α1,3Fucosyltransferases in cystic fibrosis airway epithelial cells
Introduction
Altered terminal fucosylation and/or sialylation are characteristics of cystic fibrosis (CF) and have been found in membrane glycoproteins, mucins and glycolipids [1]. In the airway epithelial cell membranes the glycophenotype of CF has been defined as decreased sialic acid (NeuAc) and increased fucosyl residues in α1,3/4 linkage to N-acetylglucosamine (Fucα1,3/4GlcNAc). Since the airways are responsible for the morbidity and mortality of CF patients, it was important to determine that the CF airway cells had this characteristic phenotype and in fact, glycosylation of the CF airway cells has been shown to be modulated by the expression of wild type (wt) CFTR [2]. That is, when wtCFTR was expressed in CF airway epithelial cells in culture, the membrane glycoproteins had increased NeuAc content and the Fucα1,3/4GlcNAc content was decreased. When wtCFTR was no longer expressed, the cells returned to the CF phenotype of decreased NeuAc and increased Fucα1,3GlcNAc. A similar result was observed in primary airway cells [2]. Mucins from CF patients contain sialylated forms of the same carbohydrate epitopes [3] but thus far have not been related to CFTR dysfunction [4].
We previously reported the activity and mRNA expression of α1,2fucosyltransferase (FucT), and the interesting lack of fucosyl residues in α1,2 linkage among the membrane glycopeptides of CF cells although present among non CF cells. These studies provided another link in describing the CF glycophenotype and allowed the proposal of a hypothesis for the glycophenotype of membrane glycoconjugates on the surface of CF airway epithelial cells [5].
The hypothesis proposed [5] was based on the chemical/biochemical data of the glycophenotype of the membrane glycoproteins in CF airway epithelial cells and these cells corrected with wtCFTR [2]. The CFTR gene encodes for a transmembrane glycoprotein with Cl- channel activity and when mutated causes CF [6], the most common lethal genetic disease of Caucasians. Homozygous ΔF508 is the most prevalent mutation [7]. The hypothesis proposes that in addition to its role as a Cl- channel, wtCFTR participates in compartmentalization and transport of the terminal glycosyltransferases through the Golgi. When mutated, CFTR causes faulty compartmentalization and α1,3FucT moves to a position where it acts prior to NeuAcT or α1,2FucT, thus giving the glycophenotype in CF airway epithelial cells [5], [8]. To further our studies on the glycophenotype in CF, we examined the activity and mRNA expression of α1,3FucT. The experiments further supported our hypothesis that the activity or mRNA expression of the α1,3FucT was not the direct cause of the glycophenotype in CF airway epithelial cells. Both the CF and non CF airway cells had significant mRNA and enzyme activity for α1,3FucT.
Section snippets
Cell culture and harvest
CF/T43, nasal epithelial cells from a CF patient homozygous for the most common mutation, ΔF508, were immortalized with SV40 T antigen [9] and obtained from Dr. J.R. Yankaskas, University of North Carolina. Tracheal epithelial cells, BEAS-2B, immortalized with SV40 virus, were obtained from Dr. J.F. Lechner, NIH [10]. CF/T43 and BEAS-2B cells are cloned airway epithelial cells whose characteristics have been described [9], [10]. They were cultured as described previously [11]. CF/T1 cells were
Activity of fucosyltransferases
Extracts of airway epithelial cells, CF/T43 and BEAS-2B were examined with a library of substrates for the ability to transfer [14C]fucose from GDP-[14C] fucose. The pattern of transfer was characteristic of not only α1,3FucT but also α1,4FucT and/or α1,2FucT (Fig. 1).Table 1 defines the substrates used for the enzymes. All of the substrates that were available supported the presence of α1,2FucT, including the lack of activity with LNFPI, 3’SNAL and 2’FL. α1,2FucT was reported previously using
Discussion
In defining the glycophenotype of the surface membranes of CF airway epithelial cells we examined the activity of α1,3FucT and the mRNA expression of several FucTs. The results showed that the biosynthetic components for fucosylated membrane glycoproteins were present in both CF and non CF cells and were consistent with previous results for α1,2FucT [5]. Therefore, the cause of the CF glycophenotype, increased Fucα1,3GlcNAc, on the membrane glycoconjugates has to be attributed to a cause other
Acknowledgements
We thank Dr. J. Yankaskas for the CF cell lines and Dr. J.F. Lechner for the non CF cells. We thank Dr. Winifred Watkins for her generous supply of oligosaccharides. We are indebted to Dr. John Lowe who provided the probes for α1,2FucT and FucTIII, IV, and VII. Supported by the CF Foundation, SCANLI00G0 and SCANLI00Z0.
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