential diagnosis of infants and children presenting with hypoelectrolytemia and metabolic alkalosis. Patients with CF are at risk of developing severe hypochloremia in hot weather and during intercurrent illness. An abrupt diminution of oral intake is an important suggestive sign, alerting the physician to look for the development of hypoelectrolytemia.

Clinical evidence that V456A is a Cystic Fibrosis causing mutation in South Asians.

BACKGROUND: Cystic Fibrosis (CF) genotypes in South Asians are variable with a decreased incidence of Delta F508 and an increased incidence of novel mutations. The objective of this study is to provide clinical evidence that V456A, a novel mutation in South Asian Cystic Fibrosis patients, can cause significant lung disease. METHODS: We extracted clinical data from a retrospective chart review of 2 CF patients of South Asian descent. RESULTS: Patient 1, a 10 year and 11 month old Pakistani female at her initial clinic visit, required multiple hospitalizations for bronchiectasis and pulmonary infections. She was pancreatic sufficient but had slow weight gain. Genetic testing revealed that she is homozygous for the CFTR V456A mutation. Patient 2, an Indian female diagnosed with CF on newborn screening, is compound heterozygous for V456A/R709X. She had slow weight gain with BMI ranging from 12.9 to 13.4 kg/m(2) from 3 to 5 years of age and was 14.2 kg/m(2) at 6 years of age. At 6 years of age, pulmonary function tests revealed mild lung disease with FVC of 71%, FEV(1) of 75%, FEF(25-75) of 119%, and FEV(1)/FVC of 86% predicted. Sputum cultures were intermittently positive for Staphylococcus aureus and Haemophilus influenza. CONCLUSIONS: We provide evidence that V456A can cause significant pulmonary disease in South Asian Cystic Fibrosis patients.

Glycosylation and the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of DeltaF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.