Mary Catherine Glick

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.

A rapid assay for neuraminidase The detection of two differences in activity associated with virus transformation

Neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) activity in fibroblast homogenates was measured by a rapid and simple assay with a synthetic substrate. The activity of neuraminidase in virus transformed hamster fibroblasts was increased over the normal counterpart. In addition, the differential activity seen using the synthetic substrate and fetuin made it possible to detect an enzyme activity hitherto not described. The advantages of this assay for metabolic screening are discussed.

Purification and characterization of GDP L -Fuc: N-acetyl β D -glucosaminide α1→6fucosyltransferase from human blood platelets

Abstractα-6 L -Fucosyltransferase (α1,6FucT; EC 2.4.1.68) from human platelets, the enzyme that is released into serum during coagulation of blood, was purified 100,000-fold. The purification required three sequential chromatographic steps: chromatofocusing, affinity column chromatography on GnGn-Gp(asialo-aglacto-transferrin glycopeptide)-CH-Sepharose, and gel filtration of Sephadex G-200. The final preparation contained a protein that migrated as a single discrete band Mr of 58,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and as a single enzymatically active peak Mr of 58,000 in gel filtration. Although the purified enzyme utilized the biantennary GnGn-Gp as substrate, it was twice as active with the triantennary oligosaccharide when the Man α1,3 antenna was substituted with GlcNacβ1,4. On the other hand the tetraantennary oligosaccharide was not a preferred substrate. The Km values for the substrate asialo-agalactotransferrin-glycopeptide, and GDP L -fucose were 29 and 28 μ M, respectively. The optimum pH of the enzyme was 6.0. The activity of α1,6FucT was abolished in the presence of β-mercaptoethanol. Divalent cations such as Mg2+ and Ca2+ activated, but Cu2+, Zn2+ and Ni2+ strongly inhibited the activity.

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.

Abnormal distribution of α-L-fucosidase in cystic fibrosis: Increased activity in skin fibroblasts

Abstract α-L-Fucosidase activity is elevated in skin fibroblasts from cystic fibrosis patients when compared to controls. The activities of nine other acid hydrolases including neuraminidase are similar in cystic fibrosis and control fibroblasts. The relationship of these results to the recent finding of a decreased activity of α-L-fucosidase in the serum of cystic fibrosis patients is discussed. It is proposed that an abnormal distribution of α-L-fucosidase is involved in the pathogenesis of this disease.

Abnormal distribution of α-l-fucosidase in cystic fibrosis: Decreased activity in serum

Abstract The activity of α- 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 α- l -fucosidase in cystic fibrosis.

ST8Sia IV mRNA corresponds with the biosynthesis of α2,8sialyl polymers but not oligomers in rat oligodendrocytes

As oligodendrocytes mature they progress through a series of distinct differentiation steps characterized by the expression of specific markers. One such marker, polysialic acid found on the neural cell adhesion molecule (NCAM), is detected by antibodies and is present on progenitor oligodendrocytes, but is not detected to the same extent on mature oligodendrocytes. Two closely related polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST) have been cloned previously and shown to synthesize polysialic acid on NCAM and other glycoproteins. To determine whether or not polyα2,8sialyltransferases are downregulated during the differentiation of oligodendrocytes, the enzyme activity and expression of ST8Sia II and ST8Sia IV mRNA at two stages of maturation in JS12/1 and JS3/16 oligodendrocytes were examined. Differentiation in both oligodendroglial cell lines was accompanied by more than a 50% reduction in the biosynthesis of polymers of α2,8sialic acid when fetuin was used as substrate. Most interestingly, extracts of JS12/1 mature cells synthesized 60% more short oligomers of α2,8sialic acid than the progenitor cells, whereas JS3/16 mature cells synthesized barely detectable amounts of the short oligomers. Transcripts for ST8Sia IV mRNA were present in both JS12/1 and JS3/16 and were reduced when the biosynthesis was markedly reduced. In contrast ST8Sia II mRNA was barely detectable in JS3/16 cells and although detectable in JS12/1 cells, there was no clear modulation with maturation. These results were supported by the examination of the brains of rats from embryonic to Day 21 ages. The enzyme activity and mRNA experiments show that polyα2,8sialyltransferase itself is down regulated to cause the reduction in sialyl polymers on mature oligodendrocytes. Moreover, ST8Sia IV is responsible for the polysialylation of NCAM in oligodendrocytes. J. Neurosci. Res. 66:497–505, 2001.

Perturbation of glycoprotein processing affects the neurotoxin-responsive Na+ channel in neuroblastoma cells

The activity of neurotoxin-responsive Na+ channels in mouse neuroblastoma cells, N-18, was examined after treating the cells with compounds that are reported to perturb intracellular traffic. The compounds used have been shown to either alter glycoprotein synthesis and processing, (swainsonine, castanospermine, monensin, and retinoic acid) or receptor mediated endocytosis (mevinolin, 7-ketocholesterol, and chloroquine), or both. All of these compounds inhibited the activity of the neurotoxin-responsive Na+ channel with the exception of retinoic acid which increased the activity. Na+ channel activity was measured by two methods: (a) In vivo, the efflux of 86Rb was measured by use of the cells in monolayer culture, and (b) in vitro, the flux of 86Rb was measured from artificial phospholipid vesicles containing the partially purified Na+ channel. In both cases, 86Rb flux responded to stimulating neurotoxins, veratridine and scorpion venom, and was inhibited by tetrodotoxin as characteristic of excitable membranes. One of the perturbing compounds, swainsonine, was examined in detail. Treatment of N-18 cells with 10 microM swainsonine for 24 h markedly reduced the activity of the neurotoxin-responsive Na+ channel, as shown by the neurotoxin-stimulated efflux of 86Rb in vivo. In addition, after reconstitution into phospholipid vesicles of the partially purified Na+ channel from swainsonine-treated cells, reduced 86Rb flux was observed when compared with that of nontreated cells. Furthermore, the activity was not recovered in other less purified fractions. A comparison of the glycopeptides from the treated and nontreated cells by size, charge, and lectin-binding affinities was consistent with the formation of hybrid oligosaccharides after swainsonine treatment. It is concluded that the oligosaccharide residues of the Na+ channel glycoprotein must be processed to the mature complex-type for full activity. The stimulation of channel activity by treatment with retinoic acid supported this conclusion.

S1.20 Control of membrane glycoprotein fucosylation

Human erythroleukemic (HEL) cells contain high activity for GDP-L-Fuc-N-acetyl-fl-D-glucosaminide a l~3fucosyl t rans ferase, although Fuca l~3GlcNAc residues are not found on the glycoproteins of HEL cells. To investigate these disparate results it was reasoned that differentiation of HEL ceils may bring about glycosylation changes in the membrane glycoproteins. Treatment with phorbol 12-myristate 13acetate (PMA) differentiates the HEL cells including the ability to adhere within a few hours whereas they normally grow in suspension culture. HEL cells were treated with 0.1 /aM PMA, labeled with L-[3H]fucose for two days as adherent cells and harvested. In contrast to HEL cells non PMA-treated, the glycopeptides derived from the PMAtreated cells contained a small amount of Fuca l~3GlcNAc residues as detected with almond al~3(4)fucosidase. At the same time, t~l~3fucosyltransferase activity in the cell extracts was similar with or without treatment. Therefore it is not the activity of al~3fucosyl transferase per se which controls the cell surface expression o f Fucal 3GlcNAc. A study of the requirements of al~3fucosyltransferase to fucosylate glycoproteins in HEL cells may provide information regarding the activation of ligands for Selectins as well as relate to the ability of other types of cells to form solid tumors at distal sites. Supported by NIH RO1 CA 37853 and Travel Award from Society for Complex Carbohydrates (L.I.S.)